A mycobacterium marinum infection mouse model based on t cell immune state difference and a construction method and application thereof

Abstract

The application discloses a kind of sea fish mycobacterium infection mouse model based on T cell immune state difference and its construction method and application.The method under unified infection condition (bacterial amount 1×10 4 CFU, temperature 25 DEG C, time 4 weeks), respectively, constructs the infection model of congenital T cell immune deficiency nude mouse and the thymus pentapeptide processing mouse model of acquired T cell immune regulation, by comparing and analyzing the infection phenotype and pathological changes under different immune states, for the mechanism of T cell immunity in non-tuberculous mycobacterium infection, screening immune regulation treatment drug provides standardized experimental model.

Description

Technical Field

[0001] This invention belongs to the field of animal model construction technology, specifically relating to a mouse model of marine fish infected with Mycobacterium tumefaciens based on differences in T-cell immune status and its construction method, as well as the application of this model in the study of nontuberculous mycobacterial infection mechanisms, drug screening and vaccine evaluation. Background Technology

[0002] Nontuberculous mycobacteria ( Non-tuberculous mycobacteria Nontuberculous mycobacteria (NTMs) are a class of opportunistic pathogens widely found in the natural environment, which can cause skin, soft tissue, and systemic infections. Clinical observations show that nontuberculous mycobacterial infections are more common in immunocompromised populations such as HIV patients and organ transplant recipients on long-term immunosuppressant therapy, and the infection process often exhibits a prolonged or progressive nature, making it one of the important types of opportunistic infections of clinical concern.

[0003] Mycobacterium marineis ( Mycobacterium marinum Mycobacterium tumefaciens accounts for a relatively high proportion of nontuberculous mycobacterial infections, primarily causing skin and soft tissue infections. Currently, animal models for mycobacterial infections in marine fish mainly include zebrafish, adult frogs, and mouse models. Among these, aquatic or amphibian models such as zebrafish and adult frogs have certain advantages in observing the infection process, but their immune system structure and pathophysiological characteristics differ from those of humans. In contrast, mice, as mammalian models, are closer to humans in terms of immune system composition and disease progression.

[0004] Several mouse models of Mycobacterium marinum infection have been established in the prior art, including tail vein infection models, footpad infection models, and skin infection models. For example, CN116762757B discloses a mouse model of subcutaneous infection with Mycobacterium marinum and its construction method, using 7-8 week old male C57BL / 6J mice, and administering different bacterial doses subcutaneously at 18℃ and 25℃. However, these models are mostly established in the context of immune-intact mice, focusing primarily on the regulation of infection conditions (infection route, inoculated bacterial dose, environmental temperature, and infection time), lacking a systematic description of the methods for constructing infection models under different T-cell immune states, making it difficult to conduct comparative observations of infection phenotypes corresponding to differences in immune states under uniform infection conditions.

[0005] Nude mice, due to congenital thymic hypoplasia and the absence of T-cell immune function, have been widely used in immunological research. Thymopentin (TP-5), the active fragment of thymopoietin, is an immune enhancer targeting T lymphocytes, promoting T-cell maturation and differentiation, and achieving immunomodulation in animal models with impaired immune homeostasis. However, the systematic construction and application of these two models in animal models of Mycobacterium tumefaciens infection in marine fish lack standardized descriptions, and current techniques do not cover comparative studies of immune status differences using these models under uniform infection conditions.

[0006] Existing studies and clinical observations have shown that Mycobacterium tumefaciens infection in marine fish can exhibit certain differences among different individuals, and these differences are related to the host's genetic background and immune status. The applicant has previously constructed animal models of Mycobacterium tumefaciens infection and infection models under genetic defect backgrounds. Therefore, it is necessary to provide a method for constructing animal models of Mycobacterium tumefaciens infection under different T-cell immune states to supplement the existing model system and provide experimental model support for comparing infection phenotypes under different immune states under uniform infection conditions. Summary of the Invention

[0007] In view of this, the present invention aims to overcome the problem of insufficient description of infection model construction methods under different T cell immune states in the prior art, and provides a mouse model of Mycobacterium marineis infection based on differences in T cell immune states and its construction method. The core of the present invention lies in constructing a congenital T cell immunodeficiency model and an acquired T cell immune regulation model respectively through standardized and unified infection conditions (same strain, bacterial load, infection route, environmental temperature, and observation time), thereby achieving comparability analysis of infection phenotypes under different immune states.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for constructing a mouse model of Mycobacterium marinum infection based on differences in T cell immune status, comprising the following steps: strain preparation Mycobacterium marinum ( Mycobacterium marinum The standard strain was inoculated into 7H9 broth medium containing OADC enrichment broth and cultured at 30℃, 200 rpm, and in the dark on a shaker for 5-7 days until the logarithmic growth phase (OD600 = 0.2-0.6). The bacterial concentration was then adjusted to 1×10⁻⁶. 4 CFU / 100μL, for later use; Constructing an infection model of congenital T-cell immunodeficiency Female nude mice aged 6-8 weeks were selected and divided into experimental and control groups. Under an ambient temperature of 25°C, the bacterial solution prepared in step S1 was inoculated by subcutaneous injection or tail vein injection to construct a nude mouse skin or tail vein infection model of Mycobacterium marinum in a T-cell immunodeficient background. Constructing an Acquired T-cell immune-regulated infection model Female C57BL / 6 mice aged 6-8 weeks were selected and inoculated with the bacterial culture prepared in step S1 via subcutaneous injection at an ambient temperature of 25℃. Four weeks after modeling, they were randomly divided into a thymopentin treatment group and a control group, which were injected intraperitoneally with thymopentin or an equal volume of PBS for 5 consecutive days to construct a marine fish mycobacterium infection model under the background of acquired T cell immune regulation. Model Evaluation Regularly observe and record changes in mouse skin lesions, measure the area of ​​erythema and the height of subcutaneous nodules or the depth of ulcers; after the experiment, sacrifice the mice, and take the skin lesions, spleen, liver and lungs for gross observation and pathological analysis, including HE staining and acid-fast staining, to evaluate the differences in infection phenotypes under different T cell immune states.

[0009] Preferably, the specific method of subcutaneous injection is as follows: Using a 1 mL syringe, puncture the skin on both sides of the mouse's back and near the buttocks at a 15° angle. After insertion, place the needle horizontally, lift the skin, and advance the needle slightly to the left and right to inject 100 μL of bacterial solution (1×10⁻⁶). 4 CFU).

[0010] Preferably, the specific method for tail vein injection is as follows: inoculating 200 μL of bacterial solution (1×10⁻⁶) via tail vein injection. 4 (CFU) to construct a systemic infection model.

[0011] Preferably, the dosage of the thymopentin is 4 mg / kg, and the administration volume is 200 μL.

[0012] Preferably, in the construction of each model, the ambient temperature is uniformly controlled at 25℃, and the model observation time is 4 weeks.

[0013] Secondly, the present invention provides a mouse model of Mycobacterium marinum infection based on differences in T-cell immune status, constructed by the above method, comprising: Congenital T-cell immunodeficiency models: nude mouse skin infection model and nude mouse tail vein systemic infection model; Acquired T-cell immune regulation model: C57BL / 6 mouse skin infection model with thymopentin-enhanced immunity.

[0014] Thirdly, this invention provides the application of the above-mentioned mouse model in the study of nontuberculous mycobacterial infection mechanisms, screening of anti-mycobacterial drugs, or evaluation of vaccine efficacy.

[0015] Compared with the prior art, the present invention has the following significant advantages: Standardized infection conditions enable comparability analysis: This invention is the first to perform comparability analysis under identical infection parameters (strain source, bacterial count 1×10⁻⁶). 4 Under CFU (congenital T-cell immunodeficiency) conditions, at a temperature of 25°C and an observation period of 4 weeks, two models were constructed: one for congenital T-cell immunodeficiency (nude mice) and the other for acquired T-cell immunomodulation (thymopentin treatment). This eliminated the interference of different infection conditions on the results, making the comparison of infection phenotypes between different immune states highly scientific and reliable.

[0016] Systematic construction of T-cell immunodeficiency models: This invention provides detailed construction methods for nude mouse skin infection models and tail vein systemic infection models, including key parameters such as specific bacterial load, injection volume, and observation period, filling the gap in the technology for constructing marine fish mycobacterium infection models under T-cell immunodeficiency background.

[0017] Establishment of an acquired immunomodulatory model: This invention is the first to apply thymopentin to the immune intervention of a marine fish mycobacterium infection model. By optimizing the dosage (4 mg / kg), timing of administration (4 weeks after modeling), and administration cycle (5 consecutive days), an infection model that can simulate the clinical immunomodulatory treatment scenario was established.

[0018] Clear pathological evaluation indicators: This invention establishes a comprehensive evaluation system that includes gross observation of skin lesions, spleen index measurement, HE staining histopathological analysis, and acid-fast staining bacterial load detection, which can quantitatively assess the severity of infection under different immune states.

[0019] The model has broad application prospects: It can be used to study the role of T cell immunity in nontuberculous mycobacterial infection, screen anti-infective drugs for immunocompromised populations, and evaluate the combined therapeutic effects of immunomodulators and antibacterial drugs. Attached Figure Description

[0020] Figure 1 Images show the culture and identification of Mycobacterium marinum; A: Cell pellet of Mycobacterium marinum after shaker culture; B: Acid-fast staining of Mycobacterium marinum 100× after bacterial suspension smear; C: Morphology of Mycobacterium marinum after solid culture; D: Acid-fast staining and Gram staining of Mycobacterium marinum 100× after bacterial suspension smear. Figure 2 A schematic diagram illustrating the procedure of skin injection of Mycobacterium marinum in constructing a nude mouse skin model of T-cell immunodeficiency; Figure 3 Photographs of skin lesions in the control and experimental groups within 4 weeks in a nude mouse Mm skin infection model; Figure 4Gross findings of spleen, liver, and lung in nude mice in a Mm skin infection model after 4 weeks of deterioration in the control and experimental groups (from left to right). Figure 5 Comparison of mean erythema area and mean nodule height / depth in nude mice during the modeling period in C57BL / 6 mice and nude mice with Mm skin infection models; Figure 6 A schematic diagram of the procedure for injecting Mycobacterium marinum into the tail vein of nude mice to construct a T-cell immunodeficient model of Mycobacterium marinum infection. Figure 7 Photographs of the tail injection site in nude mice in the control and experimental groups after 4 weeks of tail vein infection with Mycobacterium marinum, and gross images of the spleen, lungs, and liver after 4 weeks (from left to right). Figure 8 The skin treatment effect of mice in the acquired T cell immune regulation model during the modeling period and mice in the PBS immunization intervention period; Figure 9 The skin treatment effect of mice in the acquired T cell immune regulation model during the modeling period and the thymopentin immune intervention period; Figure 10 Gross images of the spleen, liver, and lungs of mice in the acquired T-cell immunomodulation model (control group and experimental group) one week after immunization intervention (from left to right). Figure 11 A comparison of mean erythema area and mean nodule height / depth in mice of the acquired T-cell immunomodulatory model in the control and experimental groups during the immune intervention period; Figure 12 In a nude mouse Mm skin infection model, A: Histopathological images of skin tissue after Mm skin infection in nude mice with HE staining and acid-fast staining; B: Histopathological images of liver, lung, and spleen tissues after Mm skin infection in nude mice with HE staining and acid-fast staining. Figure 13 HE staining and acid-fast staining images of liver, lung, and spleen tissues 4 weeks after nude mice were infected with Mm in the tail vein of a nude mouse model. Figure 14 Acquired T-cell immunomodulatory model mice for control and experimental groups: A: HE staining and acid-fast staining images of subcutaneous nodules (500 μm); B: Acid-fast staining images of subcutaneous nodules (50 μm); C: HE staining and acid-fast staining images of mouse liver, lungs and spleen. Detailed Implementation

[0021] To enable those skilled in the art to understand the present invention more clearly and intuitively, the present invention will be further described below with reference to the accompanying drawings.

[0022] Example 1: Culture and identification of Mycobacterium tumefaciens from marine fish Mycobacterium marinum ( Mycobacterium marinum Mn) standard strain (purchased from the standard strain library, initial concentration 1×10 6 (CFU / mL) was inoculated into 7H9 broth medium containing 10% OADC enrichment broth and cultured at 30℃, 200 rpm, and in the dark for 5-7 days on a shaker. (The OADC enrichment broth was a pale yellow transparent liquid after preparation. The 7H9 broth medium was uniformly pale amber after adding OADC. After dispensing, it was stored at 4℃ without precipitation.)

[0023] After culturing Mycobacterium tumefaciens in a shaker at 30℃ for 5-7 days, obvious yellow granular sediment was visible at the bottom of the 7H9 broth. Figure 1 A) indicates good bacterial growth. The OD600nm value of the bacterial suspension reached 0.2-0.6 (logarithmic growth phase). The colony count was verified by the plating method, and the concentration was adjusted to 1×10⁻⁶. 4 CFU / 100μL for later use.

[0024] Bacterial species identification was performed using acid-fast staining: after bacterial smears were stained with carbolic acid fuchsin, differentiated with hydrochloric acid alcohol, and counterstained with methylene blue. Under an oil microscope, red rod-shaped bacteria (2-5 μm in length and 0.2-0.5 μm in width) were observed, arranged singly or in short chains, and were positive for acid-fast staining. Figure 1 B).

[0025] After 3-4 days of solid culture of Mycobacterium marineis at 30℃, colonies can be seen forming on the culture dish. The colonies are 0.5-1 mm in diameter, round, with irregular edges, opaque, golden yellow on the front, lighter in color, raised in the middle, rough in surface, dry in texture, and easy to pick up. Gram staining shows blue-purple color. Figure 1 CD). The resuscitation success rate of *Mycobacterium marineum* after storage at -20℃ for 1 month is >90%, and the resuscitation success rate after storage at -80℃ for 3 months is >95%.

[0026] Example 2: Construction of a nude mouse skin model infected with Mycobacterium marineis with T-cell immunodeficiency Six female nude mice aged 6-8 weeks (BALB / c nude mice, SPF grade) were randomly divided into an experimental group (n=3) and a control group (n=3).

[0027] Modeling methods: such as Figure 2 As shown, the skin on both sides of the back and near the buttocks was selected as the modeling area. The experimental group was inoculated subcutaneously with 100 μL of the marine fish mycobacterium suspension from Example 1 (1×10⁻⁶). 4CFU), and the control group was injected with an equal volume of PBS. Injection method: Using a 1 mL syringe, the needle was inserted at an angle of approximately 15° to the skin. After insertion, the needle was placed horizontally, the skin was lifted, and the needle was inserted slightly to the left and right.

[0028] Feeding conditions: 25℃ ambient temperature, SPF grade feeding, modeling observation for 4 weeks.

[0029] Observation indicators: Observe and record changes in skin lesions weekly, measure the long and short diameters of erythema and calculate the area, and measure the height of subcutaneous nodules or the depth of ulcers.

[0030] Experimental Results: The PBS-treated control group showed no local infection at the injection site. In the experimental group of nude mice infected with Mycobacterium marineis (Mn), erythema and nodules appeared on the back and sacrococcygeal region one week after infection; the erythema and nodules became more pronounced two weeks after infection; some subcutaneous nodules ulcerated three weeks after infection; and by the fourth week after infection, the subcutaneous nodules had severe ulceration, with some nodules merging and causing skin erosion. Figure 3 During sample collection, punctate yellowish-white lesions appeared on the surface of the liver and lungs of the nude mice in the experimental group, and the spleen volume was slightly increased. Figure 4 ).

[0031] Compared with wild-type C57BL / 6 (Slc35e2b- / -) mice, nude mice had larger average erythema area and more pronounced average nodule height / depth, suggesting that T-cell immune deficiency led to a more severe infection. Figure 5 ).

[0032] Example 3: Construction of a nude mouse tail vein model of T-cell immunodeficient Mycobacterium marineis infection Similarly, six female nude mice aged 6-8 weeks were randomly divided into an experimental group (n=3) and a control group (n=3).

[0033] Modeling methods: such as Figure 6 As shown, the experimental group was inoculated with 200 μL of the marine fish mycobacterium suspension from Example 1 (1×10⁻⁶) via tail vein injection. 4 CFU was injected into the control group, which received an equal volume of PBS.

[0034] Feeding conditions: 25℃ ambient temperature, modeling and observation for 4 weeks.

[0035] Experimental Results: After modeling, localized redness and swelling of the tail were observed in the PBS control group, while small local lesions were observed in the experimental group infected with Mycobacterium tumefaciens. Upon sampling, localized yellowish-white lesions were observed in the spleen of the experimental group infected with Mycobacterium tumefaciens, and diffuse yellowish-white lesions were observed in the liver and lungs. Figure 7 The result indicates that the systemic infection model was successfully constructed.

[0036] Example 4: Construction of a mouse model of Mycobacterium tumefaciens infection by thymopentin immunotherapy Eight female C57BL / 6 mice aged 6-8 weeks were selected; Modeling method: Hair was removed from the back of mice using depilatory cream. Skin from both sides of the mouse's back and near the buttocks was selected as the modeling area and injected with 1×10⁻⁶ oz. 4 CFU Example 1: Mycobacterium tumefaciens from marine fish; Injection method: Use a 1ml syringe, insert the needle at an angle of about 15° to the skin, place the needle flat after insertion, lift the skin, and insert the needle slightly to the left and right.

[0037] Feeding conditions: 25℃ ambient temperature, SPF grade feeding, modeling observation for 4 weeks.

[0038] After successful model creation, the models are randomly divided into two groups (n=4): Experimental group: Thymopentin 4 mg / kg (200 μL) was injected intraperitoneally for 5 consecutive days; (It has been found that thymopentin at doses above 0.5 mg / kg can enhance the immune function of T lymphocytes in the spleen of mice. Regarding the maximum dosage in the thymopentin drug instructions, animal toxicity tests showed no adverse reactions below 10 mg / kg. Based on the conversion between human and mouse dosages, the human dose is 20 mg / 60 kg, and the mouse dose is 12 times the human dose, approximately 4 mg / kg).

[0039] Control group: PBS 200 μL was injected intraperitoneally for 5 consecutive days.

[0040] Administration method: Hold the mouse by the back of its neck with your left hand, so that its abdomen is facing up in a "head low, tail high" position. Select the lower left or lower right quadrant of the abdomen with your right hand (avoid the midline). After disinfection, insert the needle into the skin at a 30°-45° angle for 2-3 mm and then tilt it into the abdominal cavity. After aspiration shows no abnormalities, slowly inject the drug solution and quickly pull out the needle. Press with an alcohol swab to prevent leakage and put the mouse back in the cage for observation for 30 minutes.

[0041] Observation indicators: During the immune intervention, the skin lesions were observed, recorded, and photographed daily, and the area of ​​erythema and the height / depth of nodules were measured.

[0042] Experimental Results: In the control group mice, during the modeling period and the PBS immune intervention period, skin erythema and nodules gradually worsened, and some ulcers appeared. In the experimental group mice, during the thymopentin immune intervention period, erythema and nodules gradually shrank, and ulcerated skin lesions showed signs of healing. Figures 8-9 During tissue sampling, no obvious gross abnormalities were observed in the spleen, liver, and lungs of either group of mice. Figure 10 Quantitative analysis showed that the average erythema area and nodule height / depth in the experimental group were significantly smaller than those in the control group. Figure 11 ).

[0043] Example 5: Histopathological evaluation of the infection model Sample processing: After each experiment in the above embodiments, mice were euthanized by cervical dislocation, and the skin from the lesion site was cut off. The spleen, liver, and lungs were removed, and the weight and length of the spleen were measured. The tissues were fixed in 10% formalin solution for 2 days, followed by dehydration, paraffin embedding, and sectioning.

[0044] HE staining: The sections were 4-5 μm thick, dewaxed, and hydrated before being stained with hematoxylin and eosin in sequence to observe the tissue structure and inflammatory response.

[0045] Acid-fast staining: The sections were 5 μm thick, dewaxed, hydrated, and then stained with carbofuran, differentiated with hydrochloric acid alcohol, counterstained with methylene blue, and the acid-fast positive mycobacteria in the tissue were detected.

[0046] Staining results: Nude mouse skin infection model: HE staining revealed multiple infectious granulomas in the dermis, with multiple local inflammatory lesions; acid-fast staining showed a large number of pink rod-shaped acid-fast bacilli (3+) within the granulomas. Scattered caseous necrotic foci were observed in the liver and lung tissues, and the spleen contained numerous lymphocytes and macrophages, with weakly positive acid-fast staining (1+). Figure 12 AB).

[0047] Nude mouse tail vein infection model: In the experimental group of nude mice with tail vein infection, a large number of lymphocytes and macrophages infiltrated the spleen, and sheet-like caseous necrosis was observed in the liver and lungs, with acid-fast staining (2+). The PBS control group showed no obvious abnormalities and was negative for acid-fast staining. Figure 13 ).

[0048] Thymopentin immunomodulatory model: In the control group treated with PBS immunomodulatory intervention, large granulomatous structures were visible in the subcutaneous nodules, with acid-fast staining (3+); in the experimental group treated with thymopentin immunomodulatory intervention, the nodules were significantly smaller, local inflammation of the granulomas was reduced, and the lesions were absorbed, with acid-fast staining (2+). No histopathological changes were observed in the spleen, liver, and lungs of mice in both groups, and acid-fast staining was negative. Figure 14 AC).

[0049] in conclusion: 1. A model system for differential T-cell immune status under "uniform infection conditions" was successfully established. This invention achieves its goals by strictly controlling the source of the bacterial strain (standard strain) and the amount of bacteria infected (1×10⁻⁶). 4Key parameters such as CFU, ambient temperature (25℃), and observation period (4 weeks) were used to achieve, for the first time, the technical goal of constructing a comparative model by altering only the host's T-cell immune status (congenital deficiency vs. acquired enhancement) under identical exogenous infection conditions. This eliminates the interference of phenotypic variations caused by differences in infection conditions in existing technologies, making immune status the sole variable for infection differences.

[0050] 2. It was confirmed that T-cell immune status was negatively correlated with the severity of Mycobacterium tumefaciens infection in marine fish. The lower the T-cell immune function, the more severe the local skin lesions, the higher the risk of systemic dissemination, the higher the bacterial load in the tissues, and the more severe the pathological damage; conversely, after thymopentin enhances T-cell immunity, the infection is significantly controlled.

[0051] 3. A standardized model building technology solution has been developed. This invention establishes the following reproducible technical parameters: Congenital immunodeficiency model: nude mice + subcutaneous / tail vein injection + 1×10 4 CFU + 25℃ + 4 weeks; Acquired immunomodulatory model: C57BL / 6 mice + subcutaneous injection + 1×10 4 CFU + 25℃ + 4 weeks + Thymopentin 4 mg / kg intraperitoneal injection × 5 days; Evaluation index system: gross observation (area of ​​erythema, height of nodules) + organ index (spleen weight / spleen length) + HE staining (pathological damage) + acid-fast staining (bacterial load grading).

[0052] 4. Provide standardized model support for research on differences in immune status This invention overcomes the limitations of existing technologies that "only regulate infection conditions and ignore immune status," and successfully constructs: Nude mouse model: to simulate the infection characteristics of clinical T-cell immunodeficient individuals (such as those with HIV or after organ transplantation); Thymopentin model: mimicking the intervention effect of immunomodulatory therapy; Comparative analysis platform: Directly compares infection differences among different immune states under uniform conditions.

[0053] This model system can be directly applied to: Mechanism study: Elucidating the protective immune mechanism of T cells in Mycobacterium tumefaciens infection of marine fish; Drug screening: Evaluating the efficacy of anti-infective drugs against immunocompromised hosts; Vaccine evaluation: Testing the protective efficacy of the vaccine in T-cell deficiency states; Translational medicine: Providing experimental evidence for developing individualized anti-infection strategies in clinical practice.

[0054] Therefore, through systematic verification, this invention has successfully established a method for constructing a mouse model of Mycobacterium marinum infection based on differences in T-cell immune status. It has confirmed that T-cell immune function is negatively correlated with the severity of infection, formed a standardized technical solution, and achieved the invention's purpose of providing experimental model support for the study of differences in immune status. It has good scientific value and application prospects.

[0055] The above description of the embodiments is provided to enable those skilled in the art to understand and apply the present invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the embodiments described herein, and any improvements and modifications made to the present invention by those skilled in the art based on the disclosure thereof should be within the scope of protection of the present invention.

Claims

1. A mouse model of Mycobacterium tumefaciens infection based on differences in T-cell immune status, characterized in that, This includes innate T-cell immunodeficiency models and acquired T-cell immunomodulation models constructed under uniform infection parameters, respectively. The infection parameters include strain source, bacterial count (1×10⁻⁶), and bacterial quantity. 4 CFU, temperature 25℃, observation period 4 weeks; The live bacteria were derived from a standard strain of Mycobacterium tumefaciens from marine fish. The model animals used to construct the congenital T-cell immunodeficiency model were 6-8 week old female nude mice; The model animals used to construct the acquired T cell immunomodulatory model were 6-8 week old female C57BL / 6 mice, and the immunomodulatory drug was thymopentin.

2. The mouse model of Mycobacterium marinum infection based on T-cell immune status differences according to claim 1, characterized in that, The congenital T-cell immunodeficiency models include a nude mouse skin infection model and a nude mouse tail vein systemic infection model.

3. The mouse model of Mycobacterium marinum infection based on T-cell immune status differences according to claim 1, characterized in that, In the acquired T-cell immune regulation model, the dosage of thymopentin was 4 mg / kg, administered 4 weeks after modeling, and the administration period was 5 consecutive days.

4. The method for constructing a mouse model of Mycobacterium marinum infection based on differences in T-cell immune status according to any one of claims 1 to 3, characterized in that, Includes the following steps: S1: Strain Preparation The standard strain of Mycobacterium marineis was inoculated into 7H9 broth medium containing OADC enrichment broth and cultured at 30°C, 200 rpm, and in the dark on a shaker for 5-7 days until the logarithmic growth phase. The bacterial concentration was then adjusted to 1×10⁻⁶. 4 CFU / 100μL, for later use; S2: Construct two models ① Constructing an infection model of congenital T-cell immunodeficiency Female nude mice aged 6-8 weeks were selected and inoculated with the bacterial solution prepared in step S1 via subcutaneous injection or tail vein injection at an ambient temperature of 25℃. The mice were observed for 4 weeks to construct a marine fish mycobacterium infection model with congenital T cell immunodeficiency. ② Construct an acquired T cell immune regulation model Female C57BL / 6 mice aged 6-8 weeks were selected and inoculated with the bacterial culture prepared in step S1 via subcutaneous injection at an ambient temperature of 25℃. Four weeks after modeling, thymopentin was injected intraperitoneally for 5 consecutive days to construct an acquired T cell immune-regulated Mycobacterium tumefaciens infection model.

5. The construction method according to claim 4, characterized in that, The subcutaneous injection method described in step S2 is as follows: Using a 1 mL syringe, puncture the skin on both sides of the mouse's back and near the buttocks with the needle at a 15° angle to the skin. After insertion, place the needle flat, lift the skin, and insert the needle slightly to the left and right to inject 100 μL of bacterial solution.

6. The construction method according to claim 4, characterized in that, The tail vein injection method described in step S2 is as follows: 200 μL of bacterial solution is injected via tail vein to construct a systemic infection model.

7. The construction method according to claim 4, characterized in that, The dosage of thymopentin in step S3 is 4 mg / kg, and the administration volume is 200 μL.

8. The construction method according to claim 4, characterized in that, It also includes step S3: model evaluation step, which involves regularly observing and recording changes in mouse skin lesions, performing pathological analysis after the experiment, and evaluating the differences in infection phenotypes under different T cell immune states.

9. The construction method according to claim 8, characterized in that, The pathological analysis described in step S3 includes HE staining and acid-fast staining, and the evaluation indicators include gross observation of skin lesions, spleen index, histopathological changes, and bacterial load.

10. The application of the marine fish mycobacterium infection mouse model based on T cell immune status differences as described in any one of claims 1 to 3 in the study of nontuberculous mycobacterial infection mechanisms, screening of anti-mycobacterial drugs, or evaluation of vaccine efficacy.

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