Method for extracting nocardia seriolae mycolic acid and inducing foam cell model by using nocardia seriolae mycolic acid

Abstract

The invention discloses a nocardia seriolae mycotic acid extraction and foam cell model induction method, and belongs to the crossing field of aquatic disease control and pathogenic biology, and the nocardia seriolae mycotic acid extraction and foam cell model induction method comprises the following steps: S1, mycotic acid extraction, and S2, foam cell model induction. A reliable material basis is provided for follow-up research from thallus culture to high-purity extract obtaining, when an induced foam cell model is constructed, detailed and normative operation steps cover macrophage separation, microsphere coating and co-culture detection, the in-vivo infection process can be accurately simulated, all links are closely connected, the steps are clear, repeatability is high, and the method is suitable for large-scale popularization and application. The method has the advantages that the method is convenient for other researchers to use for reference and verification, multi-dimensional research design can comprehensively explore the action mechanism of mycolic acid from cell and living body levels, deep understanding of nocardia seriolae nocardia pathogenesis is facilitated, and powerful theoretical basis and technical support are provided for prevention and control of fish nocardia diseases.

Description

Technical Field

[0001] The present invention relates to the intersection of aquatic disease prevention and control and pathogenic biology, and more particularly to a method for extracting mycolic acid from yellowtail Nocardia scaly and inducing a foam cell model thereof. Background Art

[0002] Nocardiosis in fish causes a focal granulomatous lesion composed of aggregates of foam cells. In recent years, it has frequently broken out in a variety of freshwater and marine farmed fish, causing serious economic losses. Nocardia bacteria can infect a variety of animals, among which Nocardia seriola is the main pathogen of nocardiosis in fish, which can infect more than 42 species of farmed economic fish. With the expansion of the scale of fish farming in my country and the increase in species, this disease has become one of the main threats to the aquaculture industry.

[0003] Nocardia seriola is the main pathogen of nocardiosis in fish. Currently, there is relatively little research on its cell wall mycolic acids. Although it is known that it may be related to the formation of granulomatous lesions, there is a lack of in-depth research on mycolic acids, including the optimization of their extraction methods and their specific mechanisms of action in inducing foam cell aggregation to form granulomatous lesions. There are also deficiencies in the construction of related cell foaming models, making it difficult to systematically reveal the pathogenic mechanism of Nocardia seriola infection, which limits the effective prevention and control of the disease. Therefore, developing a method for extracting mycolic acids from Nocardia seriola and inducing a foam cell model has important theoretical and practical significance. Summary of the Invention

[0004] The present invention mainly provides a method for extracting mycolic acid from Nocardia seriola and inducing a foam cell model thereof, which can solve the problems of limited research depth and imperfect model construction proposed in the above background technology.

[0005] To achieve the above object, the present invention provides the following technical solution: a method for extracting mycolic acid from Nocardia seriola and inducing a foam cell model thereof, comprising the following steps: The virulent strain ZJ0503 of Nocardia serrata was cultured in liquid BHI medium at 28°C for 7 days. The cells were then saponified, acidified, and extracted with n-hexane. Mycolic acid extracts were then evaporated to dryness to obtain the mycolic acid extracts. The extracts were then derivatized and analyzed by gas chromatography-mass spectrometry to identify the chemical structure and fingerprint of the mycolic acids. Next, mycolic acids were injected intraperitoneally into snakehead fish and stimulated into spleen-derived macrophages to establish in vitro and in vivo cell foaming models. Finally, total cholesterol (TC) and free cholesterol (FC) levels were measured to calculate cholesterol ester (CE) and the CE / TC ratio. Cell slides and tissue sections were prepared and stained with Oil Red O to observe intracellular lipid accumulation and comprehensively assess the level of cell foaming.

[0006] Furthermore, the virulent strain ZJ0503 of Nocardia spp. is inoculated into liquid BHI culture medium, and after shaking culture at 28°C for 7 days, the bacteria are multiplied in large quantities and transferred to a borosilicate glass culture tube with a polytetrafluoroethylene cap, and then potassium hydroxide saponification solution is added and thoroughly shaken and mixed, and high-temperature treatment is performed at 121°C for 1 hour to promote saponification reaction of lipids in the bacterial cell wall and convert them into potassium salts of fatty acids, which facilitates subsequent separation operations.

[0007] Furthermore, the acidification is to cool the saponified bacteria to room temperature, then slowly add 3.75 mL of 1N HCl solution for acidification, then add 5 mL of n-hexane, and through vigorous shaking and standing to separate the layers, the fatty acid potassium salt is converted into fatty acid and dissolved in n-hexane, and then this step is repeated until both layers of solution are clear, and then the upper organic layer is collected and transferred to a rotary evaporator, and concentrated to dryness under reduced pressure using a rotary evaporator to obtain a yellowtail Nocardia mycobacterial acid extract.

[0008] Furthermore, the derivatization treatment is to add 0.2 mL of 2% KHCO3 solution to the mycolic acid extract sample of Nocardia serrata concentrated to dryness by rotary evaporation, and then concentrate to dryness under reduced pressure again, followed by adding 2 mL of chloroform solvent containing 100 μL of derivatization solution, heating at 90°C for 30 minutes to cause the mycolic acid to undergo a derivatization reaction to generate bromobenzoyl fatty acid ester compounds. After the sample is cooled to room temperature, 2 mL of purification solution is added for extraction, vigorously shaken, and allowed to stand for stratification. The chloroform layer is transferred to a 2 mL sample vial and blown dry using a nitrogen blower. The dried sample is stored in a refrigerator at 4°C away from light for use. When used, the sample is dissolved in 500 μL of CH2Cl2 and filtered through a 0.22 μm filter head in preparation for subsequent gas chromatography-mass spectrometry analysis.

[0009] Further, the gas chromatography-mass spectrometry analysis uses Rxi-5Sil MS / DB-5 chromatographic column, the inlet temperature is set to 300℃, the column temperature is changed according to a specific program: the initial temperature is 80℃ and maintained for 3 minutes, then increased to 300℃ at a rate of 20℃ / min, and then increased to 325℃ at a rate of 3℃ / min, the carrier gas is helium, the flow rate is 1.2mL / min, the scan range is set to 30-450 Amu / Da, the ion source temperature is 230℃, the quadrupole temperature is 150℃, and the transfer line temperature is 300℃. Through the analysis, the chemical structure composition of mycolic acid can be determined, and key data for further study of mycolic acid is provided.

[0010] Further, the polystyrene microspheres are sterilized, washed, diluted, and coated with different concentrations of mycolic acid. The mixture of polystyrene microspheres is sterilized by water bath at 70℃ for 24 hours, then washed with filtered acetate buffer, centrifuged at room temperature at 2200rpm / min to discard the supernatant, repeated 3-4 times, and the volume of the microspheres is recovered. If the microspheres are diluted to 5×10 7 mg / mL, chloroform and methanol are mixed at a volume ratio of 9:1 to prepare a solution with a final concentration of 10mg / mL, and then diluted with acetate buffer to working solutions of 0, 25, 50, 75, and 100μg / mL. The working solutions are mixed with the diluted microspheres, and coated at 37℃ water bath for 4 hours, with shaking every 30 minutes.

[0011] Further, the macrophages are separated by selecting healthy snakeheads, then anesthetizing with 0.04% tricaine, then dissecting the fish to obtain spleen tissue, carefully removing muscle tissue and fat clumps, washing the residual blood with RPMI-1640 cell culture medium containing 1% double antibody, and transferring the spleen tissue to a small sterilized glass reagent bottle, adding an appropriate amount of culture medium, then repeatedly blowing with a 1.0mL syringe to make it into a homogenate, and filtering out tissue debris through a cell filter with a pore size of 70μm. The cell suspension is transferred to a sterile centrifuge tube and diluted, and then the diluted cell suspension is gently overlaid on 1.077g / mL cell separation medium using density gradient centrifugation, then slowly increasing the speed and horizontally centrifuging at 500×g and 4℃ for 40 minutes, and then aspirating the middle white blood cell flocculation, washing three times with RPMI-1640 cell culture medium, and then counting the cells, diluting to 1×10 7 cells / mL with RPMI-1640 complete cell culture medium containing 1% double antibody and 10% fetal bovine serum, and placing in a 24-well cell culture plate, then culturing in a cell culture incubator containing 5% carbon dioxide at 25℃ for 7 days, aspirating the non-adherent cells, and finally washing off the adherent cells and diluting to 1×10 6 cells / mL for standby.

[0012] Further, the mycolic acid induced foam and granuloma formation is taken as an action factor of different concentrations of Nocardiopsis dassonvillei mycolic acid, on the one hand, an in vitro cell foam model is constructed: the spleen macrophages of Ophicephalus argus are separated by gradient density centrifugation, and the extracted Nocardiopsis dassonvillei mycolic acid (0, 25, 50 and 100 μg / mL) is coated on polystyrene microspheres as phagocytic particles, and is respectively placed with macrophages (5 × 10 6 On the other hand, an in vivo cell foam model is constructed: according to the proportion of 1 μg mycolic acid / 1 g fish weight, the mycolic acid is injected into Ophicephalus argus by intraperitoneal injection, and the head kidney, spleen and liver tissue samples of 0.1 g fish are collected at 0, 1, 3, 5 and 7 d after the attack, respectively; finally, the CE and CE / TC ratio are calculated by measuring the TC and FC content of the cells, and the intracellular lipid accumulation is observed by oil red O staining of the cell climbing sheet and the tissue section, so as to explore the cell foam process induced by mycolic acid and comprehensively reveal the action mechanism of mycolic acid from the cell and the living tissue level.

[0013] The method has the beneficial effects that: By using the specific strain and the strict extraction process, from the cultured bacteria to the high-purity extract, a reliable material basis is provided for the subsequent research, in the construction of the induced foam cell model, the detailed and standardized operation steps cover the macrophage separation, the microsphere coating and the co-culture detection, which can accurately simulate the in vivo infection process, and each link is closely connected and the steps are clear, not only the repeatability is strong, but also other researchers can refer and verify, and the multi-dimensional research design can comprehensively explore the action mechanism of mycolic acid from the cell and the living level, which helps to deeply understand the pathogenic principle of Nocardiopsis dassonvillei, and provides a strong theoretical basis and technical support for the prevention and control of fish Nocardia disease. BRIEF DESCRIPTION OF DRAWINGS

[0014] The application will be further described in detail below in combination with the drawings and specific implementation methods.

[0015] Figure 1 The figure is a process schematic diagram of the method for extracting mycolic acid of Nocardiopsis dassonvillei and inducing foam cell model of the application; Figure 2 The figure is a mass spectrum analysis schematic diagram of mycolic acid of the method for extracting mycolic acid of Nocardiopsis dassonvillei and inducing foam cell model of the application; Figure 3 The figure is a fingerprint schematic diagram of mycolic acid of the method for extracting mycolic acid of Nocardiopsis dassonvillei and inducing foam cell model of the application; Figure 4A cell foaming analysis schematic diagram of a method for extracting mycolic acid of Nocardia seriolae and inducing a foam cell model according to the present application; Figure 5 A granuloma cell foaming phenomenon schematic diagram of a method for extracting mycolic acid of Nocardia seriolae and inducing a foam cell model according to the present application; Figure 6 A clinical symptom schematic diagram of a method for extracting mycolic acid of Nocardia seriolae and inducing a foam cell model according to the present application; DETAILED DESCRIPTION

[0016] In order to make the technical solutions of the present application clearer, the present application will be further described in detail below in combination with the drawings and specific embodiments. EMBODIMENT

[0017] As shown in the drawings, Figures 1-6 According to one aspect of the present application, a technical solution is provided: a method for extracting mycolic acid of Nocardia seriolae and inducing a foam cell model, comprising the following steps: Step 1: Mycolic acid extraction and identification: Strain culture and saponification: select a virulent strain ZJ0503 of Nocardia seriolae, inoculate it into liquid BHI culture medium, and shake culture at 28°C for 7 days (as shown in the drawings Figure 1 The subsequent mycolic acid related experiments for research are based on the strain cultured in this way), after the culture is completed, the bacterial cells are transferred to a borosilicate glass culture tube with a polytetrafluoroethylene cover, potassium hydroxide saponification solution is added and mixed thoroughly by shaking, and then it is treated at 121°C for 1 hour to promote the saponification reaction of the lipids in the cell wall of the bacterial cells, converting them into potassium salt of fatty acids, preparing for the subsequent separation operation.

[0018] Acidification separation and extraction: after the saponification of the bacterial cells is completed, the solution is cooled to room temperature, 3.75mL of 1N HCl solution is slowly added for acidification treatment, and then 5mL of n-hexane is added. By means of vigorous shaking and static layering, the potassium salt of fatty acids is converted into fatty acids and dissolved in n-hexane. This step is repeated until both layers of the solution are clear. Then the upper organic layer is collected and transferred to a rotary evaporation flask. The rotary evaporator is used to reduce the pressure and concentrate to dryness, thereby obtaining the mycolic acid extract of Nocardia seriolae.

[0019] After the bacterial cells of Nocardia seriolae are cultured, saponified and acidified, the mycolic acid extract is obtained by extraction. The chemical composition of the mycolic acid extract is analyzed by HPLC, as shown in Table 1: Table 1 Chemical composition of mycolic acid of Nocardia seriolae ZJ0503 strain Derivatization treatment: 0.2 mL of 2% KHCO3 solution was added to the mycobacterium marinum mycolic acid extract sample concentrated to dryness by rotary evaporator under reduced pressure, and then reduced pressure concentration to dryness was performed again, followed by adding 2 mL of chloroform solvent containing 100 μL of derivatization solution, heating at 90°C for 30 minutes to allow the mycolic acid to undergo derivatization reaction to generate bromobenzoyl fatty acid ester compounds. After the sample cooled to room temperature, 2 mL of purification solution was added for extraction, and the chloroform layer was removed to a 2 mL sample bottle after vigorous shaking and layering. The dried sample was stored in a 4°C refrigerator in the dark for later use. When used, 500 μL of CH2Cl2 was used to dissolve the sample, and a 0.22 μm filter was used for filtration to prepare for subsequent gas chromatography-mass spectrometry analysis.

[0020] Gas chromatography-mass spectrometry analysis: Gas chromatography-mass spectrometry analysis was performed using Rxi-5Sil MS / DB-5 column, the inlet temperature was set to 300°C, and the column temperature was varied according to the specific program: the initial temperature was 80°C for 3 minutes, then increased to 300°C at a rate of 20°C / min, and then increased to 325°C at a rate of 3°C / min. The carrier gas was helium with a flow rate of 1.2 mL / min, the scan range was set to 30-450 Amu / Da, the ion source temperature was 230°C, the quadrupole temperature was 150°C, and the transfer line temperature was 300°C. Through this analysis, the chemical structure of mycolic acid could be determined, providing key data for further study of mycolic acid. The analysis results are shown in Figure 2 and Figure 3 wherein Figure 2 A is the GC-MS total ion chromatogram of mycolic acid of virulent strain ZJ0503 of Nocardia seriolae, Figure 2 B is a partial potential chemical structure diagram, Figure 3 which is the fingerprint of mycolic acid. From the figure, the relevant chemical information of mycolic acid can be directly obtained, providing an important basis for subsequent experiments.

[0021] Step 2: Induction of foam cell model construction: Polystyrene microsphere treatment and mycolic acid coating: The polystyrene microsphere mixture was sterilized by 70°C water bath for 24 hours, then washed with filtered sterile acetate buffer, centrifuged at room temperature at 2200 rpm / min to discard the supernatant, repeated 3-4 times, and the volume of the microspheres was recovered. If PBS is used to dilute the microspheres to 5×10 7 mg / mL, mix chloroform and methanol at a volume ratio of 9:1, add mycolic acid powder to prepare a solution with a final concentration of 10 mg / mL, then dilute with acetate buffer to working solutions of 0, 25, 50, 75, and 100 μg / mL, mix with the diluted microspheres respectively, and coat at 37°C water bath for 4 hours, with inversion and shaking every 30 minutes.

[0022] Macrophage isolation and culture: healthy snakehead were selected, then anesthetized with 0.04% tricaine, then the fish body was dissected to obtain the spleen tissue, the muscle tissue and fat mass were carefully removed, the residual blood was washed with 1% double-antibody-containing RPMI-1640 cell culture solution, and the spleen tissue was transferred to a small sterilized glass reagent bottle, an appropriate amount of culture solution was added, then a 1.0 mL syringe was used to repeatedly blow and beat it into a homogenate, and the tissue fragments were filtered out through a cell filter with a pore size of 70 μm. The cell suspension was transferred to a sterile centrifuge tube and diluted, and then the density gradient centrifugation method was used. The diluted cell suspension was gently overlaid on 1.077 g / mL cell separation liquid, then slowly increased speed horizontal centrifugation at 500xg and 4°C for 40 minutes, the middle white blood cell flocculation layer was aspirated, and washed with RPMI-1640 cell culture medium for three times. After cell counting, the RPMI-1640 complete cell culture medium containing 1% double antibody and 10% fetal bovine serum was diluted to 1x10 7 cells / mL, and placed in a 24-well cell culture plate, then cultured in a cell culture incubator containing 5% carbon dioxide at 25°C for 7 days, the non-adherent cells were aspirated, and finally the adherent cells were washed off and diluted to 1x10 6 cells / mL for standby.

[0023] Mycolic acid induces cell foam and granuloma formation: Induction of cell foam: polystyrene microspheres coated with different concentrations (0, 25, 50 and 100 μg / mL) of Nocardia seriolae mycolic acid were used as phagocytic particles, and were co-cultured with snakehead macrophages (5x10 6 cells / mL) at 25°C for 0, 1, 3, 5 and 7 days. After the culture ended, according to the total cholesterol (TC) enzyme method detection kit and free cholesterol (FC) enzyme method detection kit instructions, the TC and FC contents of each group of cells were detected, and the cholesterol ester (CE) and CE / TC ratio were calculated to quantitatively evaluate the foam level of macrophages. The experimental results are shown in Figure 3 , wherein Figure 4 A shows the cell foam level of spleen-derived macrophages stimulated by different concentrations of mycolic acid at different time points. Within the same time, the cell foam level increases with the increase of the coating concentration of mycolic acid; under the same coating concentration of mycolic acid, it increases with the extension of the action time; Figure 4 B and Figure 4 C are the morphological changes of mycolic acid-stimulated macrophages before and after cell foam, which can be directly observed that the cell morphology changes due to the action of mycolic acid, showing a foam state rich in lipid droplets; Inducing granuloma formation: mycolic acid was injected into the abdominal cavity of the snakehead at a proportion of 1 μg mycolic acid per 1 g fish weight, and 0.1 g of head kidney, spleen and liver tissues of the fish were collected at 0, 1, 3, 5 and 7 days after the attack, and the level of tissue cell foaming was determined by the ratio of CE / TC, as shown in Figure 4 D shows the level of cell foaming of the spleen, liver and kidney tissues of the snakehead attacked by different concentrations of mycolic acid at different time points, indicating that the level of tissue cell foaming is positively correlated with the time and concentration of mycolic acid attack; in addition, the head kidney, spleen and liver tissues (0.5×0.5×0.5 cm) at each time point were collected for histopathological section analysis, and the accumulation of lipid droplets in the granuloma cells was observed by oil red O staining, as shown in Figure 5 The same lipid accumulation phenomenon is shown in the cells in the granuloma induced by the N. seriolae bacteria and mycolic acid; the results of the pathological analysis experiment are shown in Figure 6 The mycolic acid attack of the N. seriolae causes the same clinical symptoms as the infection of the fish by the bacteria, such as abdominal swelling caused by ascites increase ( Figure 6 A), red and inflamed cloaca ( Figure 6 B), and white nodular structures in the liver, spleen and kidney tissues ( Figure 6 C).

[0024] The above examples only express several embodiments of the present application, and the description is relatively specific and detailed, but it cannot be understood as a limitation on the scope of the patent of the present application. It should be noted that for ordinary skilled persons in the art, several modifications and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. A method for extracting mycolic acid from Nocardia seriola and inducing a foam cell model thereof, characterized in that: The following steps are involved: The virulent strain ZJ0503 of Nocardia serrata was first cultured in liquid BHI medium at 28°C for 7 days. The cells were then saponified, acidified, and extracted with n-hexane. Mycolic acid extracts were then evaporated to dryness to obtain the mycolic acid extracts. The extracts were then derivatized and analyzed by gas chromatography-mass spectrometry to identify the chemical structure and fingerprint of the mycolic acids. Next, mycolic acids were injected intraperitoneally into snakehead fish and stimulated into spleen-derived macrophages to establish in vitro and in vivo cell foaming models. Finally, total cholesterol (TC) and free cholesterol (FC) levels were measured to calculate cholesterol esters (CE) and the CE / TC ratio. Cell slides and tissue sections were prepared and stained with Oil Red O to observe intracellular lipid accumulation and comprehensively assess the level of cell foaming.

2. The method for extracting mycolic acid from yellowtail Nocardia serrata and inducing a foam cell model thereof according to claim 1, characterized in that: The method comprises inoculating the Nocardia serrata ZJ0503 strain into a liquid BHI culture medium, and culturing the culture at 28° C. for 7 days to allow the bacteria to multiply in large quantities. The culture tube is then transferred to a borosilicate glass culture tube with a polytetrafluoroethylene cap, and then adding a potassium hydroxide saponification solution and fully vortexing and mixing the culture tube. The culture tube is then treated at a high temperature of 121° C. for 1 hour to promote a saponification reaction of lipids in the bacterial cell wall and convert them into potassium salts of fatty acids, thereby facilitating subsequent separation operations.

3. The method for extracting mycolic acid from yellowtail Nocardia serrata and inducing a foam cell model thereof according to claim 1, characterized in that: The acidification comprises cooling the saponified bacterial cells to room temperature, then slowly adding 3.75 mL of 1N HCl solution for acidification, then adding 5 mL of n-hexane, and performing a vigorous shaking and standing stratification operation to convert the fatty acid potassium salt into fatty acid and dissolve it in the n-hexane, and then repeating this step until both layers of solution are clear, then collecting the upper organic layer and transferring it to a rotary evaporator, and concentrating it to dryness under reduced pressure using a rotary evaporator, thereby obtaining the Nocardia seriola mycobacterium acid extract.

4. The method for extracting mycolic acid from yellowtail Nocardia serrata and inducing a foam cell model thereof according to claim 1, characterized in that: The derivatization treatment comprises adding 0.2 mL of 2% KHCO3 solution to a sample of the mycolic acid extract of Nocardia seriola that has been concentrated to dryness by a rotary evaporator under reduced pressure, and then concentrating to dryness again under reduced pressure. Subsequently, 2 mL of chloroform solvent containing 100 μL of the derivatization solution is added, and the sample is heated at 90°C for 30 minutes to cause a derivatization reaction of the mycolic acid to generate bromobenzoyl fatty acid ester compounds. After the sample is cooled to room temperature, 2 mL of a purification solution is added for extraction, and the sample is vigorously shaken and allowed to stand for stratification. The chloroform layer is transferred to a 2 mL sample vial and blown dry using a nitrogen blower. The dried sample is stored in a refrigerator at 4°C away from light until use. When used, the sample is dissolved in 500 μL of CH2Cl2 and filtered through a 0.22 μm filter head in preparation for subsequent gas chromatography-mass spectrometry analysis.

5. The method for extracting mycolic acid from yellowtail Nocardia serrata and inducing a foam cell model thereof according to claim 1, characterized in that: The gas chromatography-mass spectrometry analysis uses an Rxi-5SilMS / DB-5 chromatographic column, with the injection port temperature set at 300°C. The column temperature is varied according to a specific program: the initial temperature is maintained at 80°C for 3 minutes, then increased to 300°C at a rate of 20°C / min, and then increased to 325°C at a rate of 3°C / min. Helium is used as the carrier gas with a flow rate of 1.2 mL / min. The scan range is set to 30–450 Amu / Da, the ion source temperature is 230°C, the quadrupole temperature is 150°C, and the transfer line temperature is 300°C. Through this analysis, the chemical structure composition and fingerprint of mycolic acid can be determined, providing key data for in-depth research on mycolic acid.

6. The method for extracting mycolic acid from yellowtail Nocardia serrata and inducing a foam cell model thereof according to claim 1, characterized in that: The polystyrene microspheres are sterilized, washed and diluted, and coated with mycolic acid at different concentrations. The polystyrene microsphere mixture is sterilized in a 70°C water bath for 24 hours, and then washed with acetate buffer after filtration and sterilization, centrifuged at 2200 rpm / min at room temperature and the supernatant is discarded. This is repeated 3-4 times to restore the volume of the microspheres. If the microspheres are diluted to 5×10 7 To prepare microspheres / 100 mL, mix chloroform and methanol in a volume ratio of 9:1, add mycolic acid powder to prepare a solution with a final concentration of 10 mg / mL, and then dilute it with acetate buffer to 0, 25, 50, 75, and 100 μg / mL working solutions, mix them with the diluted microspheres respectively, and coat them in a 37°C water bath for 4 hours, shaking them upside down every 30 minutes.

7. The method for extracting mycolic acid from yellowtail Nocardia serrata and inducing a foam cell model thereof according to claim 1, characterized in that: The macrophages were isolated by selecting healthy snakehead fish, anesthetizing them with 0.04% tricaine, dissecting the fish to obtain spleen tissue, carefully removing muscle tissue and fat masses, and using RPMI-1640 cell culture medium containing 1% double antibody to clean residual blood. The spleen tissue was transferred to a small sterilized glass reagent bottle, an appropriate amount of culture medium was added, and then repeatedly blown with a 1.0 mL syringe to make it homogenous, and filtered through a 70 μm pore cell strainer to remove tissue fragments. The cell suspension was transferred to a sterile centrifuge tube and diluted, and then density gradient centrifugation was used to gently superimpose the diluted cell suspension on top of 1.077 g / mL cell separation solution, and then slowly increased the speed of horizontal centrifugation at 500 × g and 4 ° C for 40 minutes. The middle white blood cell flocculent layer was aspirated and washed three times with RPMI-1640 cell culture medium. After cell counting, it was diluted to 1×10 7 cells / mL and placed in a 24-well cell culture plate. The cells were then cultured at 25°C in a cell culture incubator containing 5% carbon dioxide for 7 days. Non-adherent cells were aspirated, and finally adherent cells were washed off and diluted to 1×10 6 cells / mL for future use.

8. The method for extracting mycolic acid from yellowtail Nocardia spp. and inducing a foam cell model thereof according to claim 1, characterized in that: The mycolic acid-induced foaming and granuloma formation is based on different concentrations of mycolic acid from Nocardia seriola as the acting factor. On the one hand, an in vitro cell foaming model is constructed: spleen macrophages of Snakehead argus are isolated by gradient density centrifugation, and the extracted mycolic acid from Nocardia seriola (0, 25, 50 and 100 μg / mL) are coated on polystyrene microspheres as phagocytic particles, which are then incubated with macrophages (5×10 6 / mL) were co-cultured at 25°C for 0, 1, 3, 5, and 7 days, and cell samples were collected. Secondly, an in vivo cell foaming model was constructed: mycolic acid was injected into the argus snakehead fish via intraperitoneal injection at a ratio of 1 μg mycolic acid per 1g fish weight, and 0.1g fish head kidney, spleen, and liver tissue samples were collected on days 0, 1, 3, 5, and 7 after infection. Finally, the CE and CE / TC ratios were calculated by measuring the TC and FC contents of the cells. Cell slides and tissue sections were prepared and stained with Oil Red O to observe the accumulation of intracellular lipids. This explored the process of mycolic acid-induced cell foaming and fully revealed the mechanism of action of mycolic acid at the cellular and living tissue levels.

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