Application of Mycobacterium bovis polysaccharide extract in anti-inflammatory or anti-endotoxemia

Abstract

The present application provides the use of Mycobacterium bovis ManLAM in the preparation of anti-inflammatory and anti-endotoxemia drugs. The Mycobacterium bovis ManLAM described in the present application is obtained by chemical degradation method or biological enzymatic hydrolysis method, and then purified by gel separation. It has significant differences in molecular weight and mannose content compared with Mycobacterium tuberculosis ManLAM, and its immunomodulatory effect is significantly better than that of Mycobacterium tuberculosis ManLAM, and it can continuously and efficiently control the inflammatory factor TNF-α at the physiological level. The results of animal experiments show that Mycobacterium bovis ManLAM can inhibit the replication of Mycobacterium bovis and Staphylococcus aureus in mice, significantly reduce the secretion of inflammatory factors TNF-α and IL-1β induced by bacteria or LPS, alleviate systemic inflammatory response and inflammatory pathological damage, and reduce the mortality of mice infected with bacteria or suffering from endotoxemia.

Description

Technical Field

[0001] This application belongs to the field of polysaccharides and the treatment of inflammatory diseases. Specifically, this application provides the use of polysaccharide extracts of Mycobacterium bovis in anti-inflammatory or anti-endotoxemia. Background Art

[0002] When the body is infected with pathogens such as bacteria, viruses, chlamydia, mycoplasma or parasites, as the infection intensifies, the body's immune system overreacts, producing a large number of cytokines, such as TNF-α and IL-1β, causing a "cytokine storm". When these cytokines attack regardless of friend or foe, they can damage the body's immune system and important organs such as the heart, kidneys, and liver, and are an important cause of acute respiratory distress syndrome and multiple organ failure. The inflammatory response is an extremely complex pathophysiological process. Antagonizing only a certain inflammatory pathway cannot change the prognosis of patients. Overall antagonism of the inflammatory response may be an option. However, in the control of the inflammatory response, hormone therapy is a controversial topic. Hormones can effectively control inflammation, but there are also adverse risks such as secondary infections and long-term complications. Therefore, there is an urgent need to develop drugs with anti-inflammatory, anti-infective and low side effects.

[0003] Mannose-capped lipoarabinomannan (ManLAM) is a high-molecular-weight amphiphilic lipoglycan present in pathogenic Mycobacterium tuberculosis and Mycobacterium bovis. ManLAM is both an immunogen and a modulator of the host immune system, and its key role during mycobacterial infection has been well demonstrated. ManLAM can be recognized by various types of receptors on innate and adaptive immune cells, including macrophages, dendritic cells, neutrophils, natural killer T cells, T cells and B cells. MamLAM has been shown to affect phagocytosis, cytokine production, antigen presentation, T cell activation and polarization, and antibody production, but the biological activities of ManLAM in inhibiting bacterial replication and cytokine storm, and reducing systemic inflammatory response and endotoxemia have not been reported yet.

[0004] Chinese Patent CN201710937104.9 relates to the extraction of Mycobacterium tuberculosis ManLAM and its application in the preparation of drugs for treating autoimmune diseases. However, there are significant differences between Mycobacterium tuberculosis and Mycobacterium bovis in this invention in terms of genome, pathogenicity, and transmission ability. There are also significant differences between Mycobacterium tuberculosis ManLAM and Mycobacterium bovis ManLAM in this invention in terms of molecular weight, mannose content, and immunomodulation. In addition, the patent claims only relate to the application of ManLAM in regulating B cell immunity and the preparation of drugs for treating autoimmune diseases, without explaining the biological function of ManLAM in regulating macrophage immunity, nor claiming the application right of ManLAM in the preparation of drugs for preventing and treating target organ damage caused by bacterial infection, systemic inflammation, and sepsis. Summary of the Invention

[0005] On the one hand, the present application provides the use of Mycobacterium bovis ManLAM in the preparation of drugs for treating inflammation.

[0006] On the other hand, the present application provides the use of Mycobacterium bovis ManLAM in the preparation of drugs for treating endotoxemia.

[0007] On the other hand, the present application provides the use of Mycobacterium bovis ManLAM in the preparation of drugs for treating tuberculosis.

[0008] On the other hand, the present application provides the use of Mycobacterium bovis ManLAM in the preparation of drugs for treating pneumonia.

[0009] On the other hand, the present application provides the use of Mycobacterium bovis ManLAM in the preparation of drugs for treating cytokine storm.

[0010] Furthermore, Mycobacterium bovis ManLAM reduces the levels of TNF-α and / or IL-1β in the administered subject.

[0011] Furthermore, the Mycobacterium bovis ManLAM is prepared using the Mycobacterium bovis N strain or the C68004 strain.

[0012] Furthermore, the molecular weight of the Mycobacterium bovis ManLAM is about 32 kDa; the mannose content is 1.53% to 2.14%.

[0013] Furthermore, the Mycobacterium bovis ManLAM is obtained by chemical degradation method or biological enzymatic hydrolysis method, and then purified by gel separation.

[0014] Furthermore, the preparation method of Mycobacterium bovis ManLAM is as follows: inactivate Mycobacterium bovis at high temperature and collect the bacterial cells by centrifugation; degrease the cells by shaking with chloroform:methanol at a volume ratio of 1:1 at 37°C for 12 h; collect the bacterial cells by centrifugation, add DNaseⅠ and RNaseA, and degrade nucleic acids by water bath at 37°C for 4 h; add Triton X-114 to a final concentration of 8%, and let it stand at 4°C for 12 hours; aspirate the upper liquid phase, place it in a sterile tube and put it in an incubator at 60°C until obvious stratification occurs, and take the lower Triton X-114 liquid phase; add ice-cold ethanol and place it in an environment at -80°C for 12 h until precipitation occurs; collect the precipitate by low-temperature centrifugation, add a protease K solution with a final concentration of 2 mg / mL, and perform a water bath at 58°C for 2 h; dialyze 3 times with distilled water, and the liquid in the dialysis bag is the mycobacterial crude sugar mixture; add the crude sugar mixture to the loading buffer for SDS-PAGE electrophoresis, stain the separating gel with pre-cooled 3% potassium chloride solution, cut out the milky white band near 32-34 kDa, add deionized water, grind, soak, centrifuge and take the supernatant, and concentrate and freeze-dry the obtained polysaccharide solution to obtain the Mycobacterium bovis ManLAM monomer.

[0015] The present invention provides the application of Mycobacterium bovis ManLAM in the preparation of antibacterial, anti-inflammatory and anti-endotoxemia drugs. The ManLAM is a lipoglycan and is one of the main components of the cell wall of pathogenic Mycobacterium bovis.

[0016] In the present invention, the molecular weight of the Mycobacterium bovis ManLAM is about 32 kDa, while the molecular weight of the Mycobacterium tuberculosis ManLAM is about 34 kDa.

[0017] In the present invention, the mannose content of ManLAM derived from the Mycobacterium bovis C68004 strain is 1.53%, the mannose content of ManLAM derived from the Mycobacterium bovis N strain is 2.14%, while the mannose content of ManLAM derived from the Mycobacterium tuberculosis H37Rv strain is only 1.39%, which is significantly lower than that of ManLAM derived from Mycobacterium bovis.

[0018] In the present invention, the ManLAM derived from the Mycobacterium bovis C68004 strain can control the TNF-α level in the macrophage culture supernatant at 229.23 pg / mL, the ManLAM derived from the Mycobacterium bovis N strain can control the TNF-α level in the macrophage culture supernatant at 126.19 pg / mL, while the ManLAM derived from the Mycobacterium tuberculosis H37Rv strain can only control the TNF-α level in the macrophage culture supernatant at 3233.50 pg / mL, and its immunomodulatory function is significantly weaker than that of ManLAM derived from Mycobacterium bovis.

[0019] In the present invention, the dosage of Mycobacterium bovis ManLAM is 1-10 mg / kg, preferably 4-6 mg / kg.

[0020] In the present invention, the Mycobacterium bovis ManLAM is obtained from Mycobacterium bovis cultured in 7H9 liquid medium through chemical degradation method and biological enzymatic hydrolysis method, and then the ManLAM monomer is obtained through gel separation and purification.

[0021] In the present invention, the bacterial infection refers to the infection of Mycobacterium bovis and Staphylococcus aureus, and the inflammatory reaction refers to the inflammatory reactions caused by all mycobacteria, Gram-positive bacteria and Gram-negative bacteria.

[0022] In the present invention, the Mycobacterium bovis ManLAM exerts its function by reducing the secretion of inflammatory factors TNF-α and IL-1β in the serum.

[0023] In the present invention, the Mycobacterium bovis ManLAM exerts its function by reducing the expression of mRNA of inflammatory factors TNF-α and IL-1β.

[0024] The present invention provides the application of Mycobacterium bovis ManLAM in the preparation of drugs for preventing and treating target organ damage caused by systemic inflammation and sepsis, and the target organs include the heart, liver, spleen, lungs, kidneys, brain, lymphatic system and blood system.

[0025] The present invention also provides the application of Mycobacterium bovis ManLAM in the preparation of drugs, and the drugs also include its acceptable carriers and excipients.

[0026] In the present invention, the dosage form of the drug is an oral or injection dosage form, including but not limited to aqueous injection, oily injection, powder injection, suspension, emulsion, pill, tablet, capsule or powder, as well as various known and studied controlled release preparations or sustained release preparations.

[0027] The ManLAM in the present invention is derived from pathogenic Mycobacterium bovis. Compared with Mycobacterium tuberculosis ManLAM, it has a smaller molecular weight but a higher mannose content, and can continuously and efficiently control the inflammatory factor TNF-α at the physiological level.

[0028] The present invention adopts classical macrophage inflammatory cell models, tuberculosis animal models, Staphylococcus aureus pneumonia animal models, and LPS-induced endotoxemia animal models, and finds that Mycobacterium bovis ManLAM has significant antibacterial, anti-inflammatory and anti-endotoxemia effects at the cellular level and animal level.

[0029] The ManLAM in the present invention is derived from pathogenic Mycobacterium bovis and includes three domains: a mannan-phosphatidyl-inositol anchor, a polysaccharide backbone composed of D-mannan and D-arabinan, and a mannan cap. ManLAM has many advantages such as safety, high efficiency, and stability, and has broad development and application prospects in preventing and treating various bacterial infections, cytokine storms, systemic inflammation, and endotoxemia.

[0030] The Mycobacterium ManLAM in this application can be used interchangeably with Mycobacterium polysaccharide, representing the same meaning - mannosylated lipoarabinomannan in Mycobacterium.

[0031] The use of "about" to limit the molecular weight in this application is restricted by the detection method and the analyte. For polysaccharide products, it is impossible to accurately measure their molecular weight, and only the approximate molecular weight or molecular weight range can be used for limitation. Brief Description of the Drawings

[0032] Figure 1 Showing the basic information and anti-inflammatory effects of ManLAM from different Mycobacterium sources, where part A is the molecular weight of ManLAM from different Mycobacterium sources; part B is the mannose content from different Mycobacterium sources; part C is the effect of ManLAM from different Mycobacterium sources on the viability of RAW264.7 cells; part D is the effect of ManLAM from different Mycobacterium sources on the secretion of TNF-α by RAW264.7 cells.

[0033] Figure 2 Showing the effects of ManLAM from different Mycobacterium sources on tuberculosis mice, where part A is the trend of mouse body weight change; part B is the statistics of mouse mortality; part C is the statistics of the bacterial load in the mouse lungs; part D is the statistics of the number of nodules and inflammatory area in the mouse lungs.

[0034] Figure 3 Showing the effects of ManLAM from different Mycobacterium sources on mice with Staphylococcus aureus pneumonia, where part A is the trend of mouse body temperature change; part B is the statistics of mouse mortality; part C is the statistics of the bacterial load in the mouse lungs; part D is the statistics of the inflammatory area in the mouse lungs.

[0035] Figure 4 Showing the effects of Mycobacterium bovis ManLAM on mice with LPS-induced systemic inflammation, where part A is the trend of mouse body temperature change; part B is the statistics of mouse mortality; part C is the statistics of the contents of serum inflammatory factors TNF-α and IL-1β in mice. Detailed Description of the Invention

[0036] The following examples facilitate a better understanding of the present invention, but are not limited thereto. These examples are for illustrative purposes only and in no way limit the protection scope of the present invention.

[0037] Preparation of Mycobacterium ManLAM in Example 1

[0038] (1) Preparation of Mycobacterium crude sugar mixture

[0039] Mycobacterium tuberculosis (Mtb) H37Rv strain, Mycobacterium bovis (M.bovis) C68004 strain and N strain were inactivated at high temperature, and the bacterial cells were collected by centrifugation; the bacterial cells were degreased by shaking with chloroform:methanol (1:1) at 37 °C for 12 h; the bacterial cells were collected by centrifugation, and DNaseⅠ and RNase A were added, and then the nucleic acids were degraded by water bath at 37 °C for 4 h; Triton X-114 was added to a final concentration of 8%, and the mixture was allowed to stand at 4 °C for 12 hours; the upper liquid phase was aspirated, placed in a sterile tube and placed in an incubator at 60 °C until obvious stratification occurred, and the lower Triton X-114 liquid phase was taken; ice ethanol was added and the mixture was placed in an environment at -80 °C for 12 h until precipitation occurred; the precipitate was collected by low-temperature centrifugation, and a protease K solution with a final concentration of 2 mg / mL was added, and the mixture was incubated in a water bath at 58 °C for 2 h; dialysis was performed 3 times with distilled water, and the liquid in the dialysis bag was the Mycobacterium crude sugar mixture. (2) Preparation of Mycobacterium ManLAM monomer

[0040] The Mycobacterium crude sugar mixture obtained in the above step was separated and purified by SDS-PAGE electrophoresis. The crude sugar mixture was added with loading buffer for SDS-PAGE electrophoresis, and the separating gel was stained with pre-cooled 3% potassium chloride solution. The milky white band near 32 - 34 kDa was cut out, ground and soaked with deionized water, and the supernatant was taken by centrifugation. The obtained polysaccharide solution was concentrated and freeze-dried to obtain the Mycobacterium ManLAM monomer.

[0041] Example 2 Detection of the molecular weight and mannose content of ManLAM from different Mycobacterium sources

[0042] (1) Kits

[0043] The PAS glycogen staining kit was purchased from Solarbio Science & Technology Co., Ltd.; the D-mannose content detection kit was purchased from Shanghai Future Industry Co., Ltd.

[0044] (2) Experimental methods

[0045] The Mycobacterium ManLAM monomer obtained in Example 1 was added with loading buffer and then subjected to SDS-PAGE electrophoresis. The separating gel was stained with the PAS glycogen staining kit to observe the molecular weight of ManLAM from different Mycobacterium sources. The results are as Figure 1 shown in -A. The molecular weight of ManLAM from Mycobacterium bovis is smaller than that from Mycobacterium tuberculosis. The mannose content in ManLAM from different Mycobacterium sources was detected with the D-mannose content detection kit. The detection results showed ( Figure 1-B), the ManLAM from Mycobacterium bovis strain C68004 has a mannose content of 1.53%, the ManLAM from Mycobacterium bovis strain N has a mannose content of 2.14%, while the ManLAM from Mycobacterium tuberculosis strain H37Rv has a mannose content of only 1.39%, which is significantly lower than that of ManLAM from Mycobacterium bovis.

[0046] Example 3 Comparison of anti-inflammatory activities of ManLAM from different mycobacteria

[0047] Using RAW264.7 cells, the effects of ManLAM from different mycobacteria on the secretion of inflammatory factors by macrophages induced by Mycobacterium bovis were compared. The specific experimental procedure is as follows:

[0048] (1) Cells and reagents

[0049] The mouse macrophage cell line RAW264.7 was purchased from the Basic Medical Cell Center of the Basic Medical College of Peking Union Medical College; the mouse TNF-α ELISA detection kit was purchased from Xinbosheng Biotechnology Co., Ltd.

[0050] (2) Experimental methods

[0051] Seed RAW264.7 cells in a 96-well cell culture plate, 10 4 cells / well, culture at 37 °C for 12 h until the cells are completely adherent. At the same time, add ManLAM from different mycobacteria at a final concentration of 10 μg / mL. After incubation for 48 h, add CCK-8 to detect the cell viability.

[0052] Seed RAW264.7 cells in a 96-well cell culture plate, 10 4 cells / well, culture at 37 °C for 12 h until the cells are completely adherent. Divide them into a blank group, a model group, and a drug administration group. Among them, the blank group contains only maintenance medium (DMEM containing 2% fetal bovine serum), the model group and the drug administration group are infected with Mycobacterium bovis (MOI = 10). After pre-incubation for 3 h, the drug administration group adds ManLAM from different mycobacteria at a final concentration of 10 μg / mL. After co-incubation for 48 h, collect the supernatant, and use the ELISA kit to determine the content of TNF-α in the cell supernatant.

[0053] The results are as Figure 1 shown. ManLAM from different mycobacteria does not affect the viability of RAW264.7 cells at a concentration of 10 μg / mL ( Figure 1-C); ManLAMs from different Mycobacterium species can significantly reduce the secretion of the inflammatory factor TNF-α at a concentration of 10 μg / mL. However, compared with ManLAM from Mycobacterium tuberculosis, ManLAM from Mycobacterium bovis can more significantly reduce the secretion of TNF-α at the same concentration. Among them, ManLAM from the N strain of Mycobacterium bovis can control the content of TNF-α at 126.19 pg / mL( Figure 1 -D). In summary, the anti-inflammatory effect of ManLAM from Mycobacterium bovis is better than that of ManLAM from Mycobacterium tuberculosis, and the anti-inflammatory effect of ManLAM from the highly pathogenic strain of Mycobacterium bovis (M. bovis N) is better than that of ManLAM from the low-pathogenic strain of Mycobacterium bovis (M. bovis C68004).

[0054] ManLAM of M. bovis C68004 was selected for the experiments in Examples 4-6 below

[0055] Example 4 Mycobacterium bovis ManLAM inhibits the production of serum inflammatory factors TNF-α and IL-1β in tuberculosis mice, alleviates pulmonary tissue pathological damage, reduces bacterial load and mouse mortality.

[0056] The experimental animals were 4-6-week-old female C57BL / 6 mice. The mice were divided into a blank group, a model group, and a Mycobacterium bovis ManLAM administration group (5 mg / kg). Both the model group and the administration group were infected by intranasal instillation with 200 CFU of Mycobacterium bovis. The administration group was instilled with Mycobacterium bovis ManLAM (5 mg / kg) intranasally, and the blank group was instilled with the corresponding volume of normal saline intranasally. Administration was performed once a week starting from 1 week after infection. The body weight and mortality of mice in each group were counted weekly. Four weeks after intranasal infection with Mycobacterium bovis, blood was collected from the eye socket. The collected whole blood was allowed to stand at 4 °C for 12 h, centrifuged at 4000 rpm for 20 min, the supernatant was collected, diluted by an appropriate multiple, and the contents of serum inflammatory factors TNF-α and IL-1β were measured. The left lung lobes of mice in each group were fixed with 10% neutral formalin. After fixation for 1 week, paraffin sections of lung tissues were made, stained with H&E, and observed under an optical microscope. The remaining lung tissues of the same weight were weighed, ground aseptically, serially diluted, and evenly spread on 7H10 solid medium. After incubation at 37 °C for 3-4 weeks, the number of colonies was counted, and the bacterial load in the lungs of mice in each group was calculated.

[0057] The results are as Figure 2 shown. The body weight of mice in the administration group did not show a significant downward trend 4 weeks after infection( Figure 2 -A); the mortality of mice in the administration group was significantly lower than that of model mice( Figure 2 -B); the contents of serum inflammatory factors TNF-α and IL-1β in mice in the administration group were significantly lower than those in model group mice( Figure 2 -C); the bacterial load in the lungs of mice in the administration group was significantly lower than that of model group mice(Figure 2 -D); The number of nodules and the inflammatory area in the lungs of the mice in the administration group were significantly smaller than those in the model group mice ( Figure 2 -E).

[0058] Example 5 Mycobacterium bovis ManLAM inhibits the production of serum inflammatory factors TNF-α and IL-1β in mice with Staphylococcus aureus pneumonia, reduces pathological damage to lung tissue, and decreases bacterial load and mouse mortality

[0059] The experimental animals were female BALB / c mice aged 4 - 6 weeks. The mice were divided into a blank group, a model group, and a Mycobacterium bovis ManLAM administration group (5 mg / kg). Both the model group and the administration group were infected by intranasal instillation with 1×10 8 CFU of Staphylococcus aureus. The administration group was intraperitoneally injected with Mycobacterium bovis ManLAM (10 mg / kg), and the blank group was intraperitoneally injected with the corresponding volume of normal saline. Medication was given once at 1 h after infection, and the body temperature changes of the mice in each group were recorded, and the mortality of the mice in each group was counted. At 96 h after intranasal instillation of Staphylococcus aureus, blood was collected from the orbital cavity. The collected whole blood was left standing at 4°C for 12 h, centrifuged at 4000 rpm for 20 min, the supernatant was collected, diluted by an appropriate multiple, and the contents of serum inflammatory factors TNF-α and IL-1β were measured. The left lung lobes of the mice in each group were fixed with 10% neutral formalin. After fixation for 1 week, paraffin sections of lung tissue were made, stained with H&E, and observed under an optical microscope. The remaining lung tissue of the same weight was weighed, ground aseptically, diluted in gradients, and evenly spread on Columbia blood agar plates. After incubation at 37°C for 12 h, the number of colonies was counted, and the bacterial load in the lungs of the mice in each group was calculated.

[0060] The results were as Figure 3 shown. Compared with the model group, the downward trend of the body temperature of the mice in the administration group was significantly improved at 24 h after infection ( Figure 3 -A); The mortality of the mice in the administration group was significantly lower than that of the model group mice ( Figure 3 -B); The contents of serum inflammatory factors TNF-α and IL-1β in the mice in the administration group were significantly lower than those in the model group mice ( Figure 3 -C); The bacterial load in the lungs of the mice in the administration group was significantly lower than that of the model group mice ( Figure 3 -D); The inflammatory area in the lungs of the mice in the administration group was significantly smaller than that of the model group mice ( Figure 3 -E).

[0061] Example 6 Mycobacterium bovis ManLAM inhibits the production of serum inflammatory factors TNF-α and IL-1β in mice with LPS-induced systemic inflammation and decreases mouse mortality

[0062] The experimental animals were 4-6-week-old female BALB / c mice, which were divided into a blank group, a model group, and a Mycobacterium bovis ManLAM administration group (5 mg / kg). The administration group was intraperitoneally injected with Mycobacterium bovis ManLAM (10 mg / kg), and the blank group was intraperitoneally injected with the corresponding volume of normal saline. The administration was continuously given 3 times before infection at an interval of 12 h. At 12 h after the last administration, both the model group and the administration group were intraperitoneally injected with LPS (30 mg / kg). The body temperature changes of the mice in each group were recorded, and the mortality rates of the mice in each group were statistically analyzed. At 96 h after nasal infection with Mycobacterium bovis, blood was collected from the eyeballs. The collected whole blood was left standing at 4 °C for 12 h, centrifuged at 4000 rpm for 20 min, the supernatant was collected, diluted to an appropriate multiple, and the contents of serum inflammatory factors TNF-α and IL-1β were measured.

[0063] The results are as Figure 4 shown. The body temperature of the mice in the administration group did not show a significant downward trend at 18 h after infection ( Figure 4 -A); the mortality rate of the mice in the administration group was significantly lower than that of the model mice ( Figure 4 -B); the contents of serum inflammatory factors TNF-α and IL-1β in the mice in the administration group were significantly lower than those in the model group ( Figure 4 -C).

[0064] As can be seen from the above examples, the present invention provides the differences between Mycobacterium bovis ManLAM and Mycobacterium tuberculosis ManLAM in terms of molecular weight, mannose content, and immunomodulation, and proves that Mycobacterium bovis ManLAM has a stronger immunomodulatory effect. The present invention also provides the application of Mycobacterium bovis ManLAM in the preparation of drugs for inhibiting cytokine storms, reducing systemic inflammation, and endotoxemia. The Mycobacterium bovis ManLAM can significantly reduce the secretion of the inflammatory factor TNF-α by RAW264.7 cells induced by Mycobacterium bovis, significantly reduce the production of serum inflammatory factors TNF-α and IL-1β in endotoxemic mice induced by Mycobacterium bovis, Staphylococcus aureus, or LPS, reduce histopathological damage, and reduce the mortality rate of mice.

Claims

1. Use of Mycobacterium bovis ManLAM ( Mycobacterium bovis ) in the preparation of a medicament for treating inflammation, wherein the Mycobacterium bovis ManLAM is prepared using Mycobacterium bovis strain N or strain C68004.

2. Use of Mycobacterium bovis ManLAM in the preparation of a medicament for treating endotoxemia, wherein the Mycobacterium bovis ManLAM is prepared using Mycobacterium bovis strain N or strain C68004.

3. Use of Mycobacterium bovis ManLAM in the preparation of a medicament for treating tuberculosis caused by Mycobacterium bovis, wherein the Mycobacterium bovis ManLAM is prepared using Mycobacterium bovis strain N or strain C68004.

4. Use of Mycobacterium bovis ManLAM in the preparation of a medicament for treating pneumonia caused by Staphylococcus aureus, wherein the Mycobacterium bovis ManLAM is prepared using Mycobacterium bovis strain N or strain C68004.

5. The use according to any one of claims 1-4, wherein the Mycobacterium bovis ManLAM has a molecular weight of 32 kDa; the mannose content is 1.53% to 2.14%.

6. The use according to any one of claims 1-4, wherein the Mycobacterium bovis ManLAM is obtained by chemical degradation method or biological enzymatic hydrolysis method, and then purified by gel separation.

7. The preparation method of the Mycobacterium bovis ManLAM according to any one of claims 1-4 is as follows: inactivate Mycobacterium bovis at high temperature, centrifuge to collect the bacterial cells; defat with chloroform:methanol at a volume ratio of 1:1 at 37 °C with shaking for 12 h; centrifuge to collect the bacterial cells, add DNaseⅠ and RNase A, and degrade nucleic acids in a water bath at 37 °C for 4 h; add Triton X-114 to a final concentration of 8%, and let stand at 4 °C for 12 hours; aspirate the upper liquid phase, place it in a sterile tube and place it in an incubator at 60 °C until obvious stratification occurs, and take the lower Triton X-114 liquid phase; add ice ethanol and place it in an environment at -80 °C for 12 h until precipitation occurs; centrifuge at low temperature to collect the precipitate, add a protease K solution with a final concentration of 2 mg / mL, and incubate in a water bath at 58 °C for 2 h; dialyze with distilled water 3 times, and the liquid in the dialysis bag is a crude sugar mixture of mycobacteria; add the crude sugar mixture to the loading buffer for SDS-PAGE electrophoresis, stain the separation gel with pre-cooled 3% potassium chloride solution, cut out the milky white band near 32-34 kDa, add deionized water, grind and soak, centrifuge to take the supernatant, concentrate and freeze-dry the obtained polysaccharide solution to obtain the monomer of mycobacterium ManLAM.

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