A strain of mycobacterium intracellulare with strong immunogenicity and cross-immune reaction with mycobacterium tuberculosis and its application

Abstract

The present invention relates to the field of microbial technology, and in particular to a strain of intracellular mycobacterium with strong immunogenicity and cross-immune reaction with Mycobacterium tuberculosis and its application. The present invention discloses a strain of intracellular mycobacterium with strong immunogenicity and cross-reaction with Mycobacterium tuberculosis, which can well stimulate the body's cellular immunity and humoral immunity. The strain is isolated from a sputum specimen of a suspected pulmonary tuberculosis patient, obtained after cloning, culture, purification and identification, has good immunogenicity, can produce a strong cross-immune reaction with Mycobacterium tuberculosis, and can effectively activate the body's cellular immunity and humoral immunity, produce a variety of cytokines and activate related lymphocytes, thereby resisting the infection of Mycobacterium tuberculosis. The strain involved in the present invention has the potential to be used for the immune prevention and treatment of tuberculosis, and has potential practical application value.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to a strain of intracellular mycobacterium with strong immunogenicity and cross immune reaction with mycobacterium tuberculosis and application thereof. Background Art

[0002] Mycobacterium intracellulare ( Mycobacterium intracellular ) is a Mycobacterium spp. Mycobacterium Mycobacterium avium complex ( Mycobacterium avium It is a species of MAC, also commonly known as non-tuberculosis mycobacterium (NTM). It is widely found in nature and is an opportunistic pathogen. However, it can cause severe respiratory infections in patients with AIDS (Acquired immunodeficiency syndrome).

[0003] In terms of growth rate characteristics, mycobacteria are mainly divided into slow-growing mycobacteria and rapidly growing mycobacteria. In bacterial taxonomy, mycobacteria are divided into tuberculosis mycobacteria and non-tuberculosis mycobacteria and their different species. For mycobacterial antigen analysis, a study used agarose bidirectional immune diffusion experiment to divide antigens into four categories. The first category of antigens are common antigens for all mycobacteria, the second category of antigens only exist in slow-growing mycobacteria, the third category of antigens only exist in rapidly growing mycobacteria, and the fourth category of antigens are specific antigens for each species. Among them, common antigens play an important role in the immune prevention and treatment of tuberculosis. They mainly include proteins, lipids, carbohydrates and their complexes, which stimulate the body to produce cellular and humoral immune responses, but there are also some antigens that can only participate in cell-mediated immune responses and cannot produce antibodies.

[0004] Taking advantage of the low or even non-toxicity of non-tuberculous mycobacteria, strong immunogenicity, and the presence of common antigens with Mycobacterium tuberculosis, some non-tuberculous mycobacteria are widely used in the regulation of body immunity, prevention and treatment of tuberculosis, etc. Mycobacterium Phlei ) have been developed as immune enhancers and are widely used in immunotherapy for tuberculosis and other diseases. Also due to the presence of common antigens, some non-tuberculous mycobacteria can enhance the body's immune response to Mycobacterium tuberculosis and play an immune protective role. Some modified non-tuberculous mycobacteria, such as Mycobacterium smegmatis ( Mycobacterium smegmatis ) is used in the development of anti-tuberculosis vaccines.

[0005] Most mycobacteria have diverse antigens and good immunogenicity. Taking Mycobacterium phlei as an example, the content of its different components is 47.69% crude protein, 2.3% crude fat, 39.6% carbohydrates, 3.7% ash, 0.11% calcium, 0.96% phosphorus and 6.68% crude water. Some of these components have certain immunomodulatory effects, laying the foundation for mycobacterial immunomodulation. For example, some single or mixed components such as trehalose dimycolate, nucleic acid, and polysaccharides have good immunoactivity and immunomodulatory properties. Mycobacterium polysaccharides are also one of the important applications of mycobacteria to produce immunomodulatory effects. Their ingredients are simpler than whole bacterial preparations and have fewer adverse reactions. At present, a variety of BCG polysaccharide preparations and Mycobacterium phlei polysaccharide preparations have been launched or are in the clinical trial stage.

[0006] Tuberculosis (TB) is a systemic chronic infectious disease mainly caused by infection with Mycobacterium tuberculosis, mainly pulmonary tuberculosis. It is also a serious respiratory infectious disease and the disease with the highest mortality rate caused by a single infection, which seriously affects people's health, public health security and social and economic development. In order to better treat tuberculosis, it is urgent to screen more mycobacteria with strong immunogenicity and cross-immune reaction with Mycobacterium tuberculosis. Summary of the invention

[0007] The present invention provides an intracellular mycobacterium with strong immunogenicity and cross-immune reaction with Mycobacterium tuberculosis and an application thereof. The intracellular mycobacterium provided by the present invention has cross-reaction with Mycobacterium tuberculosis and strong immunogenicity, can immunosuppress the intracellular proliferation of Mycobacterium tuberculosis, has the potential to be used for the immune prevention and treatment of tuberculosis, and has good application value.

[0008] The present invention provides a Mycobacterium intracellulare ( Mycobacterium intracellulare ), which is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration Committee with the deposit number of CGMCC NO. 28620 and the classification name of intracellular Mycobacterium Mycobacterium intracellulare . The deposit date is October 12, 2023. The address of the depository unit is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101. The intracellular mycobacterium described in the present invention was isolated by the inventor from a sputum specimen of a suspected tuberculosis patient in China, and was determined after further cloning, culture, purification, identification and related functional studies, and has completely independent intellectual property rights. Compared with other intracellular mycobacteria, the serum antibodies of this strain can produce cross-immune reactions with more Mycobacterium tuberculosis proteins after immunization. The present invention provides an intracellular mycobacterium isolate with good immunogenicity and potential for immune prevention and treatment of tuberculosis. The present invention provides a new technical means for studying and screening non-tuberculosis vaccines, immune preparations, etc. that are beneficial to the prevention and treatment of tuberculosis.

[0009] According to the intracellular mycobacterium, the 16S rDNA sequence of the intracellular mycobacterium is shown as SEQ ID NO.3.

[0010] The invention provides a product, comprising the intracellular mycobacterium or its fermentation and extraction product.

[0011] According to the product, the product is an immunological preparation or a diagnostic kit.

[0012] Preferably, the diagnostic kit is used for diagnosing mycobacteria.

[0013] The mycobacterium is one or both of Mycobacterium intracellulare and Mycobacterium tuberculosis.

[0014] The present invention provides the use of the intracellular mycobacterium in preparing immune preparations or diagnostic kits.

[0015] Preferably, the immune preparation is a vaccine or an immunotherapeutic preparation.

[0016] Further preferably, the vaccine is an injectable vaccine or an oral vaccine.

[0017] Further preferably, the vaccine is any one of an inactivated vaccine, a live attenuated vaccine, a toxoid, a subunit vaccine, a polypeptide vaccine, a genetically engineered vaccine, and a DNA vaccine.

[0018] According to the application, the fermentation liquid of Mycobacterium intracellulare is treated by high pressure homogenization.

[0019] Preferably, the fermentation liquid of Mycobacterium intracellulare is treated by a high pressure homogenizer.

[0020] Preferably, the pressure of the high-pressure homogenization method is 800-1500 MPa, more preferably 1250-1350 MPa, further preferably 1300 MPa.

[0021] According to the application, the vaccine has any one of the following functions:

[0022] 1) Regulate immune function;

[0023] 2) Prevent or treat tuberculosis;

[0024] 3) Produce cross-immune reactions with Mycobacterium tuberculosis;

[0025] 4) Promote Th1 and / or Th2 immune response;

[0026] 5) triggering humoral immune response;

[0027] 6) Enhance the level of cellular immune response;

[0028] 7) Increase the number of cells positive for the surface marker CD11b;

[0029] 8) Inhibit the proliferation of Mycobacterium tuberculosis;

[0030] Preferably, 5) includes promoting the secretion of non-specific antibodies and disease-specific antibodies;

[0031] Further preferably, the non-specific antibody comprises one or more of IgG, IgG1, and IgG2a;

[0032] Preferably, 6) includes promoting the proliferation of CD4 T cells and CD8 T cells;

[0033] Preferably, 6) includes increasing the secretion of Th17 cytokines represented by IL-17.

[0034] The present invention also provides the use of the intracellular mycobacterium or the product in any of the following:

[0035] 1) Produce cross-immune reaction with Mycobacterium tuberculosis;

[0036] 2) Promote Th1 and / or Th2 immune response;

[0037] 3) triggering humoral immune response;

[0038] 4) Enhance the level of cellular immune response;

[0039] 5) Increase the number of cells positive for the surface marker CD11b;

[0040] 6) Inhibit the proliferation of Mycobacterium tuberculosis.

[0041] According to the application, 3) includes promoting the secretion of non-specific antibodies and disease-specific antibodies.

[0042] Preferably, the non-specific antibodies include one or more of IgG, IgG1, and IgG2a.

[0043] or, 4) including promoting the proliferation of CD4 T cells and CD8 T cells.

[0044] Preferably, 4) also includes increasing the secretion of Th17 cytokines represented by IL-17.

[0045] The intracellular mycobacterium described in the present invention can be used to promote Th1 and Th2 immune response reactions, but is more inclined to promote Th1 reactions and increase the secretion levels of Th1 and Th2 cytokines.

[0046] After the body is immunized with the intracellular mycobacterium preparation of the present invention, antibodies having cross-immune reactions with mycobacterium tuberculosis proteins can be produced, and cytokines having the ability to resist tuberculosis infection can be produced, thereby enhancing the body's cellular immunity and humoral immunity against mycobacterium tuberculosis. The present invention has the potential for preparing tuberculosis vaccines.

[0047] The present invention also provides the use of the intracellular mycobacterium or the product in any of the following:

[0048] 1) Enhance the body's immunity;

[0049] 2) For the immunization prevention and / or treatment of tuberculosis.

[0050] Beneficial effects of the present invention:

[0051] Compared with the prior art, the present invention has discovered a strain of intracellular mycobacterium with strong immunogenicity and cross-immune reaction with mycobacterium tuberculosis, which can inhibit the proliferation of mycobacterium tuberculosis in vitro, showing potential for the prevention of tuberculosis.

[0052] The present invention starts with the analysis of cross-immune reactions with Mycobacterium tuberculosis and expands to immunological evaluation, lymphocyte induction, etc. It comprehensively evaluates the intracellular mycobacterium isolate involved in the present invention, and evaluates in detail the immunological response generated against Mycobacterium tuberculosis after immunization, and can inhibit the proliferation of Mycobacterium tuberculosis in vitro, illustrating the potential application value of the intracellular mycobacterium involved in the present invention from multiple angles. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0054] Figure 1 The rpoB-PRA identification (A) of intracellular mycobacterium provided in Example 1 of the present invention, as well as the colony morphology (B) and acid-fast staining results (C) of mycobacterium grown on 7H9 medium. A: M: 20 bp DNA Ladder; 1: Mycobacterium abscessus ( Mycobacterium abscessus ) (control sample); 2: Mycobacterium intracellulare.

[0055] Figure 2 This is an SDS-PAGE detection diagram of the whole bacterial protein after high-pressure homogenization and fragmentation provided in Example 2 of the present invention.

[0056] Figure 3This is a graph showing the results of IgG, IgG1 and IgG2a antibody titer detection after mice were immunized with whole bacterial proteins processed by high-pressure homogenization in Example 2 of the present invention.

[0057] Figure 4 This is the KEGG functional analysis of the cross-immune reactive proteins produced in Example 2 of the present invention.

[0058] Figure 5 This is the GO function analysis of the cross-immune reactive proteins in Example 2 of the present invention.

[0059] Figure 6 This is a graph showing the results of analyzing IL-4 and IFN-γ produced by mouse spleen lymphocytes using Elispot technology in Example 3 of the present invention.

[0060] Figure 7 Example 3 of the present invention CHPC 1.5701 whole bacterial fragmented protein antigen immunization induced mouse spleen cell resistance CD4 + and CD8 + Cell proliferation results ( : P <0.05, : P <0.01).

[0061] Figure 8 This is a diagram showing the effect of immunization in Example 3 of the present invention on the proliferation of Tcm and Tem cells in mouse spleen lymphocytes.

[0062] Fig. 9 This is a graph showing changes in cytokine secretion levels of mouse lymphocytes after immunization in Example 3 of the present invention after antigen stimulation ( : P <0.05).

[0063] Fig.10 Example 3 of the present invention: CHPC 1.5701 whole bacterial fragmented protein antigen can recruit a large number of neutrophils ( : P <0.05). DETAILED DESCRIPTION

[0064] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0065] Example 1. Isolation and identification of strains

[0066] 1.1 Experimental methods

[0067] 1.1.1 Strain isolation

[0068] First, transfer 5 mL of sputum sample to the spiral tube mouth, add 1~2 times the volume of sputum sample pretreatment solution, tighten the lid, vortex and let it stand for 15 minutes. Add pH 6.8 phosphate buffer to a total volume of 40 mL, and centrifuge at 3000g for 20 minutes. Discard the supernatant, add 1 mL of phosphate buffer to resuspend the precipitate, take 0.1 mL and inoculate it into Roche solid culture medium (LJ culture medium), and culture it in a biochemical incubator at 37°C.

[0069] 1.1.2 Identification of bacterial species

[0070] (1) Acid-fast staining

[0071] Take a clean slide, add about 50μL of the pre-treated sample or bacterial sample to the center of the slide, put it under ultraviolet light for about 30 minutes, and then take it out. Add 2 to 3 drops of carbolic acid fuchsin stain to the sample in the center of the slide, and heat it with an alcohol lamp for 3 to 5 minutes. During heating, the stain should cover the sample and do not boil the stain. After the slide cools down, rinse it with running water, decolorize it with a decolorizing solution (3% hydrochloric acid + 95% alcohol solution) for 30 seconds, rinse it with running water again, re-stain it with alkaline methylene blue stain for 1 minute, wash it with water and dry it, and observe it under an oil immersion lens (10×100). It is an acid-fast staining positive bacillus.

[0072] (2) Identification of PNB / TCH Differentiation Medium

[0073] First, p-nitrobenzoic acid (PNB) culture medium was prepared according to the following formula: p-nitrobenzoic acid was dissolved in dimethylformamide or propylene glycol to prepare a 100-fold stock solution with a concentration of 50 mg / mL, and added to the unpackaged modified LJ culture medium at a ratio of 1:100 to a final concentration of 0.5 mg / mL. The solution was thoroughly mixed and packaged, and after heat coagulation sterilization and sterility inspection, it was stored at 4°C in the dark for future use.

[0074] Preparation of Thiophene-2-carboxylic acid hydrazine (TCH) medium: Dissolve Thiophene-2-carboxylic acid hydrazine in sterile distilled water to a 100-fold stock solution with a concentration of 0.5 mg / mL; add it to LJ medium at a ratio of 1:100, with a final concentration of 5 μg / mL. Other steps are the same as the PNB medium preparation procedure.

[0075] Take fresh mycobacterium cells and prepare them into 0.01 mg / mL bacterial suspension. Take 0.1 mL and evenly spread it on the slant of PNB, TCH identification medium and modified LJ medium blank control medium. Incubate in a biochemical incubator at 37°C. Observe once a day in the first week and once a week starting from the second week.

[0076] (3) rpoB -PRA Identification

[0077] First, the whole genome DNA of the sample was extracted by CTAB method. 50-100 mg of fresh culture of intracellular mycobacterium was collected in physiological saline and placed in a spiral tube, inactivated at 80°C for 30 min, aspirated and transferred to an Eppendorf tube, centrifuged at 12000 rpm for 5 min, supernatant discarded, bacterial precipitate spun up with 400 μl TE, 50 μl lysozyme (10 mg / ml) was added, mixed, and incubated at 37°C for 16-20 hours. Preheat CTAB / NaCl at 65℃, add 75μl 10%SDS+20mg / ml proteinase K mixture (70μl 10%SDS+5μl proteinase K), mix, incubate at 65℃ for 10min, add 100μl 5M NaCl, add 100μl CTAB / NaCl, vortex mix until the liquid turns milky white, incubate at 65℃ for 10min, add 750μl chloroform / isoamyl alcohol (24:1), mix by inversion, centrifuge at 12000rpm for 8-10min, transfer the supernatant to another centrifuge tube, add 0.6 times volume (450μl) of isopropanol to precipitate DNA, place at -20℃ for 30min, centrifuge at 12000rpm for 15min. Discard the supernatant, wash the DNA precipitate with 1ml 70% ice ethanol; centrifuge at 12000rpm for 5min; remove as much liquid as possible from the tube, and dry at 37℃ for 10min. Add appropriate amount of TE to dissolve the DNA precipitate and store at -80℃ for later use.

[0078] The genomic DNA of the test sample and the control sample (Mycobacterium abscessus, Mycobacterium abscessus ) genomic DNA was used as a template and primers rpoBF:TCAAGGAGAAGCGCTACGA (SEQ ID NO.1) and rpoBR:ATGTTGATCAGGGTCTGC (SEQ ID NO.2) were used to amplify rpoB Gene. Msp I and Hae III. Two endonucleases (TaKaRa, Dalian Bao Biotechnology) rpoB Gene, the reaction system is 20μL, including Msp I enzyme digestion system includes Msp I enzyme 1 μL, purified rpoBProduct 15 μL, 10×T Buffer 2 μL, 0.1% BSA 2 μL; Hae III enzyme digestion system includes Hae III 1 μL, purified rpoB 15 μL of product, 2 μL of 10×M Buffer, 2 μL of ddH2O, all incubated at 37°C for 2 h.

[0079] Agarose (4% Metaphor) was prepared using 0.5× TBE ® Agarose gel, take 10 μL of the enzyme digestion product and add it to the sample well, use 20bp DNA Ladder Marker, electrophoresis at 80V for 2h, and use an imager to capture and save after EB staining.

[0080] The Image Lab software supporting the gel imager produced by BIO-RAD Company of the United States was used for enzyme cleavage map analysis. After determining the lane, the size of the marker bands on both sides was given, and the molecular weight standard was established. The computer calculated the electrophoretic mobility and located the position of the enzyme cleavage product bands. After all the lanes and band information were given, the computer automatically calculated the molecular weight of each band of the enzyme cleavage product and compared it with the standard strain results (Table 1) for identification.

[0081] Table 1 Standard strains of nontuberculous mycobacteria involved in the present invention rpoB -PRA table

[0082]

[0083] 1.1.3 Strain purification and monoclonal strain acquisition

[0084] Select the uniform bacterial culture with good growth status from the Roche medium, scrape it with an inoculation loop, put it into 1.5 ml of 7H9 liquid medium, and perform ultrasonic dispersion for 2 times, 25 seconds each time, ultrasonic for 5 seconds, and stop for 5 seconds. At this time, no visible particles are observed visually. Filter the dispersed bacteria through a 0.6 μm filter membrane to make a single cell bacterial suspension. Take 50 μL of the bacterial suspension to coat the solid plate and culture it in a biochemical incubator at 37°C. Select 10 single colonies with good growth status and inoculate them into 5 mL of 7H9 liquid medium containing OADC and culture them until OD 600 ≈0.8, transfer to 15 ml liquid culture medium, and expand culture to OD 600 ≈1.5, keep the sample to preserve bacteria. At the same time, take samples for acid-fast staining, whole genome sequencing, etc.

[0085] 1.1.4 Whole genome sequencing analysis

[0086] The CTAB method was used to extract the whole genome of the above strains, and the samples were sent for sequencing. The whole genome sequences were compared and analyzed to further confirm that the cloned and purified strains were intracellular Mycobacterium.

[0087] 1.2 Results

[0088] 1.2.1 Strain isolation and purification

[0089] The results showed that the sputum smear was positive after acid-fast staining, and the strain to be identified obtained by sputum culture could grow in PNB, TCH, and LJ culture media, indicating that the strain to be identified was non-tuberculous mycobacterium. rpoB -PRA identification results show that Hae The band sizes (bp) after digestion with enzyme III were 180 and 90. Msp The band sizes (bp) after digestion with I enzyme were 175, 95, and 78, which were similar to the reference band sizes of intracellular mycobacteria ( Figure 1 Middle A).

[0090] After further isolation, culture, monoclonal cloning and whole genome sequencing analysis, a single stable strain was finally obtained, which was acid-fast positive and rod-shaped ( Figure 1 B and C).

[0091] 1.2.2 Whole genome sequencing analysis

[0092] After whole genome sequencing and analysis, it was further confirmed to be Mycobacterium intracellulare by comparison with multiple standard strain sequences published by NCBI. The genome size is about 5.6MB, the total number of genes is more than 5,400, and the GC content is 68.33%. 16S rRNA The gene sequence (SEQ ID NO.3) is:

[0093] 16S rRNA :

[0094]

[0095] Example 2. Analysis of immunological characteristics of CHPC1.5701 strain based on Mycobacterium tuberculosis proteome chip

[0096] 2.1 Experimental methods

[0097] 2.1.1 Preparation of whole cell protein antigen: Take fresh intracellular mycobacteria cultured in 7H9 liquid medium, heat inactivate at 80℃, collect the bacterial precipitate by centrifugation, resuspend and wash three times with sterile PBS, resuspend the washed bacterial precipitate with PBS, and perform high-pressure homogenization. Centrifuge the crushed sample to remove the precipitate, take the supernatant, filter it through a 0.22μm filter membrane, determine the protein concentration using the BCA method, take about 2μg of sample, perform SDS-PAGE electrophoresis, and store the sample at -80℃ for later use.

[0098] 2.1.2 Mouse immunization and mouse serum IgG antibody titer determination: First, a mouse immunization experiment was conducted. Mice aged 6-7 weeks were divided into 3 groups, with 5 mice in each group, namely PBS immunization group, whole bacterial protein (whole bacterial protein in 2.1.1) immunization group, and whole bacterial protein adjuvant immunization group. The adjuvant was emulsified by mixing 25% aluminum adjuvant (Thermo #77161) with whole bacterial protein solution (aqueous solution of whole bacterial protein in 2.1.1). Each experimental group of mice was immunized 3 times with 100 μg bacterial protein by subcutaneous injection, with an interval of 2 weeks between each immunization. Two weeks after the last immunization, the mice were killed, blood was collected and serum was prepared, and stored at -80°C for later use. The CHPC1.5701 protein extract was diluted with coating buffer (Na2CO3-NaHCO3 buffer) to a final concentration of 10 μg / mL, and 100 μL was added to each well of a 96-well ELISA plate and coated overnight at 4°C. Wash the plate 3 times with PBST solution. Add 200μL of 5% skim milk blocking solution to each well and incubate at 37℃ for 2h. Wash the plate 3 times with PBST solution. Take a small amount of serum sample and dilute it with PBS, add 100μL to each well, and incubate at 37℃ for 1h. Wash the plate 3 times with PBST solution, add secondary antibody, and incubate at 37℃ for 50min. Wash the plate 3 times with PBST solution. Add TMB colorimetric solution to develop color for 15min in the dark, add 2M sulfuric acid stop solution to stop the reaction, and read the absorbance value at 450nm with an enzyme reader.

[0099] 2.1.3 Cross-immunity test between immune serum and M. tuberculosis whole proteome array

[0100] The sera with the highest antibody titer among the above mouse sera were mixed, and the mixed sera were used for cross-immunity test on the whole proteome chip of Mycobacterium tuberculosis.

[0101] 1) Blocking: Take the chip out from -80℃, add blocking solution, place it on a side-swinging shaker, and incubate at room temperature for 3h;

[0102] 2) Serum sample incubation: discard the blocking solution, quickly add the test serum diluted 200 times with the incubation solution, place on a side-swinging shaker, and incubate overnight at 4°C;

[0103] 3) Washing: discard the serum incubation solution, quickly add the washing solution, place on a horizontal shaker, wash slowly at room temperature for 10 minutes, and repeat 3 times;

[0104] 4) Secondary antibody incubation: discard the washing solution, quickly add mouse IgG or IgM antibody appropriately diluted with secondary antibody incubation solution, place on a side-swinging shaker, and incubate at room temperature for 1 hour (from this step onwards, pay attention to avoid light);

[0105] 5) Washing: Discard the secondary antibody incubation solution, quickly add the washing solution, place on a horizontal shaker, wash at room temperature for 10 minutes, repeat 3 times; after drying, scan and read the slides to collect data.

[0106] The collected data were normalized and analyzed. In this experiment, IgG-SNR>1.5 and IgM-SNR>4 were set as positive proteins. Functional clustering analysis was performed on the selected proteins according to Gene Ontology (GO) and KEGG (Kyoto Encyclopedia of Genes and Genomes).

[0107] 2.2 Results

[0108] 2.2.1 SDS-PAGE detection

[0109] Since the intracellular mycobacterium envelope is complex and thick, the conventional method of fragmentation is inefficient. In the present invention, a high-pressure homogenization fragmentation method is used, with a pressure of 1300 bar and no other auxiliary components added. The fragmentation sample is taken for SDS-PAGE detection. The results are as follows: Figure 2 The labeled lane 1 is shown.

[0110] 2.2.2 Antibody levels in mice after immunization

[0111] The results showed that the IgG antibody titer of the protein immunization group reached 1:921600±446351.3 2 weeks after immunization, and the IgG antibody titer reached 1:1638400 in the presence of aluminum adjuvant. The results are expressed in log10 ( Figure 3 ).

[0112] 2.2.3 Proteome chip reaction analysis

[0113] The Mycobacterium tuberculosis whole proteome chip contains 4262 Mycobacterium tuberculosis recombinant proteins. The use of intracellular Mycobacterium serum and tuberculosis proteome chip reaction can be used to identify the types of reactive proteins and indirectly evaluate the serum antibody status after immunization. Analysis shows that the immune serum can recognize a total of 630 Mycobacterium tuberculosis proteins.

[0114] KEGG and GO functional analysis of the reactions in which these positive proteins participated showed that they were involved in metabolic pathways, nucleic acid metabolism, protein export, amino acid synthesis, secondary metabolic biosynthesis, cytoplasmic membrane, ATP binding, protein folding, etc. Figure 4 and Figure 5 ).

[0115] Example 3. Immunological characterization analysis of CHPC 1.5701

[0116] 3.1 Experimental methods

[0117] 3.1.1 Preparation of whole cell protein

[0118] The bacterial cells were cultured according to the above-mentioned embodiments, and the cells were collected after being crushed by high-pressure homogenization. The cells were filtered through a 0.22 μm filter membrane, and the protein concentration was determined by the BCA method. The samples were stored at -80°C for future use.

[0119] 3.1.2 Immunization of mice

[0120] First, a mouse immunization experiment was conducted. Mice aged 6-7 weeks were divided into 2 groups, 5 mice in each group, namely the PBS group and the whole bacterial protein immunization group. Each immunization was performed by subcutaneous injection with 100 μg / mouse CHPC 1.5701 protein for 3 times, with an interval of 2 weeks. The PBS group was inoculated with the same amount of sterile PBS at each immunization. 14 days after the last immunization, blood was collected and the spleen was sterilely removed for subsequent analysis.

[0121] 3.1.3 Isolation of mouse spleen lymphocytes

[0122] Two weeks after the last immunization, mice were killed by cervical dislocation and immersed in 75% ethanol. The mouse spleen was removed from the clean bench and placed in a 35 mm culture dish. 4 mL-5 mL of mouse lymphocyte separation solution (restored to room temperature and shaken before use) was added, ground to form a cell suspension, and immediately transferred to a 15 mL centrifuge tube and covered with 500 μL-1000 μL of RPMI 1640 culture medium (keeping the liquid surface boundary clear). Centrifuge at room temperature with a horizontal rotor at 800 g for 30 min. Set the centrifuge (Thermo ST40R) to 3 gears. After the centrifugation, the lymphocyte layer was aspirated, and 10 mL of RPMI 1640 culture medium was added, and the cells were inverted for washing. Centrifuge at room temperature at 250 g for 10 min, collect the cells, and resuspend the cells in culture medium.

[0123] 3.1.4 Evaluation of Th1 and Th2 levels of spleen lymphocytes in immunized mice using Elispot technology

[0124] Use the mouse spleen lymphocytes isolated above and adjust the cell concentration to 1×10 6 Cells were plated at 100 μL per well, and 2 μg of protein antigen was added for stimulation, 10 μL per well, protein concentration was 200 ng / μl, and the secretion levels of IL-4 and IFN-γ were detected.

[0125] 3.1.5 Flow cytometry

[0126] The concentration of spleen lymphocytes was adjusted to 1×10 7 / ml. Splenocytes were dispensed into U-bottom 96-well plates, 100µL per well, and 100µL of stimulator and blocker mixture was added, containing 2µg protein, 2µg / mL CD28 antibody, 2µg / mL CD49d antibody, and 0.2µL BFA blocker. The cells were stimulated and cultured in a 37℃ 5% CO2 incubator for 8 hours. The supernatant was discarded after centrifugation, and an antibody mixture was added, in which CD4, CD8, CD62L and CD44 fluorescently labeled antibodies were used for cell surface antigens, and IL-2, IL-4 and IFN-γ fluorescently labeled antibodies were used for cytoplasmic antigens. After the experiment, the data were acquired and analyzed using FACS Diva8.0 software.

[0127] 3.1.6 Luminax cytokine detection

[0128] Take a 96-well cell culture plate and plate 1×10 lymphocytes per well. 5 5 μg of protein filtered through 0.22 μm was added to each cell for about 24 hours, and the liquid was collected and centrifuged. The first time, centrifuged at 300 g for 10 min at 4°C, and the supernatant was collected. The second time, centrifuged at 4°C and 3000 g for 10 min, the supernatant was collected and stored at -80°C.

[0129] The above samples were tested for cytokines according to the Luminax test requirements. The operation process is briefly described as follows. Take out the kit from the refrigerator, equilibrate at room temperature for 30 minutes, prepare the standard, prepare the cleaning solution, prepare the beads, the detection antibody, and PE-streptavidin at the same time, resuspend the beads, add 50μl of the diluted beads to each well, and wash the plate twice. According to the arrangement before the experiment, add 50μl of the standard and sample to each well respectively, shake at 800rpm at room temperature for 30 minutes, place the microplate on the magnetic rack for at least 1 minute to ensure that the beads are adsorbed, wash with cleaning solution, 100μl per well, wash 3 times, add 25μl of diluted biotin-labeled detection antibody complex to each well, also at room temperature for 30 minutes, and set the shaking speed to 800rpm. Add 50μl of diluted streptavidin-labeled PE to each well, and shake at 800rpm at room temperature for 10 minutes. Place the microplate on the magnetic rack for at least 1 minute, wash with washing solution, 100µl per well, wash 3 times. Resuspend the beads with 125µl Assay Buffer, incubate for 2 minutes, set the shaking speed to about 800rpm, and test on the machine.

[0130] 3.1.7 Functional detection of CHPC 1.5701 whole bacterial protein antigen to recruit neutrophils

[0131] The subcutaneous vacuolar model of the mouse back was established by multiple injections of sterile air subcutaneously on the back of the mouse. Briefly, first, 4 mL of sterile air was injected subcutaneously on the back of the mouse, and 3 mL of sterile air was injected again 3 days later. On the 6th day, 1 mL of 0.5% CMC (carboxymethyl cellulose) solution containing or not containing CHPC 1.5701 whole bacterial protein was injected into the vacuoles. The mice were killed 24 hours after the injection, and the vacuolar fluid was collected with cold sterile PBS (PH7.4). The cells were collected separately by centrifugation at 500g for 10 minutes at 4°C. The immune cell phenotype was detected by flow cytometry, and CD11b was defined + 6G hi F4 / 80 - The cells are neutrophils, CD11b + F4 / 80 hi 6G - The cells are macrophages.

[0132] 3.2 Results

[0133] 3.2.1 CHPC 1.5701 whole bacterial protein immunization can produce a Th1-type immune response

[0134] Th1 and Th2 immune responses are two types of immune responses produced by helper T cells. Th1 immune response is a pro-inflammatory response that can lead to cell-mediated immunity, and activated cells secrete IFN-γ and IL-2. Th2 immune response is an anti-inflammatory response that mainly causes the body to produce humoral immune responses, which can cause secretions including IL-4 and IL-10. For this reason, we chose to analyze the secretion levels of IFN-γ and IL-4 to evaluate the levels of Th1 and Th2 responses they caused. The results showed that the level of IFN-γ production was significantly higher than that of IL-4, that is, the immune response produced by the spleen lymphocytes of immunized mice after antigen stimulation tended to be Th1 type ( Figure 6 ).

[0135] 3.2.2 CHPC 1.5701 whole bacterial protein immunization can enhance the level of cellular immune response

[0136] To demonstrate the changes in T cells in mice immunized with CHPC 1.5701 whole bacterial protein, flow cytometry was used to detect the multifunctional CD4 + and CD8 + The results showed that compared with the spleen cells of the control group mice, the CHPC 1.5701 whole bacterial protein antigen immunization could effectively stimulate the CD4 + T cells and CD8 + T cell proliferation ( Figure 7 ).

[0137] The small amount of effector T lymphocytes (TEM) and central memory T cells (Tcm) produced during the immune process are of great value in maintaining the body's own cellular immune activity and ability. When the same antigen enters the body again, the activated Tcm cells can produce TEM cells under antigen stimulation, while CD4 + T CM 、CD8 + T CM 、CD4 + T EM and CD8 + T EM The production of is conducive to quickly awakening the body's immune response. Figure 8 As shown, CHPC 1.5701 whole bacterial protein antigen can enhance the proliferation of Tcm and Tem cells after immunization of mice.

[0138] Further testing of Th1, Th2, and Th17 cytokines was conducted, and the results showed that CHPC1.5701 whole bacterial protein antigen immunization could significantly increase the secretion level of cytokines that are beneficial to resisting the proliferation of Mycobacterium tuberculosis ( Fig. 9 ).

[0139] 3.2.4 CHPC 1.5701 whole bacterial protein can recruit a large number of neutrophils

[0140] Flow cytometric analysis showed that 24 hours after CHPC 1.5701 whole bacterial protein injection, compared with the control group (injection of CMC only), injection of CHPC 1.5701 whole bacterial protein can cause an increase in the proportion of cells positive for the surface marker CD11b (mainly inflammatory cells such as neutrophils, monocytes and macrophages) ( P <0.05, Fig.10 Middle A).

[0141] Neutrophils are the body's first line of defense against infections such as Mycobacterium tuberculosis. They kill pathogens by phagocytosing them and then undergoing apoptosis. They can also participate in the formation of granulomas, forming extracellular traps to limit the growth and spread of pathogens such as Mycobacterium tuberculosis. The results of this experiment showed that CHPC 1.5701 whole bacterial protein injection can recruit a large number of neutrophils ( P <0.05, Fig.10 Middle B).

[0142] The present invention provides a strain of intracellular mycobacterium that has strong immunogenicity and cross-reaction with mycobacterium tuberculosis and can immunosuppress the proliferation of mycobacterium tuberculosis in cells. The strain is isolated from a sputum specimen of a suspected pulmonary tuberculosis patient, obtained after cloning, culture, purification and identification, has good immunogenicity, can effectively activate the body's cellular immunity and humoral immunity, and can produce a strong cross-immune reaction with mycobacterium tuberculosis. In vitro antibacterial tests show that it can significantly reduce the proliferation level of mycobacterium tuberculosis in spleen cells of immunized mice. The strain involved in the present invention has the potential to be used for the immune prevention and treatment of tuberculosis and has good practical application value.

[0143] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A Mycobacterium intracellulare ( Mycobacterium intracellulare ), characterized in that, It is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration with the deposit number CGMCC NO. 28620.

2. The intracellular Mycobacterium according to claim 1, characterized in that The intracellular Mycobacterium 16S rDNA sequence is shown in SEQ ID NO.

3.

3. A product, characterized in that: The method comprises the Mycobacterium intracellulare of claim 1 or 2; The product is an immunogenic agent or a diagnostic kit.

4. Use of the intracellular mycobacterium according to any one of claims 1 to 2 in the preparation of immune preparations or diagnostic kits.

5. The application according to claim 4, characterized in that: The immune preparation has any one of the following functions: 1) Produce cross-immune reaction with Mycobacterium tuberculosis; 2) triggering humoral immune response; 3) Enhance the level of cellular immune response.

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