Application of SLAMF7 expressed CD4+T cell in preparation of tuberculosis diagnosis or treatment reagent
A technology for tuberculosis and reagents, which is applied in the field of preparation of tuberculosis diagnosis or treatment reagents, can solve problems such as drug resistance of Mycobacterium tuberculosis, and achieve the effects of reducing inflammation, easy review, and pain relief
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Embodiment 1
[0033] Example 1 The expression level of SLAMF7 on the surface of CD4+ T cells and the diagnosis of tuberculosis
[0034] Peripheral blood was collected from subjects, including 58 healthy subjects and 57 tuberculosis patients. The tuberculosis patients included 22 cases of active tuberculosis (Active TB), 16 cases of latent infection of tuberculosis (Latent TB), and 19 cases of cured tuberculosis (Cured TB).
[0035] Collect 5 mL of peripheral blood from healthy people and tuberculosis patients, lyse red blood cells to prepare single cell suspension, and adjust the total amount of cells to 5×10 6 First, add SLAMF7 antibody and CD4 antibody to 100 μL of the system, mix well and protect from light for 30 minutes, flow cytometry detection, FCS / SSC set a gate to analyze the proportion of SLAMF7 positive cells to CD4 positive cells.
[0036] The result is as figure 1 as shown, figure 1 A It can be seen that the proportion of SLMF7-positive cells in tuberculosis patients is much...
Embodiment 2
[0037]Example 2 The effect of recombinant protein activation of SLAMF7 on the secretion of IFN-γ and TNF-a by CD4+ T cells
[0038] Collect 10ml of peripheral blood from 10 active tuberculosis patients, separate PBMCs with lymphocyte separation medium; treat PBMCs with SLAMF7 recombinant protein and isotype control IgG, transfer the cells to a flow tube after 24 hours, add 1ml of reaction buffer, 1500 rpm Centrifuge for 5 min; discard supernatant.
[0039] (1) Surface staining reaction: 100 μl of reaction buffer, 2 μl of SLAMF7 antibody or isotype control antibody, and 1 μl of CD4 antibody. Incubate the above reaction tube on ice (0-4° C.) in the dark for 30 minutes. Add 1ml of reaction buffer, centrifuge at 1500 rpm for 5 minutes, repeat 3 times;
[0040] (2) Fix with 4% paraformaldehyde at room temperature for 15 minutes, and incubate for 30 minutes on ice (0-4° C.) in the dark with 1X membrane breaking agent (Permeabilization, manufacturer ebioscience).
[0041] (3) Intra...
Embodiment 3
[0044] Example 3 Effect of SLAMF7 Gene Deficiency on Cytokines Secreted by CD4+ T Cells
[0045] Splenic CD4+ T cells were sorted by magnetic beads using 4-6 week old SPF grade female C57BL / 6 wild-type (WT) mice and SLAMF7 gene deficient (SLAMF7 KO) mice, cultured in 96-well plates, and added with anti-CD3 , anti-CD28, IL-2 stimulation, cultured for three days. The expression levels of IFN-γ and TNF-a in CD4+ were detected and analyzed by flow cytometry (the method is the same as in Example 2). The concentration of anti-CD3 and CD28 is 1 ug / ml; the concentration of IL-2 is 0.25 mg / ml.
[0046] The result is as image 3 As shown in A and 3B, SLAMF7 gene deficiency significantly inhibited the expression levels of inflammatory factors IFN-γ and TNF-a produced by CD4+ T cells, indicating that the loss of SLAMF7 would inhibit the production of inflammatory factors.
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