Use of b4galt1 in preparation of drugs against mycobacterium tuberculosis infection

Abstract

The application discloses application of beta 1,4-galactosyltransferase 1 (B4GALT1) in preparation of a medicine for resisting mycobacterium tuberculosis infection and belongs to the technical field of biotechnology. The application first determines that a host functional protein B4GALT1 can improve the serum IgG galactosylation level, promote macrophage phagocytosis and killing of mycobacterium tuberculosis, and reduce the bacterial load. The B4GALT1 can be used as a new potential medicine for resisting tuberculosis infection. The application achievement can provide a new tool and thought for clinical treatment of tuberculosis, especially has a broad prospect in development and clinical application of an anti-tuberculosis medicine and can be directly applied to the scientific research field. The application has important application value for obtaining a new drug target for resisting tuberculosis and screening a new medicine.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of B4GALT1 (beta-1,4-galactosyltransferase 1), an important galactosyltransferase in eukaryotic cells, as an important target site for designing and screening anti-tuberculosis drugs in the preparation of drugs against Mycobacterium tuberculosis infection. Background Technology

[0002] Tuberculosis (TB) is a serious chronic infectious disease that severely endangers human and animal health. Caused by Mycobacterium tuberculosis (M. tb), TB is a zoonotic disease. Currently, nearly one-third of the global population is infected with Mycobacterium tuberculosis. According to the WHO's "Global Tuberculosis Report 2022," an estimated 10.6 million new TB cases were diagnosed in 2021. During 2020-2021, the TB incidence rate (new cases per 100,000 population per year) increased by 3.6%, reversing the trend of an annual decline of approximately 2% over the past 20 years. The number of deaths from TB has reached 1.6 million. my country has the third highest number of TB patients in the world.

[0003] The number of patients with multidrug-resistant tuberculosis (MDT) is increasing rapidly. Data shows that the burden of drug-resistant tuberculosis (DR-TB) also increased during 2020-2021. In 2021, there were 450,000 new cases of rifampicin-resistant tuberculosis (RR-TB) (95% CI: 399,000–501,000). Such a large number of cases underscores the urgent need to treat tuberculosis. Currently, in clinical practice, combination therapy with anti-tuberculosis drugs is often used to avoid the development of drug resistance during treatment, but this undoubtedly increases the burden on patients. Therefore, discovering new targets for anti-tuberculosis infection and developing novel anti-tuberculosis drugs have become the most pressing issues.

[0004] Immunoglobin G (IgG) is a highly abundant, multifunctional glycosylated glycoprotein in serum, secreted by plasma cells. The interaction of IgG with complement C1q and Fcγ receptors (FcγRs) on macrophages depends on the Fc region at the end of the IgG heavy chain. Each Fc region contains a conserved N-glycosylation site (Asn297). Changes in the glycosylation of this site affect its ability to bind to other proteins, thus influencing some of IgG's functions, such as opsonization and ADCC effects.

[0005] IgG N-glycosylation was quantitatively evaluated using the IgG-Gal ratio (agalactosylated (G0) vs monoalactosylated (G1) and digalactosylated (G2)G0 / (G1+G2×2)). In a mouse model of Mycobacterium tuberculosis infection, an elevated IgG-Gal ratio was observed. Furthermore, it was demonstrated that *M. tb* infection leads to degalactosylation of the host IgG Fc region. Changes in IgG Fc glycosylation affect its ability to bind to macrophage Fcγ receptors, thereby reducing the macrophage's phagocytic capacity. This suggests that *M. tb* infection can evade host immune surveillance and killing by regulating host IgG galactosylation modification.

[0006] By employing molecular biology techniques to clone and identify host genes that lead to reduced IgG galactose synthesis during Mycobacterium tuberculosis infection, important drug targets can be provided for the design and screening of novel tuberculosis drugs. This has significant theoretical and practical value for the comprehensive prevention and control of tuberculosis. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide an application of β1,4-galactosyltransferase 1 (B4GALT1), an enzyme that can significantly regulate host serum IgG galactosylation. B4GALT1 can be used as a new drug for treating tuberculosis infection.

[0008] To achieve the above objectives, the technical solution of the present invention is as follows:

[0009] This invention provides the application of B4GALT1 in the preparation of drugs against Mycobacterium tuberculosis infection. B4GALT1 is an important galactosyltransferase in eukaryotic cells. This enzyme adds UDP-galactose to the N-acetylglucosamine residue of the core glycan in N-glycoside-modified proteins (such as the antibody immunoglobulin IgG), that is, it can add one or two galactose residues to GlcNAcs (galactosylation). Subsequently, it is further modified by sialylation. B4GALT1 can be expressed in both membrane-bound and secretory forms. B4GALT1 can increase serum IgG galactosylation levels and promote macrophage phagocytosis and killing of Mycobacterium tuberculosis. The amino acid sequence of B4GALT1 is shown in SEQ ID NO:1.

[0010] Preferably, in the above applications, B4GALT1 is the active ingredient in drugs that combat Mycobacterium tuberculosis (M.tb) infection. Delivery of the B4GALT1 gene or protein into the host increases the level of B4GALT1 in the host, promotes galactosylation modification of serum IgG, thereby enhancing the opsonization and ADCC of macrophages against Mycobacterium tuberculosis.

[0011] Preferably, in the above applications, the drug includes: B4GALT1 protein itself, nucleic acid encoding B4GALT1 protein, vector expressing B4GALT1, etc.

[0012] Furthermore, this invention also provides the application of B4GALT1 as a drug screening target in screening drugs against Mycobacterium tuberculosis infection. The screening refers to selecting drugs that can promote B4GALT1 expression or enhance B4GALT1 activity, thereby promoting galactosylation modification of serum IgG, promoting macrophage opsonization, and ADCC.

[0013] In the above applications, the drug can be delivered into the host body through delivery systems such as gene delivery devices, AAVs, and liposomes to complete in vivo treatment.

[0014] This invention verified the decreased expression of B4GALT1 glycosyltransferase in peripheral blood B cells of tuberculosis-positive patients and in spleen B cells of a mouse model infected with Mycobacterium tuberculosis-H37Ra using qRT-PCR and Western blotting. Qualitative detection using specific lectin blotting confirmed the decrease in galactosylation levels of IgG in the serum of tuberculosis patients and in the serum of a mouse model infected with Mycobacterium tuberculosis-H37Ra. Primers for B4GALT1 were designed, the corresponding gene was amplified, and it was constructed into the pcDNA3.1 eukaryotic expression vector. The eukaryotic plasmid pcDNA3.1-B4GALT1 was introduced into mice using a TERESA in vivo gene delivery system, and after infecting M. tbH37Ra, mouse serum IgG and B cells were obtained. Mass spectrometry, qRT-PCR, and in vivo colony counting verified that exogenous overexpression of B4GALT1 could reverse the increase in IgG-Gal ratio induced by M. tb and reduce the bacterial load in the mouse lungs. Mycobacterium tuberculosis infection or B4GALT1 glycosyltransferase knockout leads to host IgG degalactosylation. In vitro colony counting further confirmed that IgG degalactosylation reduces the ability of macrophages to phagocytose bacteria.

[0015] The beneficial effects of this invention are as follows: This invention is the first to discover that Mycobacterium tuberculosis infection leads to a decrease in B4GALT1 and causes degalactosylation of the host IgG Fc region. This invention provides an important target gene or protein for the prevention and treatment of tuberculosis. This invention is significant in revealing the glycosylation modification of serum IgG by the host functional protein B4GALT1, thereby promoting macrophage opsonization. Using this gene or protein as a potential drug, it can promote the opsonization and killing of Mycobacterium tuberculosis by host macrophages, significantly reduce the bacterial load of Mycobacterium tuberculosis in mouse lung tissue, and control the pathogenicity of Mycobacterium tuberculosis. The results of this invention can provide new tools and ideas for the prevention and treatment of clinical tuberculosis, especially in the development and clinical application of anti-tuberculosis drugs, which have broad prospects. In addition, this achievement also has important theoretical significance for finding new drug targets and screening new drugs. Attached Figure Description

[0016] Figure 1 To compare, analyze, and verify the changes in the expression of two glycosyltransferases in B cells of tuberculosis patients and healthy volunteers.

[0017] A: The expression levels of B4GALT1 and ST6GAL1 mRNA in B cells of HDs (healthy volunteers) were significantly higher than those in tuberculosis patients.

[0018] B: The protein expression levels of B4GALT1 and ST6GAL1 in B cells of tuberculosis patients were lower than those in HDs B cells, and there were no significant changes in serum.

[0019] C: Statistical comparison of gray values ​​of target proteins B4GALT1 and ST6GAL1 with β-actin.

[0020] Figure 2 : lectin assay for glycosylation modification of IgG.

[0021] AB: Compared to HDs, TB patients (Bp (sputum-positive tuberculosis patients) and TB patients (Bn (sputum-negative tuberculosis patients)) have reduced terminal galactosylation and sialylation of IgG, and increased exposure of terminal N-acetylglucosamine of IgG.

[0022] C: Schematic diagram of IgG G0, IgG1, and IgG2 levels in TB patients compared to HDs. Red arrows indicate upregulation of IgG G0 and downregulation of IgG G1 and IgG G2 levels in TB patients.

[0023] The specific recognition of sugars by lectins is as follows:

[0024] Biotinylated Griffonia simplicifolia II (GS-II: 2 μg / mL, EY Laboratories) recognizes terminal GlcNAc.

[0025] Biotinylated Erythrina crista-galli lectin (ECL: 2 μg / mL, Vector Laboratories); recognizes β1,4-galactose;

[0026] Black elderberry lectin (biotinylated Sambucus nigra agglutinin, SNA: 2 μg / mL, Vector Laboratories) recognizes α2,6-sialic acid.

[0027] Figure 3 : To verify the expression of the eukaryotic expression vector pcDNA3.1-:B4GALT1 / ST6GAL1 in mice.

[0028] A: Schematic diagram of the mouse model.

[0029] B: WB results showed that the eukaryotic expression plasmids of B4GALT1 and ST6GAL1 could be expressed in mouse muscle cells and secreted into serum.

[0030] Seven days after plasmid injection in C:WT mice, compared with the pcDNA3.1 empty vector group, the serum IgG-Gal ratio was significantly downregulated after exogenous overexpression of B4GALT1 galactosyltransferase.

[0031] Figure 4 The effect of exogenous B4GALT1 glycosyltransferase overexpression in mouse muscle on the IgG-Gal ratio in mice infected with Mycobacterium tuberculosis-H37Ra was investigated using a gene transfer instrument.

[0032] A: Schematic diagram of the mouse infection model.

[0033] B: The introduction of eukaryotic plasmid pcDNA3.1-B4GALT1 into mice infected with H37Ra reversed the increase in IgG-Gal ratio caused by Mycobacterium tuberculosis.

[0034] C:H37Ra infection of mouse B cells decreased B4GALT1 mRNA expression, while pcDNA3.1-B4GALT1 introduction had no effect on B4GALT1 expression in B cells.

[0035] D: pcDNA3.1-B4GALT1 introduction significantly reduced the viral load of Mycobacterium tuberculosis-H37Ra in the lung tissue of H37Ra-infected mice.

[0036] Figure 5 The effect of IgG galactosylation modification on macrophage opsonization was detected by in vitro colony counting.

[0037] A: Schematic diagram of the mouse infection model.

[0038] Compared to wild-type mice, BC:H37Ra-infected and B4GALT1 knockout mice showed reduced terminal galactosylation and sialylation of serum IgG, and increased exposure of terminal N-acetylglucosamine in IgG.

[0039] D: In vitro colony counting results showed that degalactosylated IgG produced by H37Ra-infected mice and B4GALT1 knockout mice reduced the phagocytic capacity of macrophages (Mφ); grouping description: macrophage (Mφ) phagocytosis, only H37Ra, wild-type mouse IgG (IgG-WT) pre-incubated with H37Ra, H37Ra-infected mouse IgG (IgG-Ra) pre-incubated with H37Ra, B4GALT1 knockout mouse IgG (IgG-B4GALT1) + / - ) and pre-incubate with H37Ra. Detailed Implementation

[0040] The features and advantages of the present invention can be further understood through the following detailed description in conjunction with the accompanying drawings. The provided embodiments are merely illustrative of the method of the present invention and do not limit the rest of the content disclosed herein in any way.

[0041] Unless otherwise specified, all products used in the following examples are commercially available, and the operating methods are all existing conventional operating methods.

[0042] [Example 1] Quantitative RT-qPCR and Western blotting to verify changes in glycosyltransferase expression in B cells of tuberculosis patients and healthy donors.

[0043] Magnetic bead negative sorting of human peripheral blood CD19 +B cells. Take 1 mL of peripheral blood in an anticoagulant tube and dilute with an equal volume of PBS (obtained from Wuhan Medical Treatment Center); slowly add the diluted blood to a centrifuge tube containing twice the volume of lymphocyte separation medium, not exceeding 2 / 3 of the total volume of the centrifuge tube, and centrifuge at 400 g / min for 15 min (increase speed 9, decrease speed 1, 18-20℃); aspirate the cloudy liquid into a centrifuge tube and wash with PBS, centrifuge at 300 g / min for 5 min; add 3 mL of erythrocyte lysis buffer to the cell pellet for lysis for 5 min. Add 5 mL of PBS and wash 1-2 times at 300 g / 5 min; count the obtained human peripheral blood PBMCs, centrifuge at 300 g for 5 min, discarding as much supernatant as possible; resuspend the PBMCs in magnetic bead separation buffer to 5 × 10⁶. 7 Cells / mL were transferred to a 5mL sterile pre-treatment flow cytometry tube; Cocktail Enhancer (50μL / mL) was added to the sample, followed by Isolation Cocktail (50μL / mL), and the mixture was incubated at room temperature for 5 min; Magnetic beads were vortexed and added to the sample (50μL / mL), followed by magnetic bead sorting buffer, to expand the cell suspension to 2.5mL. The mixture was inverted 2-3 times to mix, and the sample tube was placed in a purple magnetic rack and incubated at room temperature for 3 min; The magnetic rack was removed and inverted once, and the cell suspension was poured into a new sterile 15mL centrifuge tube. In this cell suspension, CD19 cells were obtained through sorting. + B cells.

[0044] Real-time quantitative RT-PCR (qRT-PCR) was used to detect gene expression. Cells were washed three times with PBS, then lysed with an appropriate amount of Trizol (usually 1 mL), incubated at room temperature for 10 min, and pipetted 30-50 times (for tissues: add a certain amount of Trizol and homogenize using a homogenizer or grind the tissue with liquid nitrogen); chloroform was added at a ratio of 0.2 mL chloroform / 1 mL Trizol, vigorously shaken for about 30 seconds, and incubated at room temperature for 5-10 min; centrifuged at 4℃, 12000g for 10-15 min; 400 μL of the upper aqueous phase was aspirated, an equal volume of isopropanol was added, mixed, and incubated for 10 min; centrifuged at 4℃, 12000g for 10-15 min; 1 mL of lysate was used to lyse the cells. The RNA precipitate was washed twice with 75% ethanol prepared with DEPC water; centrifuged at 7500g for 5 minutes at 4°C; the supernatant was discarded, and the RNA precipitate was air-dried at room temperature (until it became translucent), and then dissolved in an appropriate amount of RNase-free water / DEPC water; OD260nm and OD280nm were measured with a UV spectrophotometer to calculate the RNA content and purity; the RNA was treated at 65°C for 5 minutes and then immediately placed on ice to cool; the above steps can improve the reverse transcription efficiency of RNA that easily forms higher-order structures; no 5×RT buffer or enzymes were added during the above steps.

[0045] Composition of reverse transcription reaction solution:

[0046]

[0047] First, reverse transcription was performed at 37°C for 15 minutes; then, enzyme inactivation was performed at 98°C for 5 minutes; after the reaction was completed, the product was stored at 4°C or -20°C.

[0048] qRT-PCR reaction program preparation system (20 μL):

[0049]

[0050] Amplification procedure:

[0051]

[0052] Table 1. Primers for human glycosyltransferase family qRT-PCR

[0053]

[0054] Western blotting was used to detect the expression of B4GALT1 and ST6GAL1 glycosyltransferases in B cells of TB patients. Gel preparation: Prepare the gel casting plate and mix the separating gel (10%) and stacking gel (5%). Sealing: Pour approximately 7.5 mL of separating gel into each side and immediately seal with anhydrous ethanol. When sealing with anhydrous ethanol, pour it evenly and slowly along the glass plate wall. When a dividing line appears between the water and the gel, it indicates that the gel has solidified. After solidification, pour off the top layer of sealing water and blot dry with filter paper. Pour in the stacking gel until it reaches about 1-2 mm from the top of the plate, and quickly insert a comb to avoid forming air bubbles. Allow to solidify; Assemble the electrophoresis tank: thin plates inward, thick plates outward. Remove the comb and gently blow 200μL pipette onto the sample wells to remove any flocculent precipitate, as this will affect the gel running results; Add samples: add 20μL of protein sample to each lane; Electrophoresis: place the electrophoresis tank on the electrophoresis apparatus, connect the power, and align the positive and negative electrodes. Add electrophoresis buffer, then connect the electrodes. First, stack the gel using a constant voltage of 80V. Once all samples have entered the separating gel, switch to a constant voltage of 120V. Stop electrophoresis when the bromophenol blue indicator reaches the bottom of the gel. After electrophoresis, cut the gel to a suitable size according to the marker indication and sample size, and then soak the gel in electrotransfer buffer for 15 min. For transfer: sequentially place the pre-soaked sponge, 3 thin filter papers, the cut gel, the methanol-activated PVDF membrane, 3 thin filter papers, and 1 layer of sponge into the transfer clip, ensuring each layer is properly aligned and eliminating air bubbles. For blocking: block the PVDF membrane with 5% BSA on a shaker at room temperature for 2-3 h. For primary antibody application: add a certain dilution of B4GALT1 or ST6GAL1 primary antibody; incubate overnight at 4℃, then wash the membrane 3-5 times for 10 min on a shaker using TBST. For secondary antibody application: add a certain dilution of HRP-labeled secondary antibody, react at 37℃ for 1 h, then wash the membrane 3-5 times for 10 min on a shaker using TBST. Add the mixed chromogenic solution and develop the color.

[0055] like Figure 1 The results showed that M.tb infection in humans reduced the expression levels of two glycosyltransferases, B4GALT1 and ST6GAL1, in peripheral blood B cells, but had no effect on the expression of the two glycosyltransferases in serum.

[0056] [Example 2] Determination of IgG Glycosylated Lectin

[0057] Isolate and purify serum IgG from TB patients and HDs. Turn on the UV detector and preheat for 20 min; open the software - Detection Operation - Acquisition (OM); after preheating for 20 min, adjust T% to 100%; adjust 1A to 0; equilibrate the column with starting buffer for 30 min; load the sample (serum diluted with PBS, filtered), adjusting T% to 100% and 1A to 0 again before loading. Then add the penetration buffer; after loading the sample, add the wash buffer; when the absorbance stops changing, add the elution buffer; when the absorbance rises rapidly, start collecting the purified antibody. Continue until the absorbance drops to the horizontal line; neutralize the purified liquid with 1.0M Tris-HCl at pH 9.0; ultrafilter three times, 40 min each time, at 4℃ and 5000 rpm.

[0058] Detection of glycosylation expression on the surface of IgG using lectin. SDS-PAGE electrophoresis; Primary antibody application: Add pre-prepared biotin-modified lectin primary antibody at a certain dilution ratio; incubate overnight at 4°C, remove the membrane the next day, and wash the membrane on a TBST shaker 3-5 times, 10 min each time; Secondary antibody application: After washing the membrane, add a certain dilution of HRP-labeled streptavidin secondary antibody, incubate at 37°C for 1 h, then wash the membrane on a TBST shaker 3-5 times, 10 min each time; finally, add the well-mixed chromogenic solution for development; Coomassie brilliant blue staining. At room temperature, stain the above SDS-PAGE gel with Coomassie brilliant blue staining solution for 30 min, then wash with destaining solution 3-5 times, 15 min each time, until the background becomes clear.

[0059] like Figure 2 The results showed that, compared with HDs, Bp and Bn TB patients had reduced terminal galactosylation and sialylation of IgG, and increased exposure of terminal N-acetylglucosamine in IgG. These data further demonstrate that TB patients have reduced IgG galactosylation compared with HDs.

[0060] [Example 3] Verification of the expression of the pcDNA3.1-B4GALT1 / ST6GAL1 eukaryotic expression vector in mice.

[0061] B4GALT1 and ST6GAL1 cDNA were obtained, and primers for B4GALT1 and ST6GAL1 were designed (Table 2). The corresponding genes were amplified using the following steps: The template for the mouse B4GALT1 and ST6GAL1 genes was cDNA derived from mouse spleen-derived B cell mRNA through reverse transcription. The PCR system and reaction procedure were as follows: 1 μL each of primers P1 and P2 (both 10 μM), 1 μL of genomic DNA (105 ng / μL), 25 μL of 2×Mix, and sterile deionized water to a final volume of 50 μL. The mixture was first pre-denatured at 95℃ for 3 min using Taq DNA polymerase; then denatured at 95℃ for 15 s, annealed at 60℃ for 15 s, and extended at 72℃ for 50 s, repeated for 35 cycles, and finally extended again at 72℃ for 5 min. The PCR amplification products were identified by 1% agarose gel electrophoresis and recovered and purified using a gel extraction kit. The pcDNA3.1 eukaryotic expression vector was constructed. The recovered PCR product and the empty pcDNA3.1 vector were double-digested with enzymes. The digested products were recovered using a gel extraction kit and ligated using T4 DNA ligase. The ligation products were transformed into *E. coli* DH5α competent cells using a heat shock method. The transformation products were plated on LB agar (containing 50 μg / mL ampicillin) and incubated overnight at 37°C. Single colonies were picked and inoculated into LB broth (containing 50 μg / mL ampicillin) and cultured with shaking at 37°C for 12–16 h. After preserving the bacterial strain, the plasmid was extracted using a plasmid mini-extraction kit and identified by enzyme digestion. Enzyme digestion identification and sequencing were then performed. The pcDNA3.1-B4GALT1 / ST6GAL1 plasmid was successfully constructed, double enzyme digestion identification was successful, and sequencing results showed the sequence was correct.

[0062] Table 2. PCR primers for B4GALT1 and ST6GAL1 genes

[0063]

[0064] After the pcDNA3.1-B4GALT1 / ST6GAL1 plasmid was electrotransferred into the muscle of wild-type mice using a live gene transfer instrument I (TERESA), the IgG-Gal ratio in the serum was measured. Female wild-type C57BL / 6 mice (8 weeks old) were randomly divided into 4 groups. Plasmids pcDNA3.1-B4GALT1, pcDNA3.1-ST6GAL1, or pcDNA3.1 empty vector (30 μg plasmid DNA / mouse) were electroporated into mice using a gene transducer. The mice were injected intramuscularly via the tibia, and 500 IUIL-2 was injected intramuscularly simultaneously with the plasmid injection. The mice were then electroporated with 6 pulses of 36 V / cm at 1 Hz for 50 ms, with a 1-second interval. Seven days later, blood was collected from the eyeballs of the mice and serum was separated. Western blotting was used to detect the expression of B4GALT1 and ST6GAL1 proteins in the serum, and mass spectrometry was used to detect the IgG-Gal ratio. Muscle cell lysates were separated from the mice, and the expression of B4GALT1 and ST6GAL1 proteins in the muscle cells was detected by Western blotting.

[0065] On day 7 after plasmid injection, blood and muscle tissue were collected from mice. Whole blood was incubated at 37°C for 30 min, centrifuged at 4°C for 10 min (3000 g / min), and serum was collected into new sterile EP tubes. 500 μL LRIPA was added to the muscle tissue, which was then thoroughly minced and homogenized using a grinder. Lysis was performed on ice for 30 min, with pipetting every 5 min. After centrifugation at 12000 g / min for 10 min, the supernatant was collected. Western blotting was used to detect the expression of B4GALT1 and ST6GAL1 proteins in the supernatant of the lysate.

[0066] The relative contents of IgG G0, G1, and G2 were determined by ultra-high performance liquid chromatography (UHPLC). IgG was purified using a Protein A IgG purification kit. The eluent of N-glycans extracted from IgG was dried in an Eppendorf concentrator and then labeled with 2-aminobenzamide (2-AB) as described by Maja Pucic (Pucic et al. 2011). A labeling mixture was prepared by dissolving 50 mg of 2-AB (Sigma-Aldrich) and 60 mg of sodium hydrocyanate (Sigma-Aldrich) in 1 mL of 0.7% DMSO (Sigma-Aldrich) and 0.3% glacial acetic acid (Merck) (v / v). 3 μL of the labeled mixture was added to each N-glycan sample and incubated at 60 °C for 2 h. The reaction was terminated by adding 50 μL of H2O to each sample. The labeled N-glycans were separated on a biocore system H-Class UPLC (Waters) using a fluorescence detector. The excitation wavelength was 330 nm and the emission wavelength was 420 nm. The instrument was controlled by Empower software (Waters). Labeled N-glycans were separated on a Waters BEH amino compound chromatography column (Waters), with 100 × 2.1 mm id and 1.7 μm BEH particles. 100 mm ammonium formate (pH 4.5 ± 0.05) was used as solvent A, and acetonitrile as solvent B. The separation method employed a linear gradient of 79–56% acetonitrile (v / v) at a flow rate of 0.5 mL / min for 26 min. Samples were maintained at 4 °C before injection, and the separation temperature was 60 °C. Data processing employed automated methods and a traditional integration algorithm, followed by manual correction of each chromatogram to ensure consistent integration intervals for all samples. A total of 24 sugar peaks were detected on the chromatograms. The level of each peak was normalized by dividing the area of ​​each sugar chain peak by the total area of ​​the 24 peaks (Qin et al. 2019). Glycosylation on the mass spectra was annotated using Glyco Workbench software. MALDI mass spectra were processed using Progenesis MALDI software, and the data were exported. The IgG-Gal ratio is calculated as follows: G0 / (G1+G2×2), where G0, G1, and G2 are the peak heights of the mass spectrometry peaks without galactosyl (G0) glycosides, containing one galactosyl (G1) glycoside, and containing two galactosyl (G2) glycosides, respectively. Each serum sample is analyzed in parallel three times, and the average of the three peak heights is taken and substituted into the above IgG-Gal ratio calculation formula to obtain the final IgG-Gal ratio value.

[0067] See results Figure 3 Western blot results showed that eukaryotic expression plasmids of B4GALT1 and ST6GAL1 could be expressed in mouse muscle cells and secreted into serum. Figure 3B). Furthermore, MS results showed that 7 days after injection of the B4GALT1 plasmid in WT mice, compared with the pcDNA3.1 empty vector group, exogenous overexpression of B4GALT1 galactosyltransferase significantly reduced serum IgG-Gal ratio. Figure 3 C).

[0068] [Example 4] Effects of exogenous overexpression of B4GALT1 and ST6GAL1 glycosyltransferases on serum IgG-Gal ratio and lung bacterial load in mice infected with Mycobacterium tuberculosis H37Ra.

[0069] The pcDNA3.1-B4GALT1 / ST6GAL1 plasmid was electrotransferred into the muscle tissue of mice infected with Mycobacterium tuberculosis H37Ra using a live gene transducer I (TERESA), and the serum IgG-Gal ratio was measured. Female wild-type C57BL / 6 mice (8 weeks old) were randomly divided into 5 groups. The plasmids pcDNA3.1-B4GALT1, pcDNA3.1-ST6GAL1, or the empty pcDNA3.1 vector (30 μg plasmid DNA / mouse) were electrotransferred into the mice using a gene transducer. The mice were injected intramuscularly via the tibia, and simultaneously injected with 500 IU IL-2. Six 1 Hz pulses at 36 V / cm were administered for 50 ms, with 1 second intervals between each pulse. 72 h later, live H37Ra (1 × 10⁻⁶) was injected via the tail vein. 6 Every 7 days, the plasmid was electrotransformed using a gene transfer instrument; after 14 days, blood was collected from the mouse eyeballs and serum was separated, and the IgG-Gal ratio was detected by mass spectrometry; the expression of B4GALT1 and ST6GAL1 mRNA in mouse spleen B cells was detected by qRT-PCR.

[0070] The relative contents of IgG G0, G1, and G2 were determined by ultra-high performance liquid chromatography (UHPLC). IgG was purified using a Protein A IgG purification kit. The eluent of N-glycans extracted from IgG was dried in an Eppendorf concentrator and then labeled with 2-aminobenzamide (2-AB) as described by Maja Pucic (Pucic et al. 2011). A labeling mixture was prepared by dissolving 50 mg of 2-AB (Sigma-Aldrich) and 60 mg of sodium hydrocyanate (Sigma-Aldrich) in 1 mL of 0.7% DMSO (Sigma-Aldrich) and 0.3% glacial acetic acid (Merck) (v / v). 3 μL of the labeled mixture was added to each N-glycan sample and incubated at 60 °C for 2 h. The reaction was terminated by adding 50 μL of H2O to each sample. The labeled N-glycans were separated on a biocore system H-Class UPLC (Waters) using a fluorescence detector. The excitation wavelength was 330 nm and the emission wavelength was 420 nm. The instrument was controlled by Empower software (Waters). Labeled N-glycans were separated on a Waters BEH amino compound chromatography column (Waters), with 100 × 2.1 mm id and 1.7 μm BEH particles. 100 mm ammonium formate (pH 4.5 ± 0.05) was used as solvent A, and acetonitrile as solvent B. The separation method employed a linear gradient of 79–56% acetonitrile (v / v) at a flow rate of 0.5 mL / min for 26 min. Samples were maintained at 4 °C before injection, and the separation temperature was 60 °C. Data processing employed automated methods and a traditional integration algorithm, followed by manual correction of each chromatogram to ensure consistent integration intervals for all samples. A total of 24 sugar peaks were detected on the chromatograms. The level of each peak was normalized by dividing the area of ​​each sugar chain peak by the total area of ​​the 24 peaks (Qin et al. 2019). Glycosylation on the mass spectra was annotated using Glyco Workbench software. MALDI mass spectra were processed using Progenesis MALDI software, and the data were exported. The IgG-Gal ratio is calculated as follows: G0 / (G1+G2×2), where G0, G1, and G2 are the peak heights of the mass spectrometry peaks without galactosyl (G0) glycosides, containing one galactosyl (G1) glycoside, and containing two galactosyl (G2) glycosides, respectively. Each serum sample is analyzed in parallel three times, and the average of the three peak heights is taken and substituted into the above IgG-Gal ratio calculation formula to obtain the final IgG-Gal ratio value.

[0071] In vivo bacterial colony counting in mice. Mouse lungs were harvested in a laminar flow hood, weighed, and placed in bacterial culture dishes. 2 mL of sterile PBS was added to each tissue sample, and the mixture was homogenized. This homogenate was then serially diluted 10-fold (10⁻⁶) using this stock solution. -1 10 -2 ...10 -5 Take 100 μL of bacterial suspension from each dilution, spread it on 7H10 solid medium, and incubate it in a 37℃ incubator for 15-20 days before counting the colonies.

[0072] See results Figure 4 Introducing the eukaryotic expression plasmid pcDNA3.1-B4GALT1 into mice infected with Mycobacterium tuberculosis H37Ra reversed the increase in IgG-Gal ratio caused by Mycobacterium tuberculosis infection and significantly reduced the Mycobacterium tuberculosis-H37Ra bacterial load in the lungs of H37Ra-infected mice. Figure 4 B, 4D), but had no effect on the expression of glycosyltransferases in mouse B cells ( Figure 4 C). The results showed that Mycobacterium tuberculosis H37Ra could inhibit the expression of B4GALT1 and ST6GAL1 in a mouse infection model, thereby increasing the IgG-Gal ratio, while overexpression of B4GALT1 could reduce the bacterial load in the lungs of mice.

[0073] [Example 5] In vitro colony counting assay to detect the ability of IgG galactosylation modification to induce bacterial phagocytosis in mouse bone marrow macrophages (Mφ)

[0074] Establishment of the H37Ra infection model in WT mice. Each mouse was infected with H37Ra (1×10⁻⁶) via intranasal instillation. 6 CFU / 100 μL bacteria / mouse was collected, and serum was extracted on day 15 post-infection for IgG purification and lectin blot detection. The isolation, purification, and lectin blot detection of mouse serum IgG were performed using the same method as in [Example 2].

[0075] Method for inducing and culturing mouse bone marrow macrophages. After euthanizing the mice, under aseptic conditions, collect the femur and tibia. Disinfect the tibia with 75% alcohol, generally spraying or soaking for one minute, then rinse with culture medium. Take several milliliters of ice-cold 1640 culture medium (containing serum, etc.), cut off both ends of the tibia, and wash out the bone marrow cells from the tibia with 1640 culture medium. Linear red bone marrow cells will be visible. Slowly pipette the cells to form a single-cell suspension; centrifuge at 4℃, 300G for 5 minutes, discard the supernatant, resuspend in 5 mL of AKT, and pipette to break down the red blots for no more than one minute. Centrifuge at 4℃, 300G for 3 minutes, take at least 5 times the volume of ice-cold 1640 culture medium (containing serum, etc.), wash twice at 4℃, 300G, and finally resuspend in DMEM for counting. The age of the mice should not be too old, 6-10 weeks is appropriate. After cell counting, adjust the resuspended concentration to 2×10⁻⁶. 6Add 30-50 ng / mL of mouse M-CSF, mix well, and seed 2 mL per well. Incubate for three days. Generally, a small number of macrophages will be successfully induced and adhere after 24 hours. After three days, a large number of macrophages will be observed adhering, occupying about half of the field of view. Prepare DMEM with a final concentration of 30-50 ng / mL M-CSF and preheat at 37°C for several minutes. Directly aspirate and discard the macrophage supernatant, add preheated DMEM containing M-CSF, and continue incubation for three days. After three days, discard the culture supernatant, add ice-cold DMEM to each well, gently scrape off cells with a cell scraper, and count them. If the counted macrophages are re-seeded, they will adhere after 2 hours. Related stimuli should be administered after adhesion.

[0076] In vitro colony counting assay. H37Ra (MOI=10) and 20 μg of purified IgG from mouse serum were incubated at 37°C with shaking for 30 min (60 μL / EP tube); then placed on ice for 2 min; buffer: HBSS containing 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 150 mM NaCl, and 1% BSA; the above bacterial mixture was added to macrophages (cells were also cultured in the above buffer), and cultured at 37°C for 1 h; after 1 h of infection, streptomycin (final concentration 100 μg / mL) was added, and the cells were cultured for another 1 h to kill unphagocytosed H37Ra. Colony counting: the cells were washed three times with sterile PBS, adding 2 mL of PBS to each well. 200 μL of 0.1% Triton-x-100 cell lysis buffer was added to each well of a 12-well plate, incubated for 3 min, and then centrifuged at 12000 rpm / min to collect the pellet, discarding excess lysis buffer. Resuspend the precipitate from the previous centrifugation step in 100 μL PBS, and then use this stock solution for sequential 10-fold serial dilutions (10... -1 10 -2 ......10 -5 Take 100 μL of uniform bacterial suspension from each dilution, spread it onto 7H10 solid medium, and incubate in a 37℃ incubator for 15-20 days before counting the colonies.

[0077] See results Figure 5 Compared to wild-type mice, H37Ra-infected wild-type mouse models and B4GALT1 knockout mice showed decreased terminal galactosylation and sialylation of serum IgG, and increased exposure of terminal N-acetylglucosamine in IgG. Figure 5 BC). In vitro colony counting results showed that degalactosylated IgG produced in H37Ra-infected mice and B4GALT1 knockout mice reduced the phagocytic capacity of macrophages (Mφ). Figure 5D). The above results indicate that Mycobacterium tuberculosis infection leads to host IgG degalactosylation, and IgG degalactosylation reduces the ability of macrophages to opsonize and phagocytose bacteria.

[0078] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

Claims

1. Use of B4GALT1 in the preparation of a drug for resisting Mycobacterium tuberculosis infection, characterized in that: The anti-Mycobacterium tuberculosis infection drug is a drug for promoting galactosylation modification of serum IgG and promoting opsonophagocytosis and killing effect of macrophages.

2. Use according to claim 1, characterized in that: B4GALT1 is used as an effective component of the anti-Mycobacterium tuberculosis infection drug.

3. Use according to claim 1, characterized in that: The drug comprises B4GALT1 protein, nucleic acid encoding B4GALT1 protein, and vector expressing B4GALT1, and the amino acid sequence of B4GALT1 is shown as SEQ ID NO:

1.

4. Use according to any one of claims 1 to 3, characterized in that: The drug is delivered into a host body by a delivery system, and the delivery system comprises a gene introduction instrument, AAV, and a liposome.

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