Mycobacterium tuberculosis 32C-PPE18-LS protein nanoparticle as well as preparation method and application thereof

Abstract

The invention is applicable to the field of genetic engineering, and provides a mycobacterium tuberculosis 32C-PPE18-LS protein nanoparticle, a preparation method and application thereof, the mycobacterium tuberculosis 32C-PPE18-LS protein nanoparticle is formed by sequence fusion of a C terminal of a mycobacterium tuberculosis MTB32A protein, a PPE18 protein and an LS protein, and the amino acid sequence of the mycobacterium tuberculosis 32C-PPE18-LS protein nanoparticle is shown in a sequence table SEQ ID NO: 2. According to the mycobacterium tuberculosis 32C-PPE18-LS protein nanoparticles provided by the invention, multi-fragment immunogenic protein fusion is adopted, a synergistic immune effect can be induced, and the immunostimulation ability of an antigen is remarkably enhanced. Moreover, the 32C-PPE18-LS protein nanoparticles are non-toxic and free of pathogenicity, so that the vaccine can be ensured to have good biological safety, and the 32C-PPE18-LS protein nanoparticles are suitable for clinical application.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, and in particular relates to a Mycobacterium tuberculosis 32C-PPE18-LS protein nanoparticle, its preparation method and its uses. Background Technology

[0002] Tuberculosis (TB) is a disease caused by Mycobacterium tuberculosis (Mycobacterium tuberculosis). Mycobacterium tuberculosis, M.tb Tuberculosis, a chronic infectious disease caused by tuberculosis, has been listed by the World Health Organization as one of the top ten causes of death worldwide. Although the BCG vaccine has shown significant effectiveness in preventing severe tuberculosis such as tuberculous meningitis in children, its protective efficacy against adult pulmonary tuberculosis is only 0-80%, particularly in areas with high tuberculosis prevalence. Even more serious is the fact that the recent prevalence of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) poses unprecedented challenges to existing prevention and control systems.

[0003] PPE18 (Rv1196) is M.tb MTB32A is a cell membrane protein that binds to the Toll-like receptor TLR2 on macrophages, activating the p38 mitogen-activated protein kinase (MAPK) pathway, and has potential applications in tuberculosis vaccines and diagnostics. M.tb MTB32A is a secreted protein involved in cell wall synthesis. It possesses abundant α-helical structures, which facilitate its interaction with host proteins within the cell, thereby enhancing its immunogenicity. According to relevant literature, the C-terminal domain (MTB32C, or 32C) of the MTB32A protein is immunogenic. Protein-protein interaction (PPI) network analysis revealed direct connections between nodes of MTB32A and PPE18, indicating a protein-protein interaction. This suggests that MTB32A and PPE18 may have a functional or structural relationship in certain biological processes or pathways.

[0004] Therefore, there is an urgent need to develop a new generation of highly effective vaccines against tuberculosis. Summary of the Invention

[0005] The purpose of this invention is to provide Mycobacterium tuberculosis 32C-PPE18-LS protein nanoparticles, which aim to solve the problems mentioned in the background art.

[0006] To address the above problems, the present invention provides a Mycobacterium tuberculosis 32C-PPE18-LS protein nanoparticle, which is formed by the fusion of the C-terminus of the Mycobacterium tuberculosis MTB32A protein, the PPE18 protein, and the LS protein, and its amino acid sequence is shown in SEQ ID NO:2.

[0007] Another object of the present invention is to provide a gene encoding the above-mentioned Mycobacterium tuberculosis 32C-PPE18-LS protein nanoparticles, the nucleotide sequence of which is shown in the sequence listing SEQ ID NO:1.

[0008] Another object of the present invention is to provide a recombinant expression vector comprising the above-described coding gene.

[0009] Another object of the present invention is to provide a host cell comprising at least one of the above-mentioned Mycobacterium tuberculosis 32C-PPE18-LS protein nanoparticles, the above-mentioned encoding gene, and the above-mentioned recombinant expression vector.

[0010] Another object of the present invention is to provide a method for preparing the above-mentioned Mycobacterium tuberculosis 32C-PPE18-LS protein nanoparticles, which includes the following steps: The coding gene with the nucleotide sequence shown in SEQ ID NO:1 was inserted into the expression vector to construct the recombinant expression vector. The recombinant expression vector was transformed into host cells for induced expression, and then the protein was purified to obtain the Mycobacterium tuberculosis 32C-PPE18-LS protein nanoparticles.

[0011] Another object of the present invention is to provide the use of the above-mentioned Mycobacterium tuberculosis 32C-PPE18-LS protein nanoparticles, or the above-mentioned encoding gene, or the above-mentioned recombinant expression vector, or the above-mentioned host cell in the preparation of drugs or vaccines for the prevention and treatment of tuberculosis.

[0012] Another object of the present invention is to provide a drug or vaccine for the prevention and treatment of tuberculosis, comprising a pharmaceutically acceptable carrier and the aforementioned Mycobacterium tuberculosis 32C-PPE18-LS protein nanoparticles.

[0013] This invention provides a Mycobacterium tuberculosis 32C-PPE18-LS protein nanoparticle, which employs multi-fragment immunogenic protein fusion to induce a synergistic immune effect and significantly enhance the antigen's immunostimulatory capacity. This 32C-PPE18-LS protein nanoparticle is non-toxic and non-pathogenic, ensuring good biosafety for vaccines and making it suitable for clinical application. Animal experiments show that this 32C-PPE18-LS protein nanoparticle can simultaneously activate high levels of humoral immunity (IgG antibody response) and cellular immunity, exhibiting significant protective immune effects in animals. The 32C-PPE18-LS protein nanoparticle provided by this invention not only offers a new technical route for the development of tuberculosis vaccines, but its modular design concept can also be extended to the development of vaccines for other important infectious diseases. This technology has been validated through multiple experiments, demonstrating promising translational applications and laying a solid foundation for subsequent preclinical research and industrialization development. Attached Figure Description

[0014] Figure 1 This is a diagram showing the double enzyme digestion identification of the recombinant expression vector; Figure 2 SDS-PAGE image of 32C-PPE18-LS protein nanoparticles for expression and purification; Figure 3 Western blot identification of 32C-PPE18-LS protein nanoparticles; Figure 4 Electron micrograph of 32C-PPE18-LS protein nanoparticles; Figure 5 Serum ELISA results of mice immunized with 32C-PPE18-LS protein nanoparticles; Figure 6 The results of spleen lymphocyte index detection in mice after immunization with 32C-PPE18-LS protein nanoparticles; Figure 7 The results of serum IFN-γ detection in mice after immunization with 32C-PPE18-LS protein nanoparticles. Detailed Implementation

[0015] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0016] This invention innovatively employs Lumazine Synthase (LS) protein, derived from hyperthermophilic bacteria, as a vaccine carrier platform. This LS protein possesses unique self-assembly properties, capable of forming an icosahedral symmetric structure with a diameter of approximately 15 nm under physiological conditions, displaying up to 60 antigenic epitopes on its surface. By fusing the LS protein with Mycobacterium tuberculosis protective antigen using genetic engineering techniques, a nanoparticle vaccine with high structural stability and immunogenicity can be successfully constructed.

[0017] Specifically, in one embodiment of the present invention, a Mycobacterium tuberculosis 32C-PPE18-LS protein nanoparticle is provided, which is formed by the sequence fusion of the C-terminus (referred to as 32C) of the Mycobacterium tuberculosis MTB32A protein, the PPE18 protein and the LS protein. Preferably, the C-terminus carries the LS protein sequence and adds a His6 tag. The amino acid sequence of the 32C-PPE18-LS protein nanoparticle is shown in SEQ ID NO:2 of the sequence listing.

[0018] In another embodiment of the present invention, a method for preparing the above-mentioned Mycobacterium tuberculosis 32C-PPE18-LS protein nanoparticles is also provided, which includes the following steps: S1. Insert the coding gene with the nucleotide sequence shown in SEQ ID NO:1 into the expression vector to construct the recombinant expression vector; S2. The recombinant expression vector is transformed into host cells for induced expression, and then purified to obtain the Mycobacterium tuberculosis 32C-PPE18-LS protein nanoparticles.

[0019] In practical applications, the expression vector is pET28a(+), which can be expressed in prokaryotic cells, preferably containing the T7 promoter; the host cell is Escherichia coli, preferably Escherichia coli BL21.

[0020] In this embodiment of the invention, mice were immunized with the prepared 32C-PPE18-LS protein nanoparticles, and antibody levels in the mice were measured periodically to evaluate the immunogenicity of the 32C-PPE18-LS protein nanoparticles. In another embodiment of the present invention, a drug or vaccine for the prevention and treatment of tuberculosis is also provided, comprising a pharmaceutically acceptable carrier and the aforementioned Mycobacterium tuberculosis 32C-PPE18-LS protein nanoparticles. In practical applications, the vaccine is a nanoparticle vaccine.

[0021] Example 1: This example provides a method for the expression and purification of Mycobacterium tuberculosis 32C-PPE18-LS protein nanoparticles, specifically including the following steps: The 32C-PPE18 gene was selected from GenBank (NC_000962.3), and the 32C-PPE18-LS coding gene (nucleotide sequence shown in SEQ ID NO:1) was designed as the target gene. Nde I and Hind The target gene was constructed into pET28a(+) (Invirogen, USA) by restriction enzyme III, resulting in a recombinant expression vector. 50 μL of *E. coli* BL21(DE3) competent cells (Invitrogen, USA) were added, along with 5 μL of the recombinant expression vector. The cells were incubated on ice for 30 minutes, followed by a 90-second heat shock, and then incubated on ice again for 3 minutes. 500 μL of antibiotic-free LB broth was added, and the cells were incubated at 37°C and 180 rpm for 45 minutes with shaking. 100 μL of the recombinant vector was plated on LB agar plates containing 100 μg / mL Kans and incubated overnight at 37°C. Three positive clones were picked the following day and sent to Anhui General Biotechnology Co., Ltd. for sequencing verification. The correctly recombinant strain was named pET28a(+)-32C-PPE18-LS. The recombinant expression vector was then further processed. Mlu I- Xho I double enzyme digestion identification, results are as follows Figure 1 As shown, the size of the target band is consistent with expectations, and the recombinant expression vector was successfully constructed.

[0022] A single recombinant positive clone was picked and inoculated into LB liquid medium and cultured overnight with shaking at 37°C and 180 rpm. The overnight culture was then inoculated into fresh LB medium at a 1:100 ratio and cultured until the bacterial OD reached the target value. 600 When the molecular weight reaches 0.6-0.8, IPTG at a final concentration of 1 mmol / L is added to induce expression for 3 hours. The cells are then collected by centrifugation at 12000 rpm for 10 minutes and resuspended in 20 mM Tris-HCl buffer. After sonication for 50 minutes, the cells are centrifuged at 12000 rpm for 20 minutes at 4°C, the supernatant is discarded, and the precipitate is washed. The precipitate is dissolved in 8 M urea and centrifuged again at 12000 rpm for 20 minutes at 4°C, and the supernatant is collected. The supernatant is placed in a dialysis bag with a molecular weight cutoff of 3.5 kDa for dialysis refolding. After dialysis, the cells are filtered through a 0.22 μm pore size membrane to remove residual particulate impurities. The filtrate can then be used for protein purification on a Ni-NTA affinity column. The imidazole elution peak protein is harvested, and SDS-PAGE electrophoresis shows the target band on the PAGE gel, indicating that the purified protein is a recombinant protein, designated as 32C-PPE18-LS protein nanoparticles. Figure 2 As shown.

[0023] Example 2: This example describes a Western blot identification experiment for protein nanoparticles. The specific steps are as follows: Bacterial cells induced in Example 1 were ultrasonically disrupted and centrifuged. Soluble or inclusion body protein samples were collected. Quantitative protein loading of 20-30 μg per well was performed, followed by 12% SDS-PAGE electrophoresis. After electrophoresis, the protein was transferred to a PVDF membrane (0.45 μm) and blocked at room temperature for 1 hour using 5% skim milk powder or BSA in TBST buffer. Primary antibody (such as anti-His tag antibody or specific anti-MTB protein antibody) was then added at the recommended dilution and incubated overnight at 4°C. The next day, the membrane was washed three times with TBST for 10 minutes each time, and HRP-labeled secondary antibody was added, followed by incubation at room temperature for 1 hour. After washing again, ECL chemiluminescent substrate was added, and the target protein bands were detected using a gel imaging system. Figure 3 As shown in the figure. The results indicate that all 32C-PPE18-LS protein nanoparticles can be specifically recognized by His antibodies.

[0024] Example 3: This example describes an electron microscopy observation experiment of protein nanoparticles, as follows: 10 μL of purified 32C-PPE18-LS protein nanoparticle solution was dropped onto the surface of a carbon film copper mesh and allowed to stand at room temperature for 1 minute to allow for particle adsorption. After gently absorbing excess liquid with filter paper, 10 μL of 2% sodium phosphotungstenate (pH=7.0) or 2% uric anhydride was immediately added as a negative stain and stained for 30 seconds. The stain was then absorbed again with filter paper and allowed to air dry at room temperature. The dried sample was then imaged under a transmission electron microscope (e.g., TEM, 80 kV). Figure 4 As shown. Figure 4 The distribution and morphology of the protein nanoparticles were observed. The 32C-PPE18-LS protein nanoparticles exhibited a highly uniform particle size distribution, a regular surface topology, and a stable tertiary structure.

[0025] Example 4: This example is a mouse immunization experiment, specifically as follows: Thirty-six 6-week-old female BALB / c mice were randomly divided into the following three groups (n=12 per group): 1. PBS control group; 2. BCG vaccine group; 3. 32C-PPE18-LS protein nanoparticle group. All groups underwent subcutaneous injection at multiple sites, with three immunizations administered on days 0, 14, and 28. Each mouse in the PBS group received 200 μL of PBS; each mouse in the BCG group received 2 × 10⁻⁶ PBS. 5CFU BCG vaccine; each mouse in the 32C-PPE18-LS protein nanoparticle group was injected with 30 μg of 32C-PPE18-LS protein nanoparticles. Serum samples were collected by ocular blood sampling on days 0, 7, 14, 21, 28, 35, and 42 post-immunization. On days 35 and 42, four mice from each group were randomly selected for ocular blood sampling and spleen collection for subsequent spleen lymphocyte proliferation assays.

[0026] I. Antibody Level Detection: The level of specific IgG antibodies against the antigen in the serum of immunized mice was detected using an indirect ELISA method. 100 μL of recombinant antigen diluted with 0.1 M carbonate buffer (pH=9.6) at a concentration of 1-2 μg / mL was added to each well of a 96-well ELISA plate and incubated overnight at 4°C. The next day, the coating solution was discarded, and the plate was washed three times with PBST (PBS + 0.05% Tween-20) for 5 minutes each time. Then, 200 μL of 5% BSA blocking buffer was added to each well, and the plate was blocked at 37°C for 1 hour. After blocking, the plate was washed again, and 100 μL of mouse serum sample diluted 1:10000 was added to each well, and the plate was incubated at 37°C for 1 hour. After washing, HRP-labeled anti-mouse IgG secondary antibody (dilution ratio 1:5000) was added, and the plate was incubated at 37°C for 1 hour. After washing, TMB substrate solution was added, and the plate was developed in the dark for 15 minutes. The reaction was stopped with stop solution, and the absorbance (OD) was read at 450 nm. 450 The result is as follows: Figure 5 As shown, the 32C-PPE18-LS protein nanoparticle vaccine can significantly induce high levels of antigen-specific IgG antibody production and significantly enhance the antibody response against antigens. M.tb Specific humoral immune response.

[0027] II. Splenic Lymphocyte Proliferation Assay: Mice were sacrificed on day 35 or 42 post-immunization. The spleen was aseptically removed and placed in a culture dish containing RPMI-1640 medium. The spleen was ground using a sterile syringe plunger to prepare a single-cell suspension. After filtration through a 70 μm cell sieve, red blood cells were removed with erythrocyte lysis buffer. The cells were washed twice with PBS and finally resuspended in RPMI-1640 complete medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. The cell concentration was adjusted to 2 × 10⁶ cells / mL. 6 per mL.

[0028] 100 μL of cell suspension was added to a 96-well cell culture plate, with 3 replicates per group. Antigen (final concentration 5 μg / mL) was added for stimulation, with the culture medium serving as a blank control. After incubating the cells at 37°C and 5% CO2 for 42 hours, 10 μL of CCK-8 reagent was added to each well, and incubation continued for another 4 hours. The absorbance (OD) of each well was then read using a microplate reader at 450 nm. 450Cell proliferation capacity was assessed using the stimulation index (SI = experimental group OD / control group OD). Results are as follows: Figure 6 As shown, there was no significant difference in the stimulation index between the PBS and BCG groups at 35 and 42 days (P > 0.05), and the proliferation level of splenic lymphocytes remained basically stable. The stimulation index of 32C-PPE18-LS protein nanoparticles was 2.86, which significantly enhanced the ability of splenic lymphocytes to proliferate.

[0029] III. Serum Cytokine Level Analysis: Serum was collected from mice on days 21 and 42 post-immunization via tail docking. The serum was separated by centrifugation at 3000 rpm for 10 minutes, and the supernatant was stored at -80℃. The levels of IFN-γ cytokines in the serum were quantitatively detected using a commercial ELISA kit. Following the kit instructions, the pre-coated antibody-containing 96-well plates were equilibrated at room temperature. Standards and diluted serum samples (100 μL per well) were added, and the plates were incubated at 37℃ for 2 hours. After discarding the liquid, the plates were washed 5 times, and biotin-labeled detection antibody was added. The plates were incubated at 37℃ for 1 hour, washed again, and HRP enzyme conjugate was added. The plates were incubated at room temperature for 30 minutes. After washing, TMB substrate was added for color development. After 15 minutes of reaction, stop solution was added, and the absorbance was read at 450 nm using a microplate reader. The concentration of cytokines in the samples was calculated based on the standard curve. Results are shown below. Figure 7 As shown, the 32C-PPE18-LS protein nanoparticle vaccine can significantly enhance IFN-γ secretion levels, effectively induce Th1 immune responses, and establish durable anti-tuberculosis immune protection for the host.

[0030] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A Mycobacterium tuberculosis 32C-PPE18-LS protein nanoparticle, characterized in that, It is formed by the fusion of the C-terminus of the Mycobacterium tuberculosis MTB32A protein, the PPE18 protein, and the LS protein, and its amino acid sequence is shown in SEQ ID NO:2 of the sequence listing.

2. The encoding gene of the Mycobacterium tuberculosis 32C-PPE18-LS protein nanoparticle as described in claim 1, characterized in that, The nucleotide sequence of the encoding gene is shown in the sequence listing SEQ ID NO:

1.

3. A recombinant expression vector, characterized in that, It includes the coding gene as described in claim 2.

4. A host cell, characterized in that, It comprises at least one of the following: the Mycobacterium tuberculosis 32C-PPE18-LS protein nanoparticles of claim 1, the encoding gene of claim 2, and the recombinant expression vector of claim 3.

5. A method for preparing Mycobacterium tuberculosis 32C-PPE18-LS protein nanoparticles as described in claim 1, characterized in that, Includes the following steps: The coding gene with the nucleotide sequence shown in SEQ ID NO:1 was inserted into the expression vector to construct the recombinant expression vector. The recombinant expression vector was transformed into host cells for induced expression, and then the protein was purified to obtain the Mycobacterium tuberculosis 32C-PPE18-LS protein nanoparticles.

6. The use of the Mycobacterium tuberculosis 32C-PPE18-LS protein nanoparticle as described in claim 1, or the encoding gene as described in claim 2, or the recombinant expression vector as described in claim 3, or the host cell as described in claim 4 in the preparation of drugs or vaccines for the prevention and treatment of tuberculosis.

7. A drug or vaccine for the prevention and treatment of tuberculosis, comprising a pharmaceutically acceptable carrier, characterized in that, It also includes the Mycobacterium tuberculosis 32C-PPE18-LS protein nanoparticles as described in claim 1.

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