Mycobacterium tuberculosis RpfB-LS protein nanoparticle as well as preparation method and application thereof

Abstract

The invention is applicable to the field of gene engineering, and provides a mycobacterium tuberculosis RpfB-LS protein nanoparticle, a preparation method and application thereof, the mycobacterium tuberculosis RpfB-LS protein nanoparticle is formed by sequence fusion of mycobacterium tuberculosis RpfB protein and LS protein, and the amino acid sequence of the mycobacterium tuberculosis RpfB-LS protein nanoparticle is shown in a sequence table SEQ ID NO: 2. The RpfB-LS protein nanoparticles provided by the invention are non-toxic and free of pathogenicity, can ensure that vaccines have good biological safety, and are suitable for clinical application. Animal experiments show that after a mouse is immunized by the RpfB-LS protein nanoparticle, strong humoral immunity (antibody generation) and cellular immunity (T cell response) can be stimulated at the same time, and it is proved that the recombinant RpfB-LS protein nanoparticle vaccine can effectively induce specific immune protection.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of genetic engineering, and particularly relates to a Mycobacterium tuberculosis RpfB-LS protein nanoparticle, a preparation method and use thereof. BACKGROUND

[0002] Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (Mtb), which is a major threat to global public health security. Although Bacillus Calmette-Guerin (BCG) has been widely used for neonatal immunization since the early 20th century and has shown significant effect in preventing severe tuberculosis in children, its protective effect on adult pulmonary tuberculosis is limited, especially in high-burden countries, making it difficult to effectively control the disease. In addition, the emergence of drug-resistant tuberculosis poses a serious challenge to existing vaccines and treatment strategies. Mycobacterium tuberculosis, M.tb RpfB (Rv1009) is a resuscitation promoting factor antigen in Mtb, which is involved in the process of bacterial dormancy resuscitation. This protein can finely regulate its activity and function in intracellular and extracellular environments, and is involved in complex biological processes. Therefore, it is a potential target for preventing latent infection from turning into active tuberculosis.

[0003] Therefore, developing new, efficient and safe tuberculosis vaccines has become a key direction for global tuberculosis prevention and control research. M.tb SUMMARY

[0004] The present application aims to provide a Mycobacterium tuberculosis RpfB-LS protein nanoparticle, which aims to solve the problems raised in the background art. DETAILED DESCRIPTION

[0005] The present application aims to provide a Mycobacterium tuberculosis RpfB-LS protein nanoparticle, which aims to solve the problems raised in the background art.

[0006] To solve the above problems, the present application is realized as follows: a Mycobacterium tuberculosis RpfB-LS protein nanoparticle is formed by sequence fusion of Mycobacterium tuberculosis RpfB protein and LS protein, and the amino acid sequence is shown in SEQ ID NO: 2.

[0007] Another object of the present application is to provide a coding gene of the above-mentioned Mycobacterium tuberculosis RpfB-LS protein nanoparticle, and the nucleotide sequence is shown in SEQ ID NO: 1.

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

[0009] Another object of the present application is to provide a host cell comprising at least one of the above-mentioned Mycobacterium tuberculosis RpfB-LS protein nanoparticle, the above-mentioned coding gene and the above-mentioned recombinant expression vector.

[0010] Another object of the present application is to provide a preparation method of the Mycobacterium tuberculosis RpfB-LS protein nanoparticle, comprising the following steps: The coding gene with the nucleotide sequence shown in SEQ ID NO: 1 is inserted into an expression vector to construct a recombinant expression vector; The recombinant expression vector is transformed into a host cell for induced expression, and then the Mycobacterium tuberculosis RpfB-LS protein nanoparticle is obtained through protein purification.

[0011] Another object of the present application is to provide a use of the Mycobacterium tuberculosis RpfB-LS protein nanoparticle, the coding gene, the recombinant expression vector or the host cell in the preparation of a drug or vaccine for preventing and treating tuberculosis.

[0012] Another object of the present application is to provide a drug or vaccine for preventing and treating tuberculosis, comprising a pharmaceutically acceptable carrier and the Mycobacterium tuberculosis RpfB-LS protein nanoparticle.

[0013] The Mycobacterium tuberculosis RpfB-LS protein nanoparticle provided by the present application adopts multi-fragment immunogenic protein fusion, can induce synergistic immune effect, and significantly enhances the immune stimulation capacity of the antigen. The RpfB-LS protein nanoparticle is non-toxic and non-pathogenic, can ensure that the vaccine has good biological safety, and is suitable for clinical application. Animal experiments show that after the RpfB-LS protein nanoparticle is used to immunize mice, strong humoral immunity (antibody production) and cellular immunity (T cell response) can be simultaneously stimulated, which proves that the recombinant RpfB-LS protein nanoparticle vaccine can effectively induce specific immune protection. The RpfB-LS protein nanoparticle provided by the present application provides a technical basis for constructing a high-efficiency, expressible and self-assembling nanoparticle vaccine, and lays a theoretical and practical basis for the research and industrialization of a new tuberculosis vaccine. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 It is a double enzyme digestion identification map of the recombinant expression vector; Figure 2 It is an expression and purification SDS-PAGE map of the RpfB-LS protein nanoparticle; Figure 3 It is a Western Blot identification of the RpfB-LS protein nanoparticle; Figure 4 It is an electron microscope photograph of the RpfB-LS protein nanoparticle; Figure 5 It is an ELISA detection result of the serum of the mice immunized with the RpfB-LS protein nanoparticle; Figure 6Results of spleen lymphocyte index detection in mice after immunization with RpfB-LS protein nanoparticles; Figure 7 The results of serum IFN-γ detection in mice after immunization with RpfB-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] The LS (Lumazine Synthase) protein used in this invention is derived from hyperthermophilic bacteria and is a shell protein capable of self-assembling to form a highly stable spherical structure. The LS protein exhibits excellent biocompatibility and expression stability, can fuse with different antigens to form nanoparticles, and achieves high-density, repetitive arrangement of antigens through self-assembly, significantly enhancing immunogenicity. This invention utilizes the LS protein as a platform to develop a method for constructing a nanoparticle vaccine fused with a key Mycobacterium tuberculosis immunogenicity.

[0017] Specifically, in one embodiment of the present invention, a Mycobacterium tuberculosis RpfB-LS protein nanoparticle is provided, which is formed by the sequence fusion of Mycobacterium tuberculosis RpfB protein and LS protein. Preferably, it carries the LS protein sequence and adds a His6 tag. The amino acid sequence of the RpfB-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 RpfB-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 RpfB-LS protein nanoparticles.

[0019] In practical applications, the expression vector is pET22b(+), 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 RpfB-LS protein nanoparticles, and antibody levels in the mice were measured periodically to evaluate the immunogenicity of the RpfB-LS protein nanoparticles. In another embodiment of the 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 RpfB-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 RpfB-LS protein nanoparticles, specifically including the following steps: The RpfB gene was selected from GenBank (NC_000962.3), and the RpfB-LS encoding 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 pET22b(+) (Invirogen, USA) by restriction enzyme site 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 another 3 minutes on ice. 500 μL of antibiotic-free LB broth was added, and the cells were incubated at 37°C and 180 rpm with shaking for 45 minutes to recover. 100 μL of the recombinant vector was plated on LB agar plates containing 100 μg / mL Amp 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 pET22b(+)-RpfB-LS. The recombinant expression vector was then further processed. Apa I and Xho Double enzyme digestion identification, results 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. 600When 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 subjected to SDS-PAGE electrophoresis. The presence of the target band on the PAGE gel indicates that the purified protein is a recombinant protein, denoted as RpfB-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 RpfB-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 RpfB-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, 80kV). Figure 4 As shown. Figure 4The distribution and morphology of the protein nanoparticles were observed. The RpfB-LS protein nanoparticles exhibited a uniform particle size distribution, regular surface morphology, and good structural integrity, demonstrating excellent monodispersity and physical stability.

[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. RpfB-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. 5 CFU BCG vaccine; each mouse in the RpfB-LS protein nanoparticle group was injected with 30 μg of RpfB-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 RpfB-LS protein nanoparticle vaccine can significantly increase the titer of specific IgG antibodies, effectively enhancing the protection against... M.tb 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. 450 Cell 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 relatively stable. The RpfB-LS protein nanoparticle vaccine can maintain a prolonged state of immune activation, significantly promote splenic lymphocyte proliferation, and induce a stronger immune response.

[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 RpfB-LS protein nanoparticle vaccine can promote the body to secrete high levels of IFN-γ, which helps the body form long-term protective immunity.

[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 RpfB-LS protein nanoparticle, characterized in that, It is formed by the fusion of Mycobacterium tuberculosis RpfB protein and 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 RpfB-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 RpfB-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 RpfB-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 RpfB-LS protein nanoparticles.

6. The use of the Mycobacterium tuberculosis RpfB-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 RpfB-LS protein nanoparticles as described in claim 1.

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