Mycobacterium tuberculosis Mce1A-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 Mce1A-LS protein nanoparticle, a preparation method and application thereof, the mycobacterium tuberculosis Mce1A-LS protein nanoparticle is formed by sequence fusion of mycobacterium tuberculosis Mce1A protein and LS protein, and the amino acid sequence of the mycobacterium tuberculosis Mce1A-LS protein nanoparticle is shown as SEQ ID NO: 2 in a sequence table. The Mce1A-LS protein nanoparticle provided by the invention is high in immunogenicity, the protein Mce1A with high immunogenicity can induce to generate a synergistic immune effect and is high in safety, and the expressed fusion protein is non-toxic and harmless, so that the biosafety is high, in addition, the immune effect of the Mce1A-LS protein nanoparticle is good, and after the Mce1A-LS protein nanoparticle is used for immunizing a mouse, the immunogenicity of the Mce1A-LS protein nanoparticle is greatly improved. Strong body fluid and / or cellular immune response can be generated, which indicates that after immunization, a specific immune effect can be generated in a mouse body.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, and in particular relates to a Mycobacterium tuberculosis Mce1A-LS protein nanoparticle, its preparation method and its uses. Background Technology

[0002] Tuberculosis (TB) is a disease caused by Mycobacterium tuberculosis (Bacillus tuberculosis). Mycobacterium tuberculosis, M.tb Tuberculosis, a chronic infectious disease caused by tuberculosis, poses a serious threat to global public health security. Although BCG has been widely used for neonatal immunization since the early 20th century and has played an important role in preventing severe childhood tuberculosis, its protective effect against adult pulmonary tuberculosis is limited, especially in countries with a high tuberculosis burden, where it remains difficult to effectively control the epidemic. Furthermore, the continued emergence of drug-resistant tuberculosis presents new challenges to existing vaccines and treatment strategies.

[0003] Mce1A is a transmembrane protein that plays a key role in the entry of Mycobacterium tuberculosis cells into host cells, and its domains have been shown in various studies to induce cellular immune responses.

[0004] Therefore, developing new, efficient, and safe tuberculosis vaccines has become a hot topic in global tuberculosis prevention and control research. Summary of the Invention

[0005] The purpose of this invention is to provide Mycobacterium tuberculosis Mce1A-LS protein nanoparticles, aiming to solve the problems mentioned in the background art.

[0006] To address the above problems, the present invention provides a Mycobacterium tuberculosis Mce1A-LS protein nanoparticle, which is induced by fusing the LS protein sequence with the Mycobacterium tuberculosis Mce1A 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 Mce1A-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-described Mycobacterium tuberculosis Mce1A-LS protein nanoparticles, the above-described encoding gene, and the above-described recombinant expression vector.

[0010] Another object of the present invention is to provide a method for preparing the above-mentioned Mycobacterium tuberculosis Mce1A-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 Mce1A-LS protein nanoparticles.

[0011] Another object of the present invention is to provide the use of the above-mentioned Mycobacterium tuberculosis Mce1A-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 Mce1A-LS protein nanoparticles.

[0013] This invention provides Mycobacterium tuberculosis Mce1A-LS protein nanoparticles with high immunogenicity. The highly immunogenic protein Mce1A can induce a synergistic immune effect. These Mce1A-LS protein nanoparticles have high safety, as the expressed fusion protein is non-toxic and harmless, thus exhibiting high biosafety. Furthermore, these Mce1A-LS protein nanoparticles demonstrate good immunogenicity; immunization of mice with these nanoparticles elicits a strong humoral and / or cellular immune response, indicating that immunization can induce a specific immune effect in mice. 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 Mce1A-LS protein nanoparticles for expression and purification; Figure 3 Western blot identification of Mce1A-LS protein nanoparticles; Figure 4 Electron micrograph of Mce1A-LS protein nanoparticles; Figure 5 The results of serum ELISA detection in mice after immunization with Mce1A-LS protein nanoparticles; Figure 6 The results of spleen lymphocyte index detection in mice after immunization with Mce1A-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 sequence used in this invention is derived from hyperthermophilic bacteria and is a naturally occurring shell protein domain capable of self-assembling into a highly stable spherical structure. The LS protein exhibits good biocompatibility and expression stability, and can fuse with different antigen sequences to form nanoparticles. After self-assembly, it displays a high-density, repetitive antigen presentation, significantly enhancing the immunogenicity of the antigen.

[0017] The antigen sequences selected in the embodiments of this invention have a sound immunological basis. Mce1A is a transmembrane protein that plays a key role in the entry of Mycobacterium tuberculosis cells into host cells, and its domains have been considered in various studies to induce cellular immune responses.

[0018] In summary, the development of LS self-assembly sequences and the fusion of Mce1A... M.tb Nanoparticle vaccines containing immunogenic antigens can achieve spatial integration and multivalent arrangement of antigens in their structure, and functionally stimulate stronger cellular and humoral immune responses. This represents an innovative strategy to address key issues such as insufficient immunogenicity and low delivery efficiency in current tuberculosis subunit vaccines. The method disclosed in this invention provides a technical foundation for constructing efficient, expressible, and self-assembling multi-antigen nanoparticle vaccines, and also lays the theoretical and practical foundation for the development and industrialization of novel tuberculosis vaccines.

[0019] Specifically, in one embodiment of the present invention, a Mycobacterium tuberculosis Mce1A-LS protein nanoparticle is provided, which is induced by fusing the LS protein sequence with the Mycobacterium tuberculosis Mce1A protein, and its amino acid sequence is shown in SEQ ID NO:2. Preferably, it carries the LS protein sequence and adds a His6 tag; the amino acid sequence of the Mce1A-LS protein nanoparticle is shown in SEQ ID NO:2.

[0020] In another embodiment of the present invention, a method for preparing the above-mentioned Mycobacterium tuberculosis Mce1A-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 the protein is purified to obtain the Mycobacterium tuberculosis Mce1A-LS protein nanoparticles.

[0021] In practical applications, the expression vector is pET22b(+), which can be expressed normally in prokaryotic cells. Preferably, the promoter type in the expression vector is T7 promoter; the host cell is Escherichia coli, preferably Escherichia coli BL21.

[0022] In this embodiment of the invention, mice were immunized with the prepared Mce1A-LS protein nanoparticles, and antibody levels in the mice were measured periodically to evaluate the immunogenicity of the Mce1A-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 Mce1A-LS protein nanoparticles. In practical applications, the vaccine is a nanoparticle vaccine.

[0023] Example 1: This example provides a method for the expression and purification of Mycobacterium tuberculosis Mce1A-LS protein nanoparticles, specifically including the following steps: The Mce1A gene was selected from GenBank (NC_000962.3), and the Mce1A-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 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 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 with shaking for 45 minutes to allow recovery. 100 μL of the recombinant vector was plated on LB agar plates containing 100 μg / mL Kanamycin / Amp and incubated upside down 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(+)-Mce1A-LS. Xba l and Xho The recombinant expression vector was identified by double enzyme digestion, and the results are as follows: Figure 1 As shown, the target band matches the expectation, indicating that the recombinant expression vector was successfully constructed.

[0024] 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 bacterial count reached 0.6-0.8, IPTG was added to a final concentration of 1 mmol / L to induce expression for 3 hours. The cells were 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 were centrifuged at 12000 rpm for 20 minutes at 4°C, the supernatant was discarded, and the precipitate was washed. The precipitate was dissolved in 8 M urea and centrifuged again at 12000 rpm for 20 minutes at 4°C, and the supernatant was collected. The supernatant was placed in a dialysis bag with a 3.5 kDa molecular weight cutoff for dialysis refolding. After dialysis, the cells were filtered through a 0.22 μm pore size membrane to remove residual particulate impurities. The filtrate was then used for protein purification on a Ni-NTA affinity column. The imidazole elution peak protein was harvested and subjected to SDS-PAGE electrophoresis. The presence of the target band on the PAGE gel indicated that the purified protein was a recombinant protein, denoted as Mce1A-LS protein nanoparticles. Figure 2 As shown.

[0025] 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 Mce1A-LS protein nanoparticles can be specifically recognized by His antibodies.

[0026] Example 3: This example describes an electron microscopy observation experiment of protein nanoparticles, as follows: 10 μL of purified Mce1A-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 4The distribution and morphology of the protein nanoparticles were observed. The Mce1A-LS protein nanoparticles had a uniform average particle size, smooth surface, and stable structure, exhibiting good uniformity and low size dispersion.

[0027] 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. Mce1A-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 Mce1A-LS protein nanoparticle group was injected with 30 μg of Mce1A-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.

[0028] 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. 96-well ELISA plates were coated with recombinant antigen diluted in 0.1 M carbonate buffer (pH=9.6) at a concentration of 1-2 μg / mL, 100 μL per well, and incubated overnight at 4°C. The next day, the coating solution was discarded, and the plates were 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 plates were blocked at 37°C for 1 hour. After blocking, the plates were washed again, and 100 μL of mouse serum sample diluted 1:10000 was added to each well, and the plates were incubated at 37°C for 1 hour. After washing, HRP-labeled anti-mouse IgG secondary antibody (dilution ratio 1:5000) was added, and the plates were incubated at 37°C for 1 hour. After washing, TMB substrate solution was added, and the plates were 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 Mce1A-LS protein nanoparticle vaccine can significantly increase IgG levels and enhance... M.tb Humoral immune response induced by candidate antigen nanoparticles.

[0029] 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.

[0030] 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 basically stable. The Mce1A-LS protein nanoparticle vaccine was able to maintain a certain level of immune activation, making the splenic lymphocyte proliferation response stronger and activating a higher level of immunity (P < 0.001).

[0031] 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 Mce1A-LS protein nanoparticle, characterized in that, It is induced by the fusion of the LS protein sequence with the Mycobacterium tuberculosis Mce1A 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 Mce1A-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 Mycobacterium tuberculosis Mce1A-LS protein nanoparticles as described in claim 1, the encoding gene as described in claim 2, and the recombinant expression vector as described in claim 3.

5. A method for preparing Mycobacterium tuberculosis Mce1A-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 Mce1A-LS protein nanoparticles.

6. The use of the Mycobacterium tuberculosis Mce1A-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 Mce1A-LS protein nanoparticles as described in claim 1.

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