Mycobacterium tuberculosis Ag85B-TB8.4-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 Ag85B-TB8.4-LS protein nanoparticle, a preparation method and application thereof, the mycobacterium tuberculosis Ag85B-TB8.4-LS protein nanoparticle is formed by sequence fusion of mycobacterium tuberculosis Ag85B protein, TB8.4 protein and LS protein, and the amino acid sequence of the mycobacterium tuberculosis Ag85B-TB8.4-LS protein nanoparticle is shown as SEQ ID NO: 2 in a sequence table. According to the invention, by virtue of a gene recombination technology, key immunogens Ag85B and TB8.4 of mycobacterium tuberculosis are combined with an LS protein fusion vector, and the Ag85B-TB8.4-LS protein nanoparticles with uniform particle size and stable structure are efficiently produced by virtue of an escherichia coli expression system. Animal experiment results show that the Ag85B-TB8.4-LS protein nanoparticle can simultaneously excite strong humoral immunity and cellular immunity response, not only brings a new idea for research and development of tuberculosis vaccines, but also can be popularized and applied to research and development of other infectious disease vaccines due to the modular design, and has important scientific significance and industrialization prospects.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, and in particular relates to a Mycobacterium tuberculosis Ag85B-TB8.4-LS protein nanoparticle, its preparation method and its uses. Background Technology

[0002] Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mycobacterium tuberculosis). Mycobacterium tuberculosis, M.tb Tuberculosis (TB) is a major global infectious disease and has been listed as a key focus of prevention and control by the World Health Organization. While the BCG vaccine is highly effective in preventing severe TB in children, its protective efficacy against adult pulmonary TB varies considerably (0-80%), a problem particularly pronounced in countries with a high TB ​​burden. Currently, with the continued spread of multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB), developing new and highly effective vaccines has become a critical task urgently needing to be addressed in global TB control efforts.

[0003] Ag85B is... M.tb One of the most extensively studied antigens, Ag85B, as an important component of the Ag85 complex, plays a crucial role in bacterial cell wall biosynthesis and possesses extremely high immunogenicity. Ag85B can effectively induce Th1-type cellular immune responses and has been widely used in various vaccine candidates. TB8.4 is a relatively small molecular weight antigen; although its individual immunogenicity may be weak, it can play an auxiliary role in multi-antigen combination vaccines. It can synergistically work with other highly immunogenic antigens to supplement the deficiencies of the overall immune defense system and improve the overall protective efficacy of the vaccine.

[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 Ag85B-TB8.4-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 Ag85B-TB8.4-LS protein nanoparticle, which is formed by the fusion of Mycobacterium tuberculosis Ag85B protein, TB8.4 protein and LS protein, and its amino acid sequence is shown in SEQ ID NO:2 of the sequence listing.

[0007] Another object of the present invention is to provide a gene encoding the above-mentioned Mycobacterium tuberculosis Ag85B-TB8.4-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 Ag85B-TB8.4-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 Ag85B-TB8.4-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 Ag85B-TB8.4-LS protein nanoparticles.

[0011] Another object of the present invention is to provide the use of the above-mentioned Mycobacterium tuberculosis Ag85B-TB8.4-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 Ag85B-TB8.4-LS protein nanoparticles.

[0013] This invention provides a Mycobacterium tuberculosis Ag85B-TB8.4-LS protein nanoparticle, which, by fusing multiple immunogenic protein fragments, can induce a synergistic immune effect, thereby significantly enhancing the antigen's ability to stimulate the immune system. This Ag85B-TB8.4-LS protein nanoparticle is non-toxic and non-pathogenic, ensuring good biosafety for the vaccine and laying a suitable foundation for its clinical application. Animal experiments show that this Ag85B-TB8.4-LS protein nanoparticle can simultaneously activate highly efficient humoral immunity and strong cellular immunity, exhibiting significant protective immune effects in animals. The vaccine development platform based on Ag85B-TB8.4-LS protein nanoparticles constructed in this invention not only opens up a new technical path for the development of tuberculosis vaccines, but its modular design can also be extended to the development of vaccines for other important infectious diseases. This technology not only brings new inspiration to the development of tuberculosis vaccines, but its universal design can also be used for the development of vaccines for other important infectious diseases, possessing broad industrial application potential. 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 Ag85B-TB8.4-LS protein nanoparticles for expression and purification; Figure 3 Western blot identification of Ag85B-TB8.4-LS protein nanoparticles; Figure 4 Electron micrograph of Ag85B-TB8.4-LS protein nanoparticles; Figure 5 The results of serum ELISA detection in mice after immunization with Ag85B-TB8.4-LS protein nanoparticles; Figure 6 The results of spleen lymphocyte index detection in mice after immunization with Ag85B-TB8.4-LS protein nanoparticles; Figure 7 The results of serum IFN-γ detection in mice after immunization with Ag85B-TB8.4-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 utilizes Lumazine Synthase (LS) protein derived from hyperthermophilic bacteria to construct a vaccine delivery platform. The LS protein possesses unique self-assembly properties, capable of forming a regular icosahedral structure with a diameter of approximately 15 nm under physiological conditions, and its surface can efficiently display 60 antigenic epitopes. Through gene recombination technology, this invention successfully developed a structurally stable and highly immunogenic nanoparticle vaccine by fusing the important protective antigens of Mycobacterium tuberculosis, Ag85B and TB8.4, with the LS protein.

[0017] Specifically, in one embodiment of the present invention, a Mycobacterium tuberculosis Ag85B-TB8.4-LS protein nanoparticle is provided, which is formed by the fusion of Mycobacterium tuberculosis Ag85B protein, TB8.4 protein and LS protein through sequence. Preferably, it carries the LS protein sequence and adds a His6 tag. The amino acid sequence of the Ag85B-TB8.4-LS protein nanoparticle is shown in the sequence listing SEQ ID NO:2.

[0018] In another embodiment of the present invention, a method for preparing the above-mentioned Mycobacterium tuberculosis Ag85B-TB8.4-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 Ag85B-TB8.4-LS protein nanoparticles.

[0019] In practical applications, the expression vector is pET22b(+), which can be expressed in prokaryotic cells, and preferably contains 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 Ag85B-TB8.4-LS protein nanoparticles, and antibody levels in the mice were measured periodically to evaluate the immunogenicity of the Ag85B-TB8.4-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 Ag85B-TB8.4-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 Ag85B-TB8.4-LS protein nanoparticles, specifically including the following steps: The Ag85B gene was selected from GenBank (NC_000962.3), and the Ag85B-TB8.4-LS coding gene (nucleotide sequence shown in SEQ ID NO:1) was designed as the target gene. Nde I and HindThe 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(+)-Ag85B-TB8.4-LS. The recombinant expression vector was then further processed. Xba 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 bacterial cell concentration 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 molecular weight cutoff of 3.5 kDa 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, designated as Ag85B-TB8.4-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 Ag85B-TB8.4-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 Ag85B-TB8.4-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). Transmission electron microscopy observation of the Ag85B-TB8.4-LS protein nanoparticles revealed (e.g.) Figure 4 As shown in the figure, its particle size distribution is uniform and its structure has good 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. Ag85B-TB8.4-LS protein nanoparticle group. All groups were immunized three times via subcutaneous injection at multiple sites 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 Ag85B-TB8.4-LS protein nanoparticle group was injected with 30 μg of Ag85B-TB8.4-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 splenic 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 Ag85B-TB8.4-LS protein nanoparticle vaccine exhibits excellent immunogenicity in animal experiments, effectively stimulating the body to produce high-titer IgG antibodies and effectively activating the host's humoral immune defense mechanism.

[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 basically stable. The Ag85B-TB8.4-LS protein nanoparticle vaccine showed a significant immunostimulatory effect, effectively enhancing the mitotic activity of splenic lymphocytes, with a stimulation index reaching 2.53. This data indicates that the nanoparticle vaccine can significantly enhance the activation and proliferation capacity of T lymphocytes.

[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 Ag85B-TB8.4-LS protein nanoparticle vaccine has excellent immunomodulatory effects. It can significantly increase the secretion level of IFN-γ, effectively activate the Th1-type immune response, and thus build a durable anti-tuberculosis immune protection system in 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 Ag85B-TB8.4-LS protein nanoparticle, characterized in that, It is formed by the fusion of Mycobacterium tuberculosis Ag85B protein, TB8.4 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 Ag85B-TB8.4-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 Ag85B-TB8.4-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 Ag85B-TB8.4-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 Ag85B-TB8.4-LS protein nanoparticles.

6. The use of Mycobacterium tuberculosis Ag85B-TB8.4-LS protein nanoparticles 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 Ag85B-TB8.4-LS protein nanoparticles as described in claim 1.

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