CN116726155A

CN116726155A - Construction, expression, purification and application of tuberculosis subunit vaccine

Info

Publication number: CN116726155A
Application number: CN202310338767.4A
Authority: CN
Inventors: 万康林; 刘海灿; 范雪亭; 李马超; 于晋杰
Original assignee: National Institute for Communicable Disease Control and Prevention of Chinese Center For Disease Control and Prevention
Current assignee: National Institute for Communicable Disease Control and Prevention of Chinese Center For Disease Control and Prevention
Priority date: 2023-03-31
Filing date: 2023-03-31
Publication date: 2023-09-12

Abstract

The invention relates to the technical field of biological medicine, in particular to construction, expression, purification and application of a tuberculosis subunit vaccine. Tuberculosis subunit vaccine comprising: ESAT6 protein shown as SEQ ID No.1, CFP10 protein shown as SEQ ID No.2, and nDnaK protein shown as SEQ ID No. 3. The invention successfully predicts the T cell epitope of the DnaK protein, obtains the nDnaK in the DnaK protein epitope concentration region through positioning and screening, and designs a subunit vaccine of a protective antigen epitope enrichment region by taking the nDnaK as a fusion fragment of a fusion protein and a single antigen component of a mixed protein, has the capability of inhibiting the growth of mycobacterium, and can induce organisms to generate strong and wide protective immune response.

Description

Construction, expression, purification and application of tuberculosis subunit vaccine

Technical Field

The invention relates to the technical field of biological medicine, in particular to construction, expression, purification and application of a tuberculosis subunit vaccine.

Background

Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium Tuberculosis (MTB) infection, causing millions of deaths each year, severely compromising human health. In recent years, with the concomitant infection of multi-drug resistant tuberculosis and Human Immunodeficiency Virus (HIV), the control of tuberculosis faces new challenges and threats. Vaccination is the most effective way to prevent tuberculosis. BCG is an attenuated live Mycobacterium tuberculosis vaccine, and is the only approved anti-tuberculosis vaccine to date. The study proves that the protective force of the composition on the children's castanopsis glaucomatous tuberculosis and tubercular meningitis is stronger, but the BCG has three obvious defects: firstly, the protective efficacy of BCG gradually decreases with the passage of time, and the protective effect on adult tuberculosis is very limited; secondly, inoculation of BCG can interfere with diagnosis of tuberculosis; thirdly, it may lead to fatal infections in immunocompromised individuals. Therefore, there is an urgent need to develop safer and more effective tuberculosis vaccines to control the occurrence and progression of tuberculosis.

It is counted that there are 14 tuberculosis candidate vaccines currently being developed for clinical trials. These candidate vaccines are largely classified into attenuated live vaccines, inactivated vaccines, protein subunit vaccines and viral vector vaccines. The common adenovirus vector vaccine has single immune path and obvious side effect, and antibodies of the virus vector possibly exist in the body, so that the immune effect of the vaccine is reduced; in the preparation process of the inactivated whole vaccine, the restriction of the preparation process can reduce the immune activity of the vaccine, and the content of active ingredients is low, so that the immune protection mechanism is unclear; attenuated live vaccines may have residual virulence and other safety issues that are not suitable for children and immunocompromised individuals. The protein subunit vaccine composed of a plurality of dominant antigens of the mycobacterium tuberculosis has the advantages of high safety, controllable production quality, lower cost and the like. Because protein subunit vaccines containing only protein components are less immunogenic and thus cannot induce a sufficient immunoprotection response, adjuvants are often added to protein subunit vaccines to increase their immunogenicity, and the only commercial adjuvant currently used domestically is aluminum adjuvant.

ESAT6 is an antigen secreted by mycobacterium tuberculosis in early growth phase, and has multiple CD4 on its surface ⁺ T cell epitope and CD8 ⁺ T cell epitope can be specifically recognized by T lymphocyte and promote Th1Cell proliferation induces a strong Th1 type immune response in humans and produces specific cytotoxic T cells. CFP10 and ESAT6 belong to ESAT6 family, are immunodominant antigens, and can induce organism to generate immune response. DnaK protein, also known as heat shock protein 70 (Heat shock protein, HSP 70), is one of the most important proteins in the heat shock protein family, and its main function is to control the correct folding of nascent polypeptide chains. The research shows that DnaK protein has the characteristic of immunodominant antigen, can induce and strengthen the occurrence of humoral immunity and cellular immunity of organisms, and has the functions of molecular chaperones and immunoadjuvants.

The recombinant tubercle bacillus subunit vaccine, especially the subunit vaccine expressed in colibacillus, has the advantages of multiple protective antigen epitopes, high expression efficiency, mature fermentation process, low production cost, easy mass production and wide use, and the subunit vaccine has higher safety and is easy to be accepted by people. At present, a plurality of tuberculosis subunit protein vaccines enter clinical stage, but different subunit protein vaccines induce organisms to generate cytokine expression profiles of immune protection reaction are quite different, and can not induce more comprehensive immune reaction. In addition, conventional subunit vaccines are often prepared with whole antigen mycoproteins or full antigen length to induce specific immune responses in the body against pathogen infection. However, the surface receptors of immune cells typically recognize only certain specific amino acid sequences on the antigen molecule, i.e., epitopes, and the remaining redundant fragments do not enhance antigenicity and protectiveness, and epitope antigen-based vaccines can effectively bring together dominant epitopes, thereby inducing higher levels of immune responses in the receptor, enhancing immune efficiency and reducing side effects of the vaccine by deleting unwanted sequences in the full-length protein. Therefore, how to design subunit vaccines with better protective epitope enrichment regions is a technical problem to be solved in the field.

Disclosure of Invention

First, the present invention provides a tuberculosis subunit vaccine comprising: ESAT6 protein shown as SEQ ID No.1, CFP10 protein shown as SEQ ID No.2, and nDnaK protein shown as SEQ ID No. 3.

The invention predicts the T cell epitope of DnaK based on the gene sequence of DnaK by using Teprdeict and IEDB bioinformatics software, screens the T cell epitope peptide segment which is combined with HLA-II molecule (including HLA-A 0201, 0202, 0203 and 0206) well, corresponds to the 260 th-381 th amino acid sequence of DnaK protein, cuts to obtain the nDnaK in the T cell epitope concentration region of DnaK protein, and the corresponding nucleotide sequence forms the gene sequence of the epitope enrichment region.

The invention discovers that tuberculosis subunit vaccine prepared by combining the full length of the antigen epitope concentrated region nDnaK protein, ESAT6 protein and CFP10 protein after screening and shearing can induce organisms to generate strong and wide protective immune response.

In some embodiments, ESAT6 protein, CFP10 protein, and nDnaK protein are present as separate proteins.

In some embodiments, it contains equimolar proportions of ESAT6 protein, CFP10 protein, and nDnaK protein.

In some embodiments, ESAT6 protein, CFP10 protein, and nDnaK protein are present as fusion proteins.

The fusion protein is constructed by utilizing the gene synthesis technology, and the three antigen components can be obtained only by expressing and purifying one fusion protein, so that the steps of recombinant protein expression and purification are simplified, the production efficiency is improved, and the production cost is reduced.

In some embodiments, the ESAT6 protein has a nucleotide sequence as set forth in SEQ ID No. 4; the nucleotide sequence of the CFP10 protein is shown in SEQ ID NO. 5; the nucleotide sequence of the nDnaK protein is shown as SEQ ID NO. 6.

In some embodiments, the amino acid sequence of the fusion protein is set forth in SEQ ID NO. 7.

In the fusion protein, ESAT6 protein, CFP10 protein and nDnaK protein are connected by flexible peptide segments shown as SEQ ID NO. 15.

The hydrophobic flexible peptide segments (linker) are connected among the antigens, so that the flexibility of the fusion protein can be increased, and the correct folding of three proteins is facilitated.

In some embodiments, the nucleotide sequence of the fusion protein is set forth in SEQ ID NO. 8.

In some embodiments, the tuberculosis subunit vaccine further comprises an adjuvant.

In some embodiments, the adjuvant is an aluminum hydroxide adjuvant.

Further, the invention provides application of the tuberculosis subunit vaccine in preparing a tuberculosis diagnosis or treatment reagent.

Compared with the prior art, the invention has the beneficial effects that:

the invention successfully predicts the T cell epitope of the DnaK protein, obtains the nDnaK in the DnaK protein epitope concentration region through positioning and screening, and designs a subunit vaccine of a protective antigen epitope enrichment region by taking the nDnaK as a fusion fragment of a fusion protein and a single antigen component of a mixed protein, has the capability of inhibiting the growth of mycobacterium, and can induce organisms to generate strong and wide protective immune response. Because the nDanK is formed by splicing a plurality of antigen epitope concentrated areas of DanK protein, stronger immune response can be provided, and the immune efficiency is improved.

Drawings

FIG. 1 is an SDS-PAGE analysis electropherogram of the purification and expression of a single protein contained in ECD003f and ECD003 m; wherein M represents a marker, and 1 to 4 represent ECD003f, ESAT6, CFP10 and nDnaK, respectively.

FIG. 2 is a graph showing the results of the determination of serum IgG, igG1 and IgG2a subtypes from ECD003f, ECD003m and BCG immunized BALB/c mice; wherein, FIG. 2A is serum IgG/IgG1/IgG2A antibody titers; FIG. 2B is a graph of the IgG1/IgG2a ratio.

FIG. 3 is a graph showing the results of extracellular antigen-specific cytokine levels released by spleen cells of immunized mice.

FIG. 4 is a graph showing the results of a Mycobacterium Growth Inhibition Assay (MGIA).

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The examples are not intended to identify the particular technology or conditions, and are either conventional or are carried out according to the technology or conditions described in the literature in this field or are carried out according to the product specifications. The reagents and instruments used, etc. are not identified to the manufacturer and are conventional products available for purchase by regular vendors.

In the following examples, the amino acid sequences of ESAT6, CFP10 and nDnaK are shown in SEQ ID NO. 1-3, and the nucleotide sequences are shown in SEQ ID NO. 4-6; the amino acid sequence of the fusion protein ECD003f is shown as SEQ ID NO.7, and the nucleotide sequence is shown as SEQ ID NO. 8; the nucleotide sequence of the linker is shown as SEQ ID NO. 15.

EXAMPLE 1 construction of recombinant plasmid of fusion protein ECD003f and Components of Mixed protein ECD003m

In the National Center for Biotechnology Information (NCBI) database, the gene sequences encoding ESAT6, CFP10 and DnaK are retrieved, T cell epitopes of DnaK are predicted by using Teprdelct and IEDB bioinformatics software, T cell epitope peptide fragments which are well combined with HLA-II molecules (including HLA-A 0201, 0202, 0203 and 0206) are screened, the amino acid sequences corresponding to the 260 th-381 th amino acid sequence of DnaK protein are obtained by cutting, and the corresponding nucleotide sequences form an epitope enrichment region gene sequence of the T cell epitope concentrated region nDnaK of DnaK protein.

Construction of fusion protein ECD003f recombinant plasmid: the ECD003f recombinant plasmid is formed by sequentially connecting three genes corresponding to ESAT6, CFP10 and nDnaK in series, a section of linker with a sequence of GGTGGTTCTGGCGGT is added between the genes in order to ensure that the spatial structures of the proteins are not mutually affected, the two ends of the outermost gene are connected with NdeI and XhoI restriction sites, and the genes are cloned in a pET43.1a vector after being connected.

Construction of a plasmid corresponding to a single protein in the mixed protein ECD003 m:

the gene sequences corresponding to ESAT6, CFP10 and nDnaK were amplified by PCR using H37Rv genomic DNA as a template, and the two ends of the primers included EcoRI and HindIII cleavage sites, and the primer information is shown in Table 1. The PCR reaction system and the reaction procedure are shown in tables 2 and 3. After the PCR products are digested by EcoRI and HindIII, the PCR products are reacted overnight at 16 ℃ by using T4 DNA ligase and then cloned into pET32a vectors respectively, so as to obtain three protein corresponding gene recombinant plasmids.

The four constructed plasmids are transformed into escherichia coli DH5 alpha competent cells, and the steps are as follows: mixing 10 μl of the ligation product with competent cells of Escherichia coli DH5 a, standing in ice bath for 30min, heat-shocking at 42deg.C for 90s, standing in ice bath for 2min, adding 800 μl of non-resistant LB liquid medium, and culturing in shaking table at 37deg.C at 180rpm for 1 hr. After the cultivation is finished, centrifuging at 4000rpm for 1min, discarding 600 mu l of supernatant, blowing and uniformly mixing the bacterial precipitate, taking 200 mu l of bacterial liquid to coat on an LB solid plate containing ampicillin, and carrying out inversion cultivation at 37 ℃ for 12-16 h. And (3) picking single bacterial colonies, culturing in an LB liquid culture medium containing ampicillin, absorbing bacterial liquid the next day, and carrying out PCR to identify whether the gene recombinant plasmid is successfully connected. DNA sequencing was then performed to verify the correctness of the insert. The recombinant plasmid was extracted from 100% of positive clone cells with correct sequencing and stored at-20 ℃.

TABLE 1 primer amplification List

Note that: __ EcoRI and HindIII cleavage sites are shown.

The upstream primer of Rv3875/ESAT-6 is shown as SEQ ID NO.9, and the downstream primer is shown as SEQ ID NO. 10; the upstream primer of Rv3874/CFP-10 is shown as SEQ ID NO.11, and the downstream primer is shown as SEQ ID NO. 12; the upstream primer of nRv0350/nDanK is shown as SEQ ID NO.13, and the downstream primer is shown as SEQ ID NO. 14.

TABLE 2 PCR amplification System

TABLE 3 PCR reaction procedure

EXAMPLE 2 prokaryotic expression and purification of fusion protein ECD003f and Mixed protein ECD003m Components

Transferring the successfully constructed recombinant plasmid in the example 1 into competent cells of escherichia coli BL21 (DE 3), picking single colonies, inoculating the single colonies into LB liquid medium containing ampicillin (Amp+) for enrichment culture at 37 ℃ and 180rpm, adding isopropyl beta-d-thiogalactoside (IPTG) with the final concentration of 1mmol/L to culture for 3 hours under the same condition to induce the expression of target protein when the absorbance (A) value reaches 0.6. After the induction was completed, the cells were collected by centrifugation at 4000rpm for 10min at 4℃and after the cells were resuspended in a lysate (20 mmol/L Tris-HCl+Triton 100X), the cells were disrupted by a sonicator (sonication parameters: 220W, 15s at intervals of 20s, total 15 min), and after the completion of sonication, the supernatant and the pellet were separated by centrifugation at 12000rpm for 10min, and the recombinant protein expression level and expression form were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Purifying target protein by Ni column affinity chromatography technique, which comprises the following steps: three individual antigen proteins in ECD003m were subjected to Ni column affinity chromatography: filling Ni column affinity chromatography filler, introducing 3 times column volume of balance buffer solution (20 mmol/LTris-HCl is adopted for soluble recombinant protein expressed by pH8.0, 8M urea is adopted for inclusion body expressed recombinant protein expressed by pH 8.0), and zeroing after the ultraviolet absorbance value of 280nm is stable. Pumping a sample, collecting penetrating fluid, and pumping balance buffer corresponding to recombinant protein for leaching until the ultraviolet absorbance value at 280nm is stable. Eluting with imidazole at different concentrations (30 mmol/L, 60mmol/L, 150mmol/L and 300 mmol/L), collecting eluates at different concentrations, and identifying the purity of target protein in each sample by SDS-PAGE, wherein the result of SDS-PAGE electrophoresis shows that: ESAT6 protein is eluted at 300Mm imidazole, CFP10 protein is eluted at 60Mm imidazole, nDnaK protein is eluted at 60Mm imidazole, the electrophoresis pattern has no obvious impurity band and the protein concentration is higher. The above eluent samples were collected for subsequent operations. The fusion protein ECD003f was purified by a hydrophobic column chromatography separation technique. The specific operation is as follows: the sample was collected and mixed with an ammonium sulfate solution to a concentration of 1M ammonium sulfate in the sample, after washing the hydrophobic column medium with 10mM Tris-HCl, the pH was adjusted to equilibrium with a 1M ammonium sulfate solution, the sample solution was loaded through the hydrophobic column, and then eluted sequentially with 10mM Tris-HCl having a final concentration of 0.5M ammonium sulfate, 10mM Tris-HCl having a final concentration of 0.25M ammonium sulfate, and 10mM Tris-HCl having a final concentration of 0M ammonium sulfate. And collecting the eluent for electrophoresis analysis. The purity of the target protein in each sample was identified by SDS-PAGE, and the sample with the best purity was selected for subsequent manipulation. And (3) after purification, filling the target protein into a dialysis bag, removing urea, imidazole and other small molecular impurities through gradient dialysis, finally, placing the target protein into Tris-HCl with pH of 7.4, dialyzing at 4 ℃ for 2 hours, ultrafiltering and concentrating after dialysis, filtering and sterilizing by using a 0.22 mu m filter, subpackaging, and preserving at-70 ℃. The SDS-PAGE identification results of the ECD003f recombinant protein and each protein component in ECD003m are shown in FIG. 1, and the expression conditions and expression forms of each protein are shown in Table 4.

TABLE 4 protein expression conditions and expression forms

EXAMPLE 3 evaluation of immunoprotection Effect

1. Immunization of animals

Construction of ECD003 f/adjuvant subunit vaccine: the fusion protein ECD003f (500. Mu.g, 1.5mL in PBS) was mixed with 500. Mu.L of an aluminum hydroxide adjuvant (aluminum hydroxide concentration 40mg/mL, magnesium hydroxide concentration 40 mg/mL). Construction of ECD003 m/adjuvant subunit vaccine: the three proteins ESAT6, CFP10 and nDnaK were mixed in equimolar ratio to prepare a mixed protein, and then the mixed protein (500. Mu.g, PBS was formulated into 1.5 mL) was mixed with 500. Mu.L of the above aluminum hydroxide adjuvant.

SPF-class 6-8 week old BABL/c female mice are selected and randomly divided into 5 groups, 6 in each group are respectively a negative control group PBS group, a blank control group adjuvant group, an ECD003 f/adjuvant group, an ECD003 m/adjuvant group and a positive control group BCG group, wherein the BCG group is immunized once only on day 0, the other 4 groups are subjected to subcutaneous multipoint immunization on days 0, 10 and 20, and the total dose is 200 mu l/dose. The specific groupings are shown in Table 5.

TABLE 5 animal immunization groups

2. Blood sample collection

Blood is collected by orbital blood collection before primary immunization and before mice sacrifice, standing for 2h at 37 ℃, centrifuging for 10min at 4000rpm, separating serum, packaging, and storing at-20 ℃ for standby.

3. Humoral immune effect detection

Serum-specific antibody titers were detected using an enzyme-linked immunosorbent assay (ELISA).

Specific IgG, igG1 and IgG2a titers in mouse serum were detected as follows:

a. the ELISA plate was coated with 2. Mu.g/ml ECD003f, ECD003m and BCG protein overnight at 4℃and washed 5 times with PBST the next day.

b. Blocking with PBS containing 2% BSA for 2 hours at 37deg.C, washing with PBST for 5 times.

c. Each group of sera was diluted 20000, 40000, 80000, 160000, 320000, 640000, 1280000, 2560000 times with PBS, 100 μl of diluted serum was added to each well, and after incubation for 1 hour at 37 ℃ it was washed 5 times with PBST.

d. 5000-fold dilutions of HRP-labeled IgG, igG1, igG2a antibodies were added, respectively, and after incubation at 37℃for 1 hour at 100. Mu.L per well, the wells were washed 5 times with PBST.

e. After TMB was added and developed at 37℃for 15 minutes, 2M sulfuric acid was added as a stop solution.

f. The microplate reader detects absorbance at a wavelength of 450 nm.

g. Judgment standard: and the OD value of the sample is more than or equal to 2.1 times of the OD value of the negative control, and the sample is judged to be positive.

4. Cell immune effect detection

4.1 isolation of spleen lymphocytes from mice (spleen was taken one week after the mice were sacrificed after the last immunization)

a. Anaesthetizing the mice with pentobarbital sodium until death, soaking the mice with 75% medical alcohol for sterilization, dissecting the mice, taking out spleen, putting the spleen into RPMI1640, and separating lymphocytes within 1 h;

b. separating lymphocytes by using a mouse lymphocyte separation liquid;

c. after separation of lymphocytes, the lymphocyte concentration was measured and adjusted to a concentration of 2X 10 ⁶ cells/ml was used for the subsequent experiments.

4.2Luminex method for detection of IL-2, IFN-gamma, TNF-alpha, IL-4, IL-6, IL-10, IL-12, GM-CSF and IL-17 nine cytokines. The Luminex multiple cytokine detection kit is a commercial kit and comprises the following specific operations:

1) The adjusted concentration of spleen lymphocytes was added to the 96-well cell culture plate at a concentration of 100. Mu.L per well, followed by 2. Mu.g of the corresponding stimulus (ECD 003f antigen protein, ECD003m antigen protein or BCG whole cell lysate). Each group was established with one of a sterile PBS-stimulated negative control well and a Canavalia (ConA) (5. Mu.g/mL) -stimulated positive control well, with the plate cover closed and at 37℃with 5% CO ₂ Co-culturing in an incubator for 16-24 hours;

2) After the completion of the culture, the 96-well cell plate was centrifuged at 4000rpm for 10min, and the supernatant was used for the Luminex multiple cytokine assay, and the assay steps were performed according to the instructions.

5. Mycobacterium tuberculosis in vitro growth inhibition assay (MGIA)

The protective properties of the ECD003f/ECD003m subunit vaccine were evaluated by the ability of splenic lymphocytes from immunized mice to kill Mycobacterium tuberculosis.

1) A total of 1mL of 500. Mu.L of spleen lymphocytes and 500. Mu.L of Mycobacterium tuberculosis standard strain H37Rv containing 50CFU were added to each well of the 24-well plate. Mixing and placing at 37deg.C and 5% CO ₂ Co-culturing in an incubator for 4 days;

2) After the incubation was completed, the co-cultures were transferred to centrifuge tubes, centrifuged at 12000rpm for 10 minutes and 900. Mu.L of supernatant was discarded;

3) Adding 500 mu L of sterile water into each hole of a 24-hole plate while centrifuging, blowing the bottom and the side wall by using a pipettor, standing for 5 minutes, transferring into a corresponding centrifuge tube, standing for 10 minutes after vortex oscillation to thoroughly lyse cells and release intracellular mycobacterium tuberculosis;

4) 50 mu L of a 7H 10-coated plate is taken, meanwhile, 50 mu L H Rv bacterial liquid is directly coated as a blank control, and the plate is inversely cultured for 2-3 weeks at 37 ℃ for colony counting.

6. Analysis of results:

1) Humoral immune Effect evaluation

The results of serum antibody titer detection are shown in figure 2. The results show that both ECD003f and ECD003m can stimulate the body to produce specific IgG; wherein the IgG, igG1 and IgG2a levels of ECD003m were higher than those of BCG group; the ECD003f group had a slightly higher IgG1 to IgG2a ratio than the BCG group, and the ECD003m group had a IgG1 to IgG2a ratio comparable to the BCG group (IgG 2a generally represents Th1 type cellular immunity, and mediated cell immunity was mainly, and IgG1 generally represents Th2 type cellular immunity, mediated liquid immunity was mainly, and the type of immune response was determined by calculating the ratio of IgG1 to IgG2 a).

The above results indicate that: the highest antibody content is produced by ECD003m stimulation, and the humoral immune response effect produced by ECD003m and ECD003f group stimulation is not lower than that of BCG.

2) Evaluation of cellular immune Effect

Spleen cells of immunized mice were stimulated with antigen in vitro and Luminex detected nine cytokines, antigen-specific IL-2, IFN-gamma, TNF-alpha, IL-4, IL-6, IL-10, IL-12, GM-CSF, and IL-17. As shown in fig. 3, the results showed that, after mice were immunized with ECD003f and ECD003m, the levels of cytokines were significantly higher than BCG except for IL-2, indicating that both could stimulate high levels of cytokines and induce a more comprehensive cellular immune response.

3) In vitro protective evaluation

As shown in fig. 4, the results show: ECD003f, ECD003m and BCG all had a strong inhibitory effect on Mycobacterium tuberculosis compared to PBS and adjuvant groups, and the ability of ECD003f group and ECD003m group to inhibit Mycobacterium tuberculosis was not lower than that of BCG immune group, indicating that both can exert the same or higher protective efficacy as BCG.

In conclusion, the ECD003 f/adjuvant and ECD003 m/adjuvant of the two mycobacterium tuberculosis subunit vaccines prepared in the invention show that the ECD003f and ECD003m can generate high-level serum antibody titer, the content of antibodies generated by stimulation of the ECD003m is highest, and the humoral immunity effect of the two mycobacterium tuberculosis subunit vaccines is not lower than that of BCG. The cellular immune effect showed that both ECD003f and ECD003m induced a more pronounced, broader cellular immune response than BCG. The in vitro protective evaluation results showed that the ability of ECD003f group and ECD003m group to inhibit Mycobacterium tuberculosis was not lower than that of BCG immune group, indicating that both can produce the same or higher protective efficacy as BCG. The above experimental results demonstrate that both have intrinsic potential as independent tuberculosis vaccines or bcg booster vaccines.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A tuberculosis subunit vaccine comprising: ESAT6 protein shown as SEQ ID No.1, CFP10 protein shown as SEQ ID No.2, and nDnaK protein shown as SEQ ID No. 3.

2. The tuberculosis subunit vaccine of claim 1 wherein ESAT6 protein, CFP10 protein and nDnaK protein are present as separate proteins.

3. Tuberculosis subunit vaccine according to claim 2, characterized in that it contains ESAT6 protein, CFP10 protein and nDnaK protein in equimolar proportions.

4. The tuberculosis subunit vaccine of claim 1 wherein ESAT6 protein, CFP10 protein and nDnaK protein are present as fusion proteins.

5. The tuberculosis subunit vaccine of claim 4 wherein the fusion protein has an amino acid sequence as shown in SEQ ID No. 7.

6. The tuberculosis subunit vaccine of claim 1 wherein the ESAT6 protein has a nucleotide sequence shown as SEQ ID No. 4; the nucleotide sequence of the CFP10 protein is shown in SEQ ID NO. 5; the nucleotide sequence of the nDnaK protein is shown as SEQ ID NO. 6.

7. The tuberculosis subunit vaccine of claim 5 wherein the fusion protein has a nucleotide sequence as shown in SEQ ID No. 8.

8. The tuberculosis subunit vaccine of any one of claims 1 to 7 further comprising an adjuvant.

9. The tuberculosis subunit vaccine of claim 8 wherein the adjuvant is an aluminum hydroxide adjuvant.

10. Use of a tuberculosis subunit vaccine of any one of claims 1-9 in the preparation of a tuberculosis diagnostic or therapeutic agent.