Application of mycobacterium tuberculosis antigen in tuberculosis subunit vaccine

Abstract

The invention discloses a mycobacterium tuberculosis deletion region protein Rv1577c and a fusion antigen LB28 containing a mycobacterium tuberculosis RD region and a latent infection related antigen, wherein the fusion antigen comprises Rv1577c and Rv1738. The invention provides a candidate antigen Rv1577c of a tuberculosis subunit vaccine through antigen screening and experimental verification. The Rv1577c is deleted in BCG, so that the immunogenicity is better, and the Rv1577c has certain consistency with ESAT6 and CFP10 in bovine tuberculosis immunodiagnosis. And the Rv1577 is combined with a latent infection associated antigen Rv1738 to construct the tuberculosis fusion protein LB28. Under the assistance of an adjuvant DDA + PolyI: C (DP), the fusion protein LB28 shows good immunogenicity in a mouse model, and has an immune protection effect of resisting mycobacterium bovis infection. The Rv1577 and the fusion protein LB28 constructed based on the Rv1577 can be applied to animal and human tuberculosis prevention and treatment.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of genetic engineering and recombinant subunit vaccine technology development, and particularly relates to application of Mycobacterium tuberculosis antigen Rv1577c in a tuberculosis subunit vaccine. BACKGROUND

[0002] Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis complex infection. Newborns vaccinated with Bacillus Calmette-Guérin (BCG) can prevent children from suffering from severe miliary tuberculosis and tuberculous meningitis, but as age increases, the immunity induced by BCG decreases, and the protective effect on adults is poor. Subunit vaccines have a wide application prospect of strengthening BCG and preventing reactivation of latent tuberculosis infection. With the in-depth study of Mycobacterium tuberculosis and the update of vaccine technology means, a large number of vaccines in different forms have been developed for the prevention of tuberculosis infection. At present, vaccines in the clinical trial pipeline include Mycobacterium inactivated vaccine, virus vector vaccine, recombinant BCG or Mycobacterium tuberculosis attenuated live vaccine, nucleic acid vaccine and protein subunit vaccine. The protein subunit vaccines currently in clinical trials include M72 / AS01E, H56 / IC31, H107e / CAF01b, ID93 / GLA-SE, GamTBVa and AEC / BC02.

[0003] Subunit vaccines aim to induce immune responses against specific antigens of pathogens, and the selection of protective antigens is particularly crucial. By using genome biology techniques such as DNA microarray, bacterial artificial chromosome array and subtractive genome hybridization, the genomes of Mycobacterium tuberculosis standard strain, Mycobacterium bovis and BCG were compared and studied, and it was found that BCG lacks 16 different regions of difference (RD), referred to as RD regions. These different regions are composed of 129 open reading frames in the genome of Mycobacterium tuberculosis, and these open reading frames are lost during the long-term in vitro culture of BCG

[10] , and the decrease in toxicity of BCG may be closely related to the loss of part of the genes.

[0004] Developing subunit vaccines based on RD region antigens can effectively enhance the immune efficacy of BCG and induce long-lived memory T cells [6] . Early secretory antigenic target 6 (ESAT6) and culture filtrate protein 10 (CFP10) in the RD1 region are widely used as tuberculosis diagnostic antigen targets due to their high immunogenicity [11,12]Screening of the antigen other than ESAT6 and CFP10 in RD region of M. tuberculosis, constructing subunit vaccine, facilitating the clinical differentiation between tuberculosis infection and vaccination, and having important significance for clinical test and popularization of subunit vaccine. SUMMARY

[0005] The technical problem solved by the present application is: on the one hand, screening and verifying the M. tuberculosis RD region antigen with high immune response in tuberculosis infected human and bovine population; on the other hand, constructing the fusion protein containing RD region and latent infection related antigen, verifying the immunogenicity and protective efficacy of the fusion protein on mouse model, and developing the tuberculosis subunit vaccine with independent intellectual property rights of China.

[0006] In order to solve the corresponding technical problems, the present application adopts the following technical solutions:

[0007] The present application screens and verifies a M. tuberculosis RD region high immune response antigen Rv1577c.

[0008] The present application provides a recombinant fusion protein containing M. tuberculosis RD region and latent infection related antigen, and the fusion protein antigen includes Rv1577c and Rv1738.

[0009] The fusion protein antigen provided by the present application contains partial sequence of Rv1577c protein and full length of Rv1738 protein, and the sequence of the fusion protein is in sequence partial sequence of Rv1577c, linker sequence and full length sequence of Rv1738 protein.

[0010] The present application verifies the immunogenicity of subunit vaccine LB28 / DP in mouse model.

[0011] The present application also provides the use of the tuberculosis subunit vaccine in preventing acute infection of M. bovis.

[0012] The use can use the fusion protein antigen alone as the tuberculosis subunit vaccine, or can be used in combination with other tuberculosis subunit vaccines.

[0013] The subunit vaccine is inoculated subcutaneously.

[0014] The application firstly screens RD3-RD16 region antigens by literature review and bioinformatics prediction, and obtains 10 candidate antigens such as Rv1575. The candidate antigens are expressed in Escherichia coli, and 6 antigens such as Rv1577c are obtained through purification. Further screening is carried out in tuberculosis infected human and bovine groups through whole blood IFN-gamma release experiment, and finally a new RD region high immunogenicity antigen Rv1577c is obtained, and a dominant T cell epitope P4 (YNEATTVRDFDG) of Rv1577c is identified. The fusion protein LB28 is constructed by combining Rv1577c and the latent infection related antigen Rv1738, which can be expressed in Escherichia coli and purified through dialysis renaturation. LB28 can induce high level of IFN-gamma production in EC-IGRA positive human groups, indicating that it has good immunogenicity. LB28 / DP immunized mice can induce long-term survival CD4 + T cells, induce high level of antigen specific cytokines, produce high level of antibodies, and show good immunogenicity. After bovine mycobacterium infection, LB28 / DP shows good ability to resist bovine mycobacterium infection, and the immune protection effect induced by the LB28 / DP group is close to BCG.

[0015] The application has the following beneficial effects:

[0016] Tuberculosis subunit vaccine has the advantages of safety and reliability, but the selection and screening of antigen components are still important problems restricting its development. At present, the only vaccine approved for the prevention of tuberculosis is BCG. Although BCG vaccination in newborns can effectively prevent children from developing severe tuberculous meningitis and systemic miliary tuberculosis, it does not protect adults well. In order to better control Mycobacterium tuberculosis infection worldwide, it is urgent to develop new tuberculosis vaccines with preventive effect and anti-latent infection. Rv1577c and Rv1738 are antigens common to Mycobacterium tuberculosis and bovine mycobacterium. The application selects Rv1577c and Rv1738 to construct fusion protein LB28, which can be efficiently expressed in Escherichia coli and effectively purified through inclusion body dialysis renaturation, and has the conditions for large-scale production. When used in combination with DP adjuvant in a mouse model infected with bovine mycobacterium, LB28 shows good protection effect. We construct a fusion protein subunit vaccine common to human tuberculosis and animal tuberculosis, evaluate the protection effect in bovine groups infected with mycobacterium, screen out fusion proteins with protection effect, lay a foundation for the popularization and application of subunit vaccine in human groups, and will certainly be beneficial to the development and application of human tuberculosis subunit vaccine, and has good development prospect. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 Table 1 is the information of 10 candidate antigens of Mycobacterium tuberculosis RD region screened in Example 1.

[0018] Figure 2 Is the expression and purification information of M. tuberculosis RD region candidate antigen.

[0019] Figure 3 Is the IFN-γ secretion level induced by RD region candidate antigen in tuberculosis patients.

[0020] Figure 4 Is the immunoreactivity of candidate antigen Rv1577c in cattle population.

[0021] Figure 5 Is the construction, expression and purification of fusion protein LB28.

[0022] Figure 6 Is the immunoreactivity of fusion protein LB28 in human population.

[0023] Figure 7 Is the antigen-specific cytokine secretion level of mouse spleen lymphocytes induced by vaccine.

[0024] Note: 6 weeks after the last immunization, spleen lymphocytes were isolated, and after 68 hours of in vitro stimulation of lymphocytes by antigens Rv1577c and Rv1738 respectively, cell culture supernatant was collected, and the levels of antigen-specific cell secretion of IFN-γ, IL-2 and granzyme B were detected by ELISA. (A) Antigen-specific cell IFN-γ secretion level after Rv1577c stimulation. (B) Antigen-specific cell IFN-γ secretion level after Rv1738 stimulation. (C) Antigen-specific cell IL-2 secretion level after Rv1577c stimulation. (D) Antigen-specific cell IL-2 secretion level after Rv1738 stimulation. (E) Antigen-specific cell granzyme B secretion level after Rv1577c stimulation. (F) Antigen-specific cell granzyme B secretion level after Rv1738 stimulation. The results are expressed as mean ± standard deviation, n = 4-5. *P <0.05, **P <0.01, ***P <0.001.

[0025] Figure 8 Is the long-term survival CD4 + T cell proliferation ability induced by vaccine.

[0026] Note: 12 weeks after the last immunization, 3 days after intraperitoneal injection of antigens Rv1577c and Rv1738, splenic lymphocytes were isolated and stimulated with a mixture of antigens Rv1577c and Rv1738 (5 μg / mL, respectively) and EdU (10 μM) for 3 days. The proliferation of lymphocytes was detected by flow cytometry. (A) Representative flow cytometry detection of CD4+ T cell proliferation. (B) Statistical analysis of the proportion of CD4+ T cell proliferation after stimulation with antigen Rv1577c. (C) Statistical analysis of the proportion of CD4+ T cell proliferation after stimulation with antigen Rv1738. Data are shown as mean ± standard error. n = 5, *P < 0.05, ***P < 0.001.

[0027] Figure 9 LB28 induced the level of serum antigen-specific antibodies in mice.

[0028] Figure 10 ELISPOT detected the number of T cells secreting IFN-γ stimulated by antigen epitopes.

[0029] Note: 6 weeks after the last immunization, splenic lymphocytes were isolated and stimulated with antigen Rv1577c and overlapping peptide epitopes in vitro for 20 hours. The number of antigen-specific T cells was detected by ELISPOT. (A) Representative ELISPOT detection. (B) Statistical analysis of ELISPOT detection.

[0030] Figure 11 LB28 / DP immunized mice were protected against M. bovis infection.

[0031] Note: Lung pathological damage and lung bacterial load of vaccine-immunized mice after M. bovis challenge. A. Whole scanning of HE-stained lung sections of M. bovis-infected mice. B. Pathological changes in the lungs of M. bovis-infected mice. Scale bar, 200 μm. C. Percentage of pathological damage area to the whole lung area. D. Lung bacterial load count. All data are shown as mean ± standard deviation, n = 5-6. *P < 0.05; **P < 0.01. DETAILED DESCRIPTION

[0032] In order for those skilled in the art to better understand the technical solutions of the present application, the preferred embodiments of the present application are described in detail below.

[0033] Example 1: Screening of M. tuberculosis RD region antigens

[0034] 1. Analysis and prediction of RD region antigens by bioinformatics method. The gene and protein sequence of candidate antigens were found and downloaded in https: / / www.ncbi.nlm.nih.gov / . The basic parameters of RD region antigens, such as molecular weight, hydrophilicity and isoelectric point, were analyzed in mycobrowser, ExPASy ProtParam tool and ExPASy ProtScale; the T cell dominant epitopes of RD region antigens were predicted by IEDB CD4episcore; the antigenicity was predicted by vaxijen.

[0035] 2. The antigen characteristics were selected as follows: the molecular weight range was between 10 to 60 kDa, the hydrophilicity was greater than 0, the immunogenicity was greater than 0.5, and at least two dominant epitopes existed, and the relative score of epitopes needed to be higher than 60. According to the above screening criteria, 8 dominant candidate antigens were identified, which were Rv1575, Rv1577c, Rv3118, Rv1507c, Rv1513, Rv2650c, Rv2653c and Rv1972. Although RD7 region antigen Rv2353c lacked obvious dominant epitopes, its immunogenicity value was the highest 0.8963, so it was also included in the screening range. RD9 region antigen Rv3619c belonged to ESAT6 family protein, the molecular weight was close to 10 kDa, and the antigenicity and dominant epitope score were higher, so it was also included in the screening.

[0036] 3. The specific information of candidate antigens was as follows Figure 1 .

[0037] Example 2: Expression and purification of RD region candidate antigens

[0038] 1. The nucleic acid sequences of 10 candidate antigens were amplified from Mycobacterium tuberculosis H37Rv and inserted into E. coli expression vector pET30a. After construction, they were transformed into E. coli expression strain BL21 for expression, and the target proteins were obtained by column chromatography and inclusion body dialysis renaturation purification.

[0039] 2. The antigen expression and purification information was as follows Figure 2 .

[0040] Example 3: Immune reactivity of RD region candidate antigens in tuberculosis infected patients

[0041] 1. To evaluate the immunoreactivity of RD region candidate antigens, the present study selected patients infected with M. tuberculosis for analysis. The 13 patients with tuberculosis enrolled in the study were all diagnosed as active infection, and the sputum smear results of all patients were positive. In the study, peripheral blood samples were collected by intravenous injection, and the peripheral blood was stimulated with RD region candidate antigens Rv1577c, Rv3118, Rv1513, Rv2353c, Rv2650c and Rv1972. At the same time, ESAT6-CFP10 was used as a positive control, and PBS was used as a negative control. The level of IFN-γ in the serum was detected 20 hours after stimulation. The results showed that the peripheral blood antigen-specific cells of patients with tuberculosis could secrete a high level of IFN-γ under the stimulation of ESAT6-CFP10 (P<0.01, Figure 3 ); RD3 region antigen Rv1577c induced a higher level of IFN-γ compared with PBS stimulation (P<0.05, Figure 3 ).

[0042] Example 4: Immunoreactivity of Rv1577c in cattle herd

[0043] 1. The immunoreactivity of Rv1577c was further evaluated in the cattle herd. A total of 224 cattle were selected, and the cattle herd was screened by whole blood IFN-γ release experiment using ESAT6-CFP10 to distinguish EC-IGRA positive cattle and EC-IGRA negative cattle. Peripheral blood samples were collected by tail vein, stimulated with Rv1577c, and the supernatant was collected by centrifugation after 20 hours to detect the level of IFN-γ. The results showed that Rv1577c induced significantly higher secretion of IFN-γ in EC-IGRA positive cattle than in EC-IGRA negative cattle (P<0.001, Figure 4 A). In addition, we found that the level of IFN-γ secreted by peripheral blood stimulated by ESAT6-CFP10 and Rv1577c was moderately correlated in all cattle samples (B). Figure 4

[0044] Example 5: Construction, expression and purification of fusion protein LB28

[0045] 1. Construction, expression and purification of fusion protein LB28. First, the present application fuses the M. tuberculosis deletion region antigens Rv1577c and Rv1738 at the genetic level. Considering the influence of protein spatial structure, a linker (GGGGS)3 sequence is added between Rv1577c and Rv1738 to assist the folding expression of the fusion protein and reduce the mutual influence of spatial structure. According to this, the recombinant plasmid pET30a(+)-LB28 (Rv1577c-linker-Rv1738) is constructed Figure 5 ​A). The plasmid pET30a (+) -LB28 was constructed by total gene synthesis, and then the pET30a (+) -LB28 plasmid vector was transformed into E. coli for expression of the fusion protein LB28. Finally, according to the protein characteristics of the protein, the fusion protein was successfully purified by urea denaturation and dialysis renaturation. The specific expression and purification steps are as follows:

[0046] (1) Expression of the fusion protein LB28

[0047] The successfully constructed pET30a (+) -LB28 (Rv1577c-linker-Rv1738) vector was transformed into the expression vector E. coli BL21 (DE3). The LB28-expressing E. coli BL21 (DE3) strain was activated, and 1 ml of the activated bacterial solution was added to 200 ml of LB medium and incubated at 37°C for 4 h with shaking. Then, 100 ul of protein inducer IPTG (1.0 mmol / L) was added, and the culture was induced and shaken at 37°C for 4 h. The bacterial cells were collected by centrifugation at 4°C at 10,000 G for 10 min. The bacterial cells were resuspended in 10 ml of PB buffer (Na2HPO4·12H2O 20 mol / L, NaH2PO4·2H2O 20 mol / L, pH 7.4) per gram of wet bacteria, and the bacteria were sonicated for 0.5 h (180-200 W, 3 s on and 3 s off) on ice. After centrifugation at 10,000 G for 15 min, the supernatant and the precipitate were collected and subjected to polyacrylamide gel electrophoresis, respectively. Compared with the E. coli BL21 (DE3) empty bacteria, there was a specific protein expression band with a molecular weight of about 28 KD, mainly in the form of inclusion bodies. Figure 5 B).

[0048] (2) Purification of the fusion protein LB28

[0049] A large amount of fusion protein LB28 was expressed, and the collected bacterial cells were resuspended in 20 mM PB buffer, and then ultrasonically broken for about 0.5 h in an ice bath. The sample was centrifuged at 10,000 G at 4°C for 10 min, and the precipitate containing LB28 protein was collected. First, the inclusion body was resuspended with a washing buffer (the washing buffer contains urea, Tris, EDTA and Triton-100, with concentrations of 2 Mol / L, 0.1 Mol / L, 0.01 Mol / L and 0.5%, respectively, and pH 8.0-8.5), and the precipitate was collected by centrifugation (10000 rpm, 2 min) for five times. ① The centrifuged sample was denatured and dissolved in 8 M urea + Tris buffer (Tris, EDTA, NaCl and glycerol, with concentrations of 50 mMol / L, 0.5 mMol / L, 50 mMol / L and 50 ml / L, respectively, and pH 8.0-8.5) at 4°C for 12 h. ② Then the sample was dialyzed and renatured in 6 M urea + Tris buffer (Tris, EDTA, NaCl and glycerol, with concentrations of 50 mMol / L, 0.5 mMol / L, 50 mMol / L and 50 ml / L, respectively, and pH 8.0-8.5) at 4°C for 12 h. ③ Then the sample was dialyzed and renatured in 4 M urea + Tris buffer (Tris, EDTA, NaCl and glycerol, with concentrations of 50 mMol / L, 0.5 mMol / L, 50 mMol / L and 50 ml / L, respectively, and pH 8.0-8.5) at 4°C for 12 h. ④ Then the sample was dialyzed and renatured in 2 M urea + Tris buffer (Tris, EDTA, NaCl and glycerol, with concentrations of 50 mMol / L, 0.5 mMol / L, 50 mMol / L and 50 ml / L, respectively, and pH 8.0-8.5) at 4°C for 12 h. ⑤ The sample was dialyzed and renatured in 1 M urea + Tris buffer (Tris, EDTA, NaCl and glycerol, with concentrations of 50 mMol / L, 0.5 mMol / L, 50 mMol / L and 50 ml / L, respectively, and pH 8.0-8.5) at 4°C for 12 h. ⑥ Finally, the sample was replaced into a Tris buffer (Tris, EDTA, NaCl and glycerol, with concentrations of 50 mMol / L, 0.5 mMol / L, 50 mMol / L and 50 ml / L, respectively, and pH 8.0-8.5) without urea to obtain a fusion protein LB28 with a purity of 95% (Figure 6). Figure 5 C).

[0050] Example 6: Immunoreactivity of fusion protein LB28 in human population

[0051] 1. To analyze the immunogenicity of fusion protein LB28, we first established a cohort of clinical human population. All participants were tested by EC-IGRA (enzyme-linked immunospot assay for interferon-gamma release after stimulation with ESAT6-CFP10 antigens) and grouped. The cohort included 38 EC-IGRA positive individuals and 30 EC-IGRA negative individuals.

[0052] 2. Peripheral blood was collected from the cohort by venous blood collection and stimulated with fusion protein LB28. After 20 hours of incubation at 37°C, the supernatant was collected by centrifugation and tested for IFN-γ levels. The results showed that LB28 induced significantly higher levels of IFN-γ production in EC-IGRA positive population than in EC-IGRA negative population. Figure 6 ).

[0053] Example 7: Preparation of fusion protein LB28 subunit vaccine

[0054] The fusion protein LB28 was diluted to 0.2 mg / ml with PBS buffer; Poly I:C was dissolved to 1 mg / ml with PBS buffer; cationic liposome dimo-thylidioctyl ammonium bromide (DDA) was prepared to 5 mg / ml with PBS, and incubated at 80°C for 10 min and then cooled to room temperature. 50 μl of Poly I:C solution was mixed with an equal amount of protein solution, and incubated at room temperature for 1 min. Then DDA solution was added dropwise to the mixed solution, and emulsified thoroughly to obtain a uniform emulsion of the vaccine, i.e. subunit vaccine LB28-DDA / Poly I:C (LB28 / DP). The vaccine was used for immunization at a dose of 200 μL per mouse.

[0055] Example 8: Analysis of immunogenicity of fusion protein LB28 in a mouse model

[0056] 1. 6-8 week old C57BL / 6 mice were selected and immunized with Rv1577c / DP, Rv1738 / DP and LB28 / DP respectively for three times with three weeks interval; phosphate buffered saline (PBS) group with the same immunization regimen was used as vaccine negative control; fusion protein LT33 subunit vaccine with the same immunization regimen was used as homologous vaccine control; and BCG was used for immunization once as vaccine positive control.

[0057] 2. Six weeks after the last immunization of mice, spleen lymphocytes were isolated and stimulated in vitro with the corresponding single antigen (5ug / ml) for 68h. The supernatant was collected by centrifugation and the secretion levels of IFN-γ, IL-2 and granzyme B in spleen lymphocytes were detected by ELISA. Twelve weeks after the last immunization of the vaccine, the proliferation capacity of long-term surviving T cells was assessed by incorporating EDU during two antigen stimulations. Serum was collected 6 weeks after the last immunization of the vaccine, and the levels of specific antibodies IgG, IgG1 and IgG2c against antigens Rv1577c and Rv1738 in the serum were analyzed by indirect ELISA.

[0058] 3. Six weeks after the last immunization of mice with the subunit vaccine LB28 / DP, upon stimulation with the antigen Rv1577c, LB28 / DP induced significantly higher secretion of antigen-specific IFN-γ, IL-2, and GzmB than the PBS and BCG groups (P < 0.001); upon stimulation with the antigen Rv1738, LB28 / DP induced secretion of antigen-specific IFN-γ, IL-2, and GzmB levels close to those of LT33 / DP ( Figure 7 12 weeks after the last immunization with subunit vaccine, the long-term survival CD4 + T cell proliferation ability was significantly higher than that of PBS and BCG control groups ( Figure 8 In addition, LB28 / DP induced high levels of antigen-specific antibodies IgG, IgG1, and IgG2c ( Figure 9 ).

[0059] Example 9: Analysis of the dominant T cell epitopes of antigen Rv1577c

[0060] 1. Rv1577c epitopes were synthesized separately using overlapping peptide methods, with six amino acids repeated between adjacent epitopes. A total of 25 Rv1577c antigen epitopes were obtained by chemical synthesis. 6-8 week old C57BL / 6 mice were selected and immunized three times with Rv1577c / DP and LB28 / DP, respectively, three weeks apart. PBS was used as a control. Six weeks after the last immunization of mice, splenic lymphocytes were isolated. The mice were stimulated in vitro with the peptide epitopes (5 μg / ml) for 20 hours, and the number of spleen IFN-γ secreting cells was measured using the ELISPOT method.

[0061] 2. The results of the study found that after LB28 / DP immunized mice, the P4, P23 and P24 peptide epitopes had good immunoreactivity. The results showed that the P4, P23 and P24 peptides were the dominant epitopes that induced cellular immune responses ( Figure 10 ).

[0062] Example 10: Protective effect of LB28 / DP immunized mice against Mycobacterium bovis infection

[0063] 1. In the development of vaccines, we selected the antigens Rv1577c and Rv1738 expressed in both Mycobacterium tuberculosis and Mycobacterium bovis to construct a subunit vaccine, increasing the LB28 / DP applicability. To evaluate the immunoprotective effect of the subunit vaccine LB28 / DP, we selected Mycobacterium bovis to infect the vaccinated mice to analyze the immunoprotective effect. We used LB28 / DP to vaccinate mice at 0-3-6 weeks, and used LT33 / DP as a control subunit vaccine to compare the immunoprotective effect of LB28 / DP and LT33 / DP in parallel to explore the potential of LB28 to replace LT33. Mycobacterium bovis contains the single antigens Rv1577c and Rv1738 that constitute LB28, so it is feasible to use it as an infectious strain.

[0064] 2. Fourteen weeks after the last vaccination of the mice with the subunit vaccine, all the experimental mice were infected with Mycobacterium bovis strain by intranasal instillation at a dose of 200 CFU per mouse. Forty-two days after infection, the mice were sacrificed and the lungs were removed for detection of lung pathological damage.

[0065] 3. BCG, as an attenuated strain of Mycobacterium bovis, showed lower lung pathological damage in mice infected with Mycobacterium bovis than the PBS group, but there was no significant difference. The lung bacterial load of the BCG group was significantly lower than that of the PBS group (P<0.05, Figure 11 D). The LB28 / DP and LT33 / DP groups had slightly better protection than BCG, but there was no significant difference. Compared with the PBS group, the percentage of lung pathological damage area in the LB28 / DP and LT33 / DP subunit vaccine groups was significantly reduced (P<0.05, Figure 11 C), and the lung bacterial load of the LB28 / DP and LT33 / DP groups was significantly lower than that of the PBS group (P<0.05, Figure 11 D); at the same time, the percentage of lung pathological damage area and lung bacterial load in the LB28 / DP immunized mice were close to those in the LT33 / DP group, indicating that LB28 / DP induced an immune protection effect close to that of LT33 / DP, and had the potential to replace LT33 for optimization of subunit vaccine antigen combination.

[0066] Example 11: Construction of recombinant gene vector pET30a(+)-LB28 (Rv1577c-linker-Rv1738)

[0067] The single antigen Rv1577c and Rv1738 are connected and constructed into the vector pET30a by the whole gene synthesis mode, the 5' end enzyme cutting site is EcoR I (CATATG), and the 3' end enzyme cutting site is Hind III (AAGCTT). Meanwhile, the single antigen Rv1577c and Rv1738 are added with a linker sequence in the middle to promote protein folding and increase stability (GGTGGTGGTGGTTCT), and the recombination gene vector pET30a (+)-LB28 (Rv1577c-linker-Rv1738) construction is completed.

[0068] The gene sequence of LB28

[0069] The italic part is an enzyme cutting site, and the part marked with "" is the nucleic acid sequence of Rv1577c; the part marked with "" is the nucleic acid sequence of Rv1738, and the bold part is the nucleic acid sequence of linker (GGGGS) 3;

[0070]

[0071]

[0072] The amino acid sequence of LB28

[0073] The italic part is an enzyme cutting site, and the part marked with "" is the amino acid sequence of Rv1577c; the part marked with "" is the amino acid sequence of Rv1738, and the bold part is the amino acid sequence of linker (GGGGS) 3;

[0074]

[0075] The tuberculosis subunit vaccine has an immune prevention and treatment effect. The ESAT6 and CFP10 antigens in the deletion region RD1 of the BCG vaccine have been developed and applied to the diagnosis of tuberculosis infection, and screening new antigens in the RD region to construct a subunit vaccine can provide convenience for future clinical trials. Through antigen screening and experimental verification, the application provides a candidate antigen Rv1577c of a tuberculosis subunit vaccine. Rv1577c is deleted in BCG and has good immunogenicity, and has certain consistency with ESAT6 and CFP10 in the diagnosis of bovine tuberculosis. The Rv1577c is combined with the latent infection related antigen Rv1738 to construct a tuberculosis fusion protein LB28. Under the assistance of the adjuvant DDA+PolyI:C (DP), the fusion protein LB28 shows good immunogenicity in a mouse model and has an immune protection effect against bovine Mycobacterium infection. Rv1577 and the fusion protein LB28 constructed based on the same can be applied to the prevention and treatment of animal and human tuberculosis.

Claims

1. A Mycobacterium tuberculosis deleted region protein Rv1577c, characterized in that The antigen has strong immunogenicity and can induce a strong cellular immune response. It can be used in the diagnosis of tuberculosis infection and the development of subunit vaccines.

2. A fusion antigen LB28 comprising the RD region of Mycobacterium tuberculosis and latent infection-related antigens, characterized in that: The fusion antigen comprises Rv1577c and Rv1738.

3. The fusion antigen LB28 according to claim 2, characterized in that The fusion antigen comprises an antigen combination in which the full length or partial sequences of Rv1577c and Rv1738 proteins are fused in various orders and connection forms, comprising Rv1577c, linker and Rv1738.

4. The fusion antigen LB28 according to claim 3, characterized in that The gene sequence of the RD region protein Rv1577c is SEQ ID NO: 1; the gene sequence of the gene linker is SEQ ID NO: 2; and the gene sequence of the gene Rv1738 is SEQ ID NO:

3.

5. A tuberculosis subunit vaccine comprising the fusion antigen according to claim 2 or 3, wherein the vaccine is in the form of a fusion protein subunit vaccine.

6. Use of the fusion antigen according to claims 2, 3, and 4 or the tuberculosis subunit vaccine according to claim 5 in the prevention and treatment of tuberculosis in humans and animals.

7. Use of the fusion antigens according to claims 2, 3, and 4 or the tuberculosis subunit vaccine according to claim 5 in the preparation of diagnostic reagents for tuberculosis in humans and animals.

8. A method for preparing the fusion antigen LB28 according to claim 2, characterized in that: The fusion antigen was purified as follows: (1) Purification of fusion antigen: The fusion antigen is expressed in inclusion bodies and is first washed with a washing buffer containing urea, NaCl, EDTA, and Triton-100, all at a concentration of 20 mMol / L, pH 7.4; the precipitate is collected by centrifugation (10,000 rpm, 10 min) and treated five times; (2) After washing, the samples containing fusion antigens were dissolved in 8 M urea + 50 mM Tris (pH 8.0-8.5) at 4°C for 12 h. Replace it with 6M urea + 50mM Tris (pH8.0-8.5) and dissolve it at 4°C for 12 hours; replace it with 4M urea + 50mM Tris (pH8.0-8.5) and dissolve it at 4°C for 12 hours; replace it with 2M urea + 50mM Tris (pH8.0-8.5) and dissolve it at 4°C for 12 hours; replace it with 1M urea + 50mM Tris (pH8.0-8.5) and dissolve it at 4°C for 12 hours; replace it with 0M urea + 50mM Tris (pH8.0-8.5) and dissolve it at 4°C for 12 hours to obtain the target protein.

9. The tuberculosis subunit vaccine according to claim 5, characterized in that The fusion antigen is mixed with an adjuvant to obtain a tuberculosis subunit vaccine, wherein the adjuvant is DP adjuvant.

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