CN104151433A - Mycobacterium tuberculosis fusion proteins and their preparation method and use - Google Patents

Abstract

The invention provides a mycobacterium tuberculosis fusion protein, which is the protein in sequence 2 and sequence 4 in the sequence list. And provide its preparation method and application. In the present invention, the fusion protein LT70 and the adjuvant DDA+PolyI:C are combined to construct a tuberculosis subunit vaccine, and the experimental results show that the vaccine can effectively induce tuberculosis antigen-specific cellular and humoral immune responses in mice, and is effective in mice. In the challenge protection efficiency test of Li strains, it has strong immune protection, the protection time is relatively long, the protection effect is stronger than BCG, and it also has the effect of strengthening BCG immunity. It is an effective candidate vaccine for tuberculosis.

Description

A kind of mycobacterium tuberculosis fusion protein and its preparation method and application

technical field

The invention relates to a mycobacterium tuberculosis fusion protein, and also relates to a preparation method of the fusion protein and its application in preparing a tuberculosis subunit vaccine.

Background technique

Tuberculosis, caused by Mycobacterium tuberculosis infection, is the most widely spread, longest lasting and most serious infectious disease in the world. Since the 1980s, with the emergence and prevalence of drug-resistant strains, especially multidrug-resistant tuberculosis, and the spread and prevalence of human immunodeficiency virus (HIV) and acquired immunodeficiency syndrome (AIDS, AIDS), in The incidence of tuberculosis around the world shows a rising trend, and the morbidity and mortality remain high. As the pathogen of tuberculosis, one of the reasons why Mycobacterium tuberculosis is difficult to control is that Mycobacterium tuberculosis can exist in the immune cells of the human body as a latent infection state for a long time, so as to evade the body's specific immune response to clear it, and can be Active tuberculosis develops when the body's immunity is weakened.

The vaccine BCG (BCG) used in the prevention and control of tuberculosis at this stage is a live attenuated Mycobacterium bovis vaccine. BCG has been used for human vaccination since 1921. It can effectively prevent severe meningeal tuberculosis and systemic miliary tuberculosis in newborns and children, but it cannot effectively prevent the occurrence of pulmonary tuberculosis in adults. Therefore, the main goal of novel vaccines and vaccination strategies is to prolong the duration of vaccine protection or to perform booster immunization on top of BCG priming. Compared with vaccines with BCG and other microorganisms as carriers, the tuberculosis subunit vaccine has the advantage of enhancing the immune effect of BCG, and has the characteristics of safety and reliability. An effective tuberculosis vaccine must not only protect against active tuberculosis, but also be able to generate an immune response against latent tuberculosis. Therefore, an ideal tuberculosis vaccine must incorporate multiple antigens from active and latent stages of Mycobacterium tuberculosis.

Studies have found that Mycobacterium tuberculosis active antigen ESAT-6 is an early secreted protein, which can stimulate the proliferation of T lymphocytes in the body, secrete interferon-γ, and play an important role in protective cellular immunity. The Ag85 complex is the main secretory protein in Mycobacterium tuberculosis and BCG, accounting for 30% of the total secreted protein in Mycobacterium tuberculosis H ₃₇ R _V strain. The activity of mycosyltransferase, which plays an important role in the final stage of mycobacterial cell wall synthesis, can induce specific cellular immune responses. In the previous study, we constructed six fusion proteins ESAT6-Ag85B-MPT64190-198-Mtb8.4(EAMM), Mtb10.4-HspX(MH), ESAT6-Mtb8.4, Mtb10.4-Ag85B, ESAT6 -Ag85B and ESAT6-RpfE were evaluated for immune protection, and it was found that EAMM (composed of the active antigen fusion of Mycobacterium tuberculosis) showed a strong immune protective effect, especially when EAMM and MH (containing the latent antigen HspX of Mycobacterium tuberculosis) were combined When used in combination (ie, EAMM+MH vaccine), the bacterial load in the spleen and lung was significantly reduced, achieving the same protective effect as BCG. The results showed that the immune protection of the protein subunit vaccine constructed in combination with growth phase and latent related antigens can promote the effective recognition of bacteria, especially dormant bacteria, and improve immunity, thereby eliminating and killing bacteria.

Contents of the invention

The invention provides a mycobacterium tuberculosis fusion protein, which is constructed by combining antigens related to growth phase and incubation period, which can promote the effective recognition of bacteria, especially dormant bacteria, and improve immunity, thereby eliminating and killing bacteria.

Mycobacterium tuberculosis Hrp1 (Rv2626c) is highly expressed in the latent period of Mycobacterium tuberculosis, and can bind to the surface of macrophages, induce the significant expression of pro-inflammatory cytokines interleukin-12 and tumor necrosis factor, and cause strong The immune response is an ideal dormant antigen.

Therefore, the present invention fuses the 190-198 amino acid polypeptide (CD8+ T cell epitope) of the active phase antigens ESAT6, Ag85B, Mtb8.4 and MPT64 of Mycobacterium tuberculosis with the latent antigen Hrp1 (Rv2626c) of Mycobacterium tuberculosis, Construct EAMM-Rv2626 fusion protein (molecular weight is about 70kD, so it is called LT70 for short).

The invention provides a mycobacterium tuberculosis fusion protein, which is the protein in sequence 2 and sequence 4 in the sequence list.

In Sequence Table 2, positions 1-95 are the amino acid sequence of ESAT6, positions 96-97 are the amino acid sequence of the restriction site, positions 98-382 are the amino acid sequence of Ag85B, positions 383-384 are the amino acid sequence of the restriction site Sequence, 385-393 is the 190-198 amino acid sequence of MPT64, 394-503 is the amino acid sequence of Mtb8.4, 504-505 is the amino acid sequence of the restriction site, 506-660 is the amino acid sequence of Rv2626c .

In Sequence Table 4, positions 1-95 are the amino acid sequence of ESAT6, positions 96-97 are the amino acid sequence of the restriction site, positions 98-412 are the amino acid sequence of Ag85B with linker, and positions 413-414 are the restriction site The amino acid sequence of the point, the 415-423 position is the 190-198 amino acid sequence of MPT64, the 424-533 position is the amino acid sequence of Mtb8.4, the 534-535 position is the amino acid sequence of the restriction site, and the 536-690 position is Rv2626c amino acid sequence.

The second object of the present invention is to provide the gene encoding the above-mentioned Mycobacterium tuberculosis fusion protein.

Preferably, it is the gene described in 1) or 2) below:

1) Its nucleotide sequence is sequence 1 or 3 in the sequence listing;

2) Nucleotide sequence complementary to sequence 1 or 3 in the sequence listing;

3) A DNA molecule that hybridizes to the DNA sequence defined in 1) under stringent conditions and encodes the fusion protein.

In Sequence Table 1, positions 1-285 are the encoding gene of ESAT6, positions 286-291 are the gene encoding the restriction site, positions 292-1146 are the gene encoding Ag85B, positions 1147-1152 are the encoding gene for the restriction site Gene, 1153-1179 is the coding gene of 190-198 amino acids of MPT64, 1180-1509 is the coding gene of Mtb8.4, 1510-1515 is the coding gene of restriction site, 1516-1980 is the coding gene of Rv2626c coding genes.

In Sequence Table 3, positions 1-285 are the encoding gene of ESAT6, positions 286-291 are the gene encoding the restriction site, positions 292-1236 are the gene encoding Ag85B, positions 1237-1242 are the encoding site for the restriction site Gene, 1243-1269 is the coding gene of 190-198 amino acids of MPT64, 1270-1599 is the coding gene of Mtb8.4, 1600-1605 is the coding gene of restriction site, 1606-2070 is the coding gene of Rv2626c coding genes.

The third object of the present invention is to provide a recombinant expression vector containing the above coding gene.

The fourth object of the present invention is to provide a host cell containing the above-mentioned recombinant expression vector.

The fifth object of the present invention is to provide a method for preparing the above-mentioned Mycobacterium tuberculosis fusion protein, including the expression and purification of the fusion protein, characterized in that: the expression of the Mycobacterium tuberculosis fusion protein is as follows: the bacteria containing the fusion protein The body was added to the LB medium for shaking culture, and the protein inducer IPTG with a final concentration of 0.5 mmol/L was added, and the shaking culture was induced at 29°C for 4 hours. The supernatant obtained after post-treatment was the expression product.

The sixth object of the present invention is to provide the application of the above-mentioned Mycobacterium tuberculosis fusion protein in the preparation of tuberculosis subunit vaccine.

The seventh object of the present invention is to provide a tuberculosis subunit vaccine containing the above-mentioned Mycobacterium tuberculosis fusion protein.

Preferably, the tuberculosis subunit vaccine is composed of the Mycobacterium tuberculosis fusion protein according to claim 1, DDA and PolyI:C.

The eighth object of the present invention is to provide the preparation method of the above-mentioned tuberculosis subunit vaccine, the preparation method of every 200 μ L tuberculosis subunit vaccine is as follows: the fusion protein is diluted to 0.2mg/ml with PBS buffer; PolyI:C uses PBS buffer Dissolved to 1mg/ml; DDA was prepared to 2.5mg/ml with PBS, cooled to room temperature after water bath; take 50μl of PolyI:C solution and the same amount of fusion protein solution, mix well, and place at room temperature; add 100μL DDA dropwise to the mixed solution Solution, and then fully emulsified, so that the vaccine is in a uniform creamy state to obtain the tuberculosis subunit vaccine.

The beneficial effects of Mycobacterium tuberculosis of the present invention are as follows:

1. The present invention utilizes genetic engineering technology to construct expression plasmids pET30a(+)-LT70 (ESAT6-Ag85B-MPT64 _190-198 -Mtb8.4-Rv2626c) and pET30a(+)-LT70 (ESAT6-linker -Ag85B- linker-MPT64 _190-198 - Mtb8.4-Rv2626c), the plasmid was transformed into Escherichia coli E. col-i BL-21(DE3), and the fusion protein was expressed in the supernatant in a soluble form. The conditions for the expression of the target protein LT70 in the supernatant soluble form were optimized.

2. The present invention uses the unlabeled protein purification technology to successfully purify the LT70 protein through two purification methods: hydrophobic column chromatography and molecular sieve chromatography.

3. In the present invention, the fusion protein LT70 and the adjuvant DDA+PolyI:C are combined to construct a tuberculosis subunit vaccine, and the immunological evaluation and protection efficiency evaluation are carried out through the C57BL/6 mouse model to comprehensively evaluate the immune protection effect of the LT70 vaccine. The experimental results show that the vaccine can effectively induce tuberculosis antigen-specific cellular and humoral immune responses in mice, and has strong immune protection in the challenge protection efficiency test of mouse virulent strains, and the protection time is relatively long. Stronger than BCG, it also has the effect of strengthening BCG immunity, and is an effective candidate vaccine for tuberculosis.

4. In order to enhance the soluble expression of the fusion protein and facilitate production, we have optimized the gene sequence and antigen structure of the fusion protein as follows: (1) Optimized the gene sequence of Ag85B and Rv2626c: optimize according to homo sapiens and use the advantage of Escherichia coli codons for expression, so that the fusion protein can be better expressed in E. coli host cells; (2) In order to keep each antigen fully folded into its natural conformation, we introduced amino acids with low hydrophobicity and low charge effect before and after Ag85B The linker of the fusion protein separates each antigen of the fusion protein, so that each antigen is fully folded, and the expression level of the supernatant is increased compared with that without the linker (see Figure 1), which enhances the expression function of the LT70 fusion protein.

Description of drawings

The accompanying drawings are used to provide a further understanding of the present invention, and constitute a part of the description, and are used together with the embodiments of the present invention to explain the present invention, and do not constitute a limitation to the present invention. In the attached picture:

Figure 1 shows the expression results of fusion protein LT70 and LT70 (with linker) in Escherichia coli E. coli BL21(DE3); among them, (a) is the expression result of fusion protein LT70. M, marker; 1, BL21 empty bacteria broken whole bacteria night; 2, LT70 bacteria broken whole bacteria liquid; 3, LT70 bacteria broken supernatant. (b) is the expression result of fusion protein LT70 and LT70 (plus linker). M, marker; 1, LT70 cell crushed whole cell liquid; 2, LT70 cell cell crushed supernatant; 3, LT70 cell cell crushed and precipitated; 4, LT70 (with linker) cell cell crushed whole cell liquid; 5 , Supernatant after crushing LT70 (with linker) cells; 6, Precipitate after crushing LT70 (with linker) cells.

Figure 2 shows the effect of different IPTG induction concentrations on the expression of the fusion protein LT70 when expressed in E. coli BL21 (DE3). Among them, M, marker, 1, 2, and 3 are the whole bacteria after crushing LT70 at a final concentration of 0.5mM IPTG, the supernatant after crushing LT70, and the precipitate after crushing LT70; 4, 5, and 6 are the final concentration of 0.2mM IPTG after crushing LT70 Whole bacteria, supernatant after crushing LT70, precipitate after crushing LT70; 7, 8, and 9 are the final concentrations of 0.1mM IPTG respectively. Whole bacteria after crushing LT70, supernatant after crushing LT70, and precipitate after crushing LT70.

Figure 3 shows the effect of different expression temperatures on the expression of fusion protein LT70 when expressed in E. coli BL21 (DE3). Among them, ( a ) M, marker; 1, 2, and 3 are the whole bacteria after LT70 crushing at 37°C, the supernatant after LT70 crushing, and the precipitate after LT70 crushing; 4, 5, and 6 are the whole cells after LT70 crushing at 34°C. Bacteria, supernatant after crushing LT70, and precipitate after crushing LT70; 7 and 8 are whole bacteria after crushing LT70 induced at 25°C, and supernatant after crushing LT70. ( b ) M, marker; 1, 2 are the supernatant after LT70 crushing induced at 31°C, and the precipitate after LT70 crushing; 3 is the supernatant after LT70 crushing induced at 31°C; 4, 5 are the supernatant after LT70 crushing induced at 29°C , LT70 was crushed and precipitated; 6 was 31°C induced LT70 and precipitated after crushing; 7 and 8 were the supernatant after 27°C induced LT70 crushing, and LT70 was crushed and precipitated. ( c ) M, marker; 1, 2 are the supernatant after LT70 crushing induced at 31°C, and the precipitate after LT70 crushing; 3 is the supernatant after LT70 crushing induced at 31°C; 4 is the precipitate after LT70 crushing induced at 31°C; 5, 6 7 and 8 are the supernatant after LT70 induction and crushing at 27°C, and the precipitation after LT70 crushing. ( d ) M, marker; 1, LT70 broken whole bacteria; 2, BL21 empty bacteria; 3, 4 respectively, 30 ℃ induced LT70 broken, supernatant after LT70 broken; 5, 6 29 ℃ induced LT70 broken, respectively After precipitation, the supernatant after LT70 crushing; 7 and 8 are the precipitation after LT70 crushing induced at 28°C, and the supernatant after LT70 crushing.

Figure 4 shows the purification and analysis results of the fusion protein LT70 in Escherichia coli E.coli BL21(DE3). Among them, (a) purification by hydrophobic chromatography. M, marker; 1, the fusion protein LT70 was salted out with 15% ammonium sulfate saturation; 2, the flow peak of the Butyl FF column of hydrophobic chromatography; 3, the elution peak of the Butyl FF column of hydrophobic chromatography; 4, the fragmentation of the fusion protein LT70 supernatant; (b) molecular sieve chromatography purification. M, marker; 1, fusion protein LT70 supernatant after crushing; 2, fusion protein LT70 was salted out with 15% ammonium sulfate and then filtered on the machine sample; 3, 4, gel Superdex 75 filtration chromatography; 5 was fusion Samples of protein LT70 salted out with 15% ammonium sulfate saturation. (c) LT70 Western blot analysis results: M, protein molecular weight standard; 1, anti-Rv2626c mouse serum; 2, mouse anti-Esat-6 monoclonal antibody; 3, negative control; 4, rabbit anti-Ag85B monoclonal antibody; 5, negative control.

Figure 5 shows the results of cell immunogenicity testing of tuberculosis subunit vaccine LT70; * p <0.05 vs PBS, BCG.

Figure 6 shows the test results of humoral immunogenicity of tuberculosis subunit vaccine LT70, where a is IgG1 and b is IgG2c; * p <0.05 vs PBS, BCG.

Figure 7 shows the evaluation of the protective effect of the tuberculosis subunit vaccine: the LT70 vaccine was immunized three times in the 0th, 2nd, and 4th weeks respectively, and 30 weeks after the last immunization, the respiratory aerosol was used to challenge the Mycobacterium tuberculosis H37Rv strain. Weekly organ CFU counts. * p <0.05 vs PBS, BCG, ** p <0.01 vs PBS, BCG, EAMM+MH.

Fig. 8 is the result of pathological response of mouse lung tissue after TB subunit vaccine immunization and strain challenge.

Fig. 9 is a histogram of the ratio of tuberculosis lesion area in lung tissue of mice to section area after tuberculosis subunit vaccine immunization and strain challenge.

Figure 10 shows the results of the cellular immune response of BCG enhanced by tuberculosis subunit vaccine LT70; * p <0.05 vs PBS, BCG.

Figure 11 shows the humoral immune response results of BCG enhanced by tuberculosis subunit vaccine LT70, where a is IgG1 and b is IgG2c ; * p <0.05 vs PBS, BCG.

Figure 12 is the evaluation of the protective effect of tuberculosis subunit vaccine LT70 to strengthen BCG; * p <0.05 vs PBS, ** p <0.01 vs PBS.

Figure 13 is the histopathological response of lung tissue in mice after enhanced BCG immunization with tuberculosis subunit vaccine and strain challenge; ** p <0.01 vs PBS.

Fig. 14 is a histogram of the ratio of tuberculosis lesion area in lung tissue of mice to section area after enhanced BCG immunization with tuberculosis subunit vaccine and strain challenge.

Detailed ways

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores.

The invention constructs a novel tag-free fusion protein, and mixes it with an adjuvant to construct a novel tuberculosis subunit vaccine.

Material:

The pre-coated ELISPOT kit was purchased from Beijing Dakwei Biotechnology Co., Ltd.;

pET30a(+)-Rv2626c was donated by Aeras;

PolyI:C was purchased from Kaiping Morning Glory Biochemical Pharmaceutical Co., Ltd.;

Cationic liposome dimethyl hexadecyl ammonium (dimo-thylidioctyl ammonium bromide, DDA) was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.;

T4 ligase was purchased from Takara Company.

Example 1

1. Construction and purification of fusion protein

1.1 Construction of fusion protein LT70 expression plasmids pET30a(+)-LT70 and pET30a(+)-LT70 (add linker)

(1) Construct the recombinant gene vector pET30a(+)-LT69 (ESAT6-Ag85B-MPT64 _190-198 - Mtb8.4-HspX).

A, construction of pET30a(+)-EA (ESAT6-Ag85B):

Using the clinical mycobacterium liquid as a template, ESAT6 and the corresponding genes of Ag85B were amplified by PCR, and cloned into plasmid pET30a.

Among them, the RCR reaction system:

Water 16.25μl

5×PCR buffer 5μl

dNTP 2 μl

Upstream primer P1 0.5μl

Downstream primer P2 0.5μl

DNA template 0.5μl

DNA polymerase (Primer STAR, takara company) 0.25μl

Total system 25μl.

PCR reaction parameters: denaturation at 98°C for 15 s, annealing at 60°C for 15 s, extension at 72°C for 50 s, a total of 30 cycles.

B, Primers designed to amplify the EA (ESAT6-Ag85B) gene fragment. The primers are: upstream (5'-3' CGG CATAT GACAGAGCAGCAGTGGAAT, where the underlined part is the Nde I restriction enzyme site); downstream (5'-3' GA AGATCT GCCGGCGCCTAACGAACTCTGGAG, where the underlined part is the Bgl II restriction enzyme site site). The EA (ESAT6-Ag85B) fusion gene product was amplified by PCR using the pET30a(+)-EA plasmid as a vector and using the above two primers.

Among them, the RCR reaction system:

Water 16.25μl

5×PCR buffer 5μl

dNTP 2 μl

Upstream primer P1 0.5μl

Downstream primer P2 0.5μl

DNA template 0.5μl

DNA polymerase (Primer STAR, takara company) 0.25μl

Total system 25μl.

PCR reaction parameters: denaturation at 98°C for 15 s, annealing at 63°C for 15 s, extension at 72°C for 50 s, a total of 35 cycles.

After the PCR product was purified, it was digested with Nde I and Bgl II, and the clone was constructed on the plasmid pET30a(+)-MMH (ie, pET30a(+)-MPT64 _190-198 - Mtb8.4-HspX), constructed as pET30a(+)-LT69 (ESAT6-Ag85B- _MPT64190-198 -Mtb8.4-HspX) vector. MPT64 _190-198 refers to the amino acid sequence at positions 190-198 of MPT64.

Wherein, the construction method of plasmid pET30a(+)-MMH is:

Use restriction sites Sac I and Hind Cut out the plasmid pET30a(+)-MH (i.e. pET30a(+)-Mtb8.4-Hspx) (the construction method has been disclosed, the invention patent: a method for the preparation and application of a fusion protein of Mycobacterium tuberculosis. Patent No.: ZL 201010613320.6) Hspx and Hspx were cut after the plasmid pET30a(+)-MM (ie pET30a(+)-MPT64 _190-198 - Mtb8.4), ligated by T4 ligase overnight at 16°C.

Among them, the plasmid pET30a(+)-MM (namely pET30a(+)-MPT64 _190-198 - Mtb8.4) was directly synthesized.

(2) Construct the recombinant gene vector pET30a(+)-LT70 (ESAT6-Ag85B- MPT64 _190-198 - Mtb8.4-Rv2626c).

Primers were designed to amplify the Rv2626c gene fragment. The primers are: upstream (5'-3' AC GAGCTC ATGACCACGG, where the underlined part is the Sac I restriction site); downstream (5'-3' CCC AAGCTT CTATGCATTTAG, where the underlined part is Hin d restriction enzyme sites). The pET30a(+)-Rv2626c plasmid was used as a template, and the above two primers were used to amplify the Rv2626c gene sequence by PCR. After the PCR product was purified, Sac I and Hin d Double digestion was performed, and the clone was constructed on the plasmid pET30a(+)-LT69, which was constructed into a pET30a(+)-LT70(ESAT6-Ag85B-MPT64 _190-198 -Mtb8.4-Rv2626c) vector.

Among them, the RCR reaction system:

Water 16.25μl

5×PCR buffer 5μl

dNTP 2 μl

Upstream primer P1 0.5μl

Downstream primer P2 0.5μl

DNA template 0.5μl

DNA polymerase (Primer STAR, takara company) 0.25μl

Total system 25μl

PCR reaction parameters: denaturation at 98°C for 15 s, annealing at 60°C for 15 s, extension at 72°C for 50 s, a total of 30 cycles.

(3) Construct the recombinant gene vector pET30a(+)-LT70 (ESAT6-Ag85B- MPT64 _190-198 - Mtb8.4-Rv2626c) (add linker).

Primers were designed to amplify the Ag85B gene fragment. The primers are: upstream primer (5'-3' CGG GAATTC GGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCT TTCTCC CGG CCGG, wherein the single underlined part is the Eco RI restriction site, and the double underlined part is the linker sequence); the downstream primer (5'-3' CCC AGATCTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTG GTGGTGGTGGTTCT ACCAGCACCCAGAGAAG, where the single underlined part is the Bgl II restriction site, and the double underlined part is the linker sequence). The pET30a(+)-LT70 plasmid was used as a vector, and the above two primers were used to amplify the Ag85B (linker) gene sequence by PCR.

RCR reaction system:

Water 16.25μl

5×PCR buffer 5μl

dNTP 2 μl

Upstream primer P1 0.5μl

Downstream primer P2 0.5μl

DNA template 0.5μl

DNA polymerase (Primer STAR, takara company) 0.25μl

Total system 25μl

PCR reaction parameters: denaturation at 98°C for 15 s, annealing at 60°C for 15 s, extension at 72°C for 50 s, a total of 30 cycles.

After the PCR product was purified, it was digested with Eco RI and Bgl II, cloned and constructed on the plasmid pET30a(+)-LT70, and constructed into pET30a(+)-LT70(ESAT6-Ag85B-MPT64 _190-198 -Mtb8. 4-Rv2626c) (plus linker) vector.

1.2 Expression of fusion protein LT70 and LT70 (with linker)

The successfully constructed pET30a(+)-LT70(ESAT6-Ag85B-MPT64 _190-198 -Mtb8.4-Rv2626c) vector was transformed into E. coli E. coli BL21(DE3). Activate E. coli BL-21(DE3) cells expressing LT70 protein, take 1ml of the activated cells and add them to 200ml LB medium for shaking culture for 4h; add protein inducer IPTG (1.0mol/L) 100μl (final concentration is 0.5mmol/L), induced shaking culture at 29°C for 4h; 4°C, 10000rpm centrifugation for 30min to collect the bacteria; the bacteria were resuspended in 20mM PB buffer (Na ₂ HPO ₄ ·12H ₂ O 20mmol/L, Na ₂ HPO ₄ ·12H ₂ O 20mmol/L, pH7.4), 10ml/g wet bacteria, sonicate the bacteria in ice bath for 1h (180～200W, every 3s, stop for 5s); centrifuge at 10000rpm for 30min, collect supernatant and precipitate respectively, and put The supernatant and the precipitate were subjected to polyacrylamide gel electrophoresis respectively; the electrophoresis diagram is shown in Figure 1, and after SDS-PAGE electrophoresis analysis, compared with E. coli BL21 (DE3) empty bacteria, there were obvious specific protein expression bands at a molecular weight of about 70KD, The expression is mainly in the supernatant form, and the protein in the precipitate is relatively small.

The expression process and conditions of the fusion protein LT70 (with linker) are exactly the same as those of the fusion protein LT70. See Figure 1 for the electropherogram. It can be seen from Figure 1 that the expression level of the supernatant after adding the linker is increased compared with that without adding the linker.

The above fusion protein expression conditions were optimized in the following aspects:

1.2.1 Inducer IPTG concentration optimization

To activate the E. coli BL-21 (DE3) cell expressing LT70 protein, take 1 ml of the activated cell and add it to 200 ml LB medium for 4 hours of shaking culture. Add three final concentrations of protein inducer IPTG (1.0mol/L): a: 100μl (final concentration is 0.5mmol/L); b: 40μl (final concentration is 0.2mmol/L); c: 20μl (final concentration is 0.1 mmol/L), induced shaking at 37°C for 4 h. Centrifuge at 10,000 rpm for 30 min at 4°C to collect the bacterial cells respectively. The bacteria were resuspended in 20mM PB buffer (Na ₂ HPO ₄ 12H ₂ O 20mmol/L, Na ₂ HPO ₄ 12H ₂ O 20mmol/L, pH7.4) 10ml/g wet bacteria, and the bacteria were sonicated in an ice bath for 1h (180-200 W, every 3s of ultrasound, stop for 5s). After centrifuging at 10000 rpm for 30 min at 4°C, the supernatant and the precipitate were collected respectively, and the supernatant and the precipitate were subjected to polyacrylamide gel electrophoresis respectively. The analysis results showed that the expression of the target protein LT70 could be induced when the final concentration of IPTG was three concentrations, and the expression of the target protein LT70 was the highest when the final concentration was 0.5mmol/L.

1.2.2 Optimization of LT70 induction expression temperature

Activate the E. coli BL-21(DE3) strain expressing LT70 protein, take 1ml of the activated bacteria and add it to 200ml LB medium for shaking culture for 4h; add protein inducer IPTG (1.0mol/L) 20ul, respectively (37°C/34°C/31°C/30°C/29°C/28°C/27°C/25°C) induced shaking culture for 4h; 4°C, 10000rpm centrifugation for 30min to collect the bacteria; the bacteria were resuspended in 20mM PB buffer ( Na ₂ HPO ₄ 12H ₂ O 20mmol/L, Na ₂ HPO ₄ 12H ₂ O 20mmol/L, pH 7.4), 10ml/g wet bacteria, sonicate the bacteria in ice bath for 1h (180～200W, every sonication 3s, stop for 5s); after centrifuging at 10000rpm for 30min, the supernatant and the precipitate were collected respectively, and the supernatant and the precipitate were subjected to polyacrylamide gel electrophoresis; SDS-PAGE electrophoresis analysis showed that under different induction temperature conditions, the LT70 target protein could be expressed on the Soluble expression in the supernatant, with the highest expression in the supernatant when the induction temperature was around 29°C.

1.3 Purification method of fusion protein LT70

  Resuspend LT70 bacteria in 20mM PB buffer, sonicate for about 1 hour in an ice bath, centrifuge at 10,000 rpm at 4°C for 10 minutes, and collect the supernatant containing LT70 protein after centrifugation.

1.3.1 Salting out crude purification: the fusion protein was salted out with 15% saturated ammonium sulfate solution; the salted out protein was resuspended in 20mM PB buffer.

1.3.2 Chromatographic purification of the fusion protein after salting out

1.3.2.1 Purification by hydrophobic column chromatography: The LT70 protein after salting out was further purified by using a hydrophobic prepacked column HiTrap Butyl FF, and the elutions from different gradients were collected for polyacrylamide gel (SDS-PAGE) electrophoresis analysis of the purified product. The finally obtained purified protein can be used after its concentration is determined by the Lorry phenol reagent method, and it is diluted to 0.2 mg/ml with PBS before each use.

1.3.2.2 Molecular sieve chromatography purification: use gel filtration medium Superdex 75 pre grade (superdex is a suitable filler for cross-linked agarose and glucose; separation range: 3000-70000) for purification, collect different gradient eluents for polypropylene The final purified product was analyzed by amide gel (SDS-PAGE) electrophoresis. The finally obtained purified protein can be used after its concentration is determined by the Lorry phenol reagent method, and it is diluted to 0.2 mg/ml with PBS before each use.

The purification process and method of fusion protein LT70 and LT70 (plus linker) are exactly the same, and the purification results of the two are the same after experimental verification.

1.4 Western Blot verification of the activity of the fusion protein LT70

(1) Run SDS-PAGE electrophoresis on the purified protein;

(2) Membrane transfer: first cut the PVDF membrane into a size of 5.3×8.3cm, and cut the filter paper into a size of 8×10cm and immerse it in methanol for 1-2min. Then, arrange in order whiteboard/sponge/four-layer filter paper/PVDF membrane/glue/four-layer filter paper/sponge/blackboard to make a sandwich solid-phase carrier. Connect the electrodes in the ice bath state, place the ice box in the transfer tank on the side of the blackboard, and fill the inner and outer tanks with transfer buffer. Film transfer conditions: 200V, 200mA (constant current fast transfer), 1h;

(3) Sealing: Rinse the PVDF membrane with TBST for a while (1-2min). Cover the membrane with blocking solution (two PVDF membranes back to back, put them in 5% skimmed milk powder crosswise), and shake gently on a horizontal shaker for 4 hours;

(4) Membrane washing: Put the sealed PVDF membrane into TBST, shake slowly and lightly on a horizontal shaker, wash 10 times, 10min/time;

(5) Incubate the primary antibody (antigen-immunized mouse serum): According to the maker, cut off the PVDF membrane where the target protein is located on the PVDF membrane, and pay attention to the corner mark. Put the trimmed PVDF membrane strips into a centrifuge tube containing the diluted primary antibody (the primary antibody covers the membrane strips, only two can be added to a centrifuge tube, back to back). Shake gently on a horizontal shaker at room temperature for 12 hours;

(6) Membrane washing: Put the PVDF membrane incubated with the primary antibody into TBST, shake gently on a horizontal shaker, wash 3 times, 10min/time;

(7) Incubate the secondary antibody (rabbit anti-mouse polyclonal antibody): put the cleaned PVDF membrane into a large centrifuge tube containing the diluted secondary antibody (1:5000) (same as the primary antibody, one centrifuge tube can only Plus two, and back to back). At room temperature, shake slowly and lightly for 3 hours on a horizontal shaker in the dark;

(8) Membrane washing: Put the PVDF membrane incubated with the secondary antibody into TBST, shake gently on a horizontal shaker, wash 5 times, 10min/time;

(9) Development (operation in darkroom): Lay the plastic wrap in the exposure box flatly, place the PVDF film flat on the lower plastic wrap, add ECL ultra-sensitive luminescent liquid to each PVDF film drop by drop, and cover the upper layer with plastic wrap PVDF membrane (pay attention to drive out air bubbles). Observe the fluorescence intensity in a dark room, and consider the thickness of the exposure film (number of sheets). Cut a piece of film, press it on the PVDF film, avoid moving, cover the exposure box, and place it in the dark for 30 minutes. Take out the film and place it in developer, ultrapure water, and fixer in sequence. Finally, wash the film with tap water and observe the results.

2.2 Evaluation of immunological activity of tuberculosis subunit vaccine LT70 and LT70 (plus linker)

Tuberculosis subunit vaccine LT70 (with linker) is a linker added at the gene level, which increases the expression of 15 amino acids during amino acid synthesis. Since this linker is non-immunogenic and will not affect the function of the protein, it will not change the protective effect of the vaccine and can be used as a vaccine. At the same time, it has been verified by experiments that the immune effects of tuberculosis subunit vaccine LT70 and LT70 (plus linker) are the same. The following will take tuberculosis subunit vaccine LT70 as an example to evaluate its immunological activity in detail.

2.2.1 Experimental materials: tuberculosis subunit vaccine LT70-DDA/PolyI:C; BCG (BCG); phosphate buffered saline (PBS).

The preparation method of tuberculosis subunit vaccine LT70-DDA/PolyI:C is as follows: fusion protein LT70 is diluted to 0.2mg/ml with PBS buffer; PolyI:C is dissolved to 1mg/ml with PBS buffer; Dimo-thylidioctyl ammonium bromide (DDA) was prepared in PBS to 2.5mg/ml, in a water bath at 80°C for 10min, and cooled to room temperature. Take 50 μl of PolyI:C solution and mix well with an equal amount of fusion protein LT70 solution, and let stand at room temperature for 1 min. Add 100 μL of DDA solution dropwise to the mixed solution, and then fully emulsify to make the vaccine in the form of a uniform cream to obtain the tuberculosis subunit vaccine LT70-DDA/PolyI:C. When the vaccine is used for immunization, the dosage is 200 μL/mouse.

2.2.2 Experimental animals: C57BL/6 mice;

2.2.3 Grouping of experimental animals (four groups in total):

Control group: PBS; BCG;

test group: (EAMM+MH)-DDA/PolyI:C; LT70-DDA/PolyI:C.

2.2.4 Time point for immunizing animals:

Week 0: subcutaneously immunize the control group with PBS and BCG (5×10 ⁶ CFU/rat) in the groin, and at the same time subcutaneously immunize the experimental group with the subunit vaccine in the groin (the group and group) animal 200μl/only;

The 2nd and 4th weeks: The same dose of subunit vaccine was subcutaneously immunized the animals in the experimental group twice.

2.2.5 Detection of immune indicators

2.2.5.1 ELISPOT method to detect the level of antigen-specific IFN-γ secreted by mice

Six weeks after the last immunization of the mice, the spleen lymphocytes were aseptically isolated, and the enzyme-linked immunospot assay (ELISPOT) technique was used to detect the IFN- The secretion level of gamma.

Specific steps: remove the spleen aseptically, filter through a 200-mesh nylon mesh after grinding, and separate lymphocytes with lymphocyte separation medium. The isolated lymphocytes were added to 96-well IFN-γ pre-coated ELISPOT plates with a final concentration of 5×10 ⁶ /well, and ESAT6 (10 ug/ml), Ag85B (5 ug/ml), and Mtb8 were given respectively. 4 (200ug/ml, a peptide synthesized by ourselves), Rv2626c (10 ug/ml) and PPD (5ug/ml) stimulation. After co-incubating for 48 hours at 37°C and 5% CO ₂ , add detection antibodies and other reagents in sequence according to the ELISPOT operating instructions, wash the plate, develop color, and count the number of spots. See Figure 5 for the results.

2.2.5.2 ELISA method to detect the level of antigen-specific antibodies (IgG1, IgG2c) secreted by mice

Coat the 96-well plate (100 μl/well) with ESAT6 (10ug/ml), Ag85B (5ug/ml) and Rv2626c (10ug/ml) respectively at 4°C overnight; wash the plate with PBST solution 300μl/well 5 times×1min/time ; Start doubling dilution from 1:100 to 1:102400, add double-diluted serum samples (serum obtained 6 weeks after the last immunization of mice), and place at 37°C for 1h. After washing the plate, 200 μl/well of 1:15000 diluted rabbit anti-mouse IgG1 and 1:10000 diluted rabbit anti-mouse IgG2c were added, and placed at 37°C for 1 hour. After washing the plate, add 100 μl/well TMB color developing solution, react in the dark for 15 minutes at room temperature, then add 50 μl/well stop solution (2N H ₂ SO ₄ ) to terminate the reaction; detect the OD value at 450 nm. See Figure 6 for the results.

2.3 Evaluation of the protective effect of tuberculosis subunit vaccine LT70

The mice were immunized with LT70 vaccine three times at the 0th, 2nd, and 4th week respectively. At the 30th week after the last immunization, the Mycobacterium tuberculosis strain H37Rv was challenged with respiratory aerosol at a dose of 50-100 CFU/mouse. At 10 weeks, lung and spleen tuberculosis loads were detected. This experiment was carried out in the ABSL-3 laboratory of Wuhan University.

Specific method: Aseptically take the lungs and spleen of mice, grind the organs in a mortar, add 2ml of PBS buffer, then dilute the liquid in five gradients, and inoculate 0.1ml of each gradient liquid into 7H11 on the culture medium. Cultured in a 37°C incubator for 18 days, and counted CFU. See Figure 7 for the results.

2.4 Histopathological damage detection of tuberculosis subunit vaccine LT70 in mice

Observation of organ tissue slices in experimental mice: After the mice were dissected, the right lung tissue was taken, and paraffin sections were stained with hematoxylin-eosin (HE) staining to observe the pathological damage of the lungs; Stain to observe the number of Mycobacterium tuberculosis in the tissue. Pathological testing was entrusted to Wuhan University ABSL-3 to complete. The results are shown in Figure 8 and Figure 9: PBS group: the average area of tuberculous lesions accounted for 17% of the slice area, BCG group: the average area of tuberculous lesions accounted for 11% of the slice area, EAMM+MH vaccine group: tuberculous lesions The average area of lesion area accounted for 14% of the section area, LT70 vaccine group: the average area of tuberculous lesion area accounted for 12%.

2.5 Evaluation of the immune activity of tuberculosis subunit vaccine LT70 to strengthen BCG

2.5.1 Experimental materials: subunit vaccine LT70-DDA/PolyI:C; BCG (BCG); phosphate buffered saline (PBS).

2.5.2 Experimental animals: C57BL/6 mice;

2.5.3 Grouping of experimental animals (three groups in total):

control group PBS; BCG; experimental group BCG primary immunization, LT70-DDA/PolyI:C vaccine booster group.

2.5.4 Time point of immunization of animals

Week 0: Bacillus Calmette-Guerin (BCG) 5×10 ⁶ CFU subcutaneously immunized the animals of the experimental group once in the groin; at the same time, immunized the control group PBS and BCG groups (5×10 ⁶ CFU/rat).

The 18th and 21st weeks: The subunit vaccine LT70 subcutaneously in the groin with 200ul/only boosted the animals in the experimental group twice.

2.5.5 Determination method of immune index

2.5.5.1 ELISPOT method to detect the level of antigen-specific IFN-γ secreted by mice

Six weeks after the last immunization, the levels of cytokines secreted by mouse splenocytes under the stimulation of antigens ESAT6, Ag85B and Rv2626c were detected. The specific method and steps are the same as 2.2.5.1. See Figure 10 for the results.

2.5.5.2 ELISA method to detect the level of antigen-specific antibodies (IgG1, IgG2c) secreted by mice

Six weeks after the last immunization, the level of antibody in spleen cells of mice stimulated by antigens ESAT6, Ag85B and Rv2626c was detected. The specific method and steps are the same as 2.2.5.2 . See Figure 11 for the results.

2.6 Evaluation of the protective effect of tuberculosis subunit vaccine LT70 on strengthening BCG

The mice were primed with BCG at the 0th week, and the LT70 vaccine was boosted twice at the 18th and 21st weeks. After the last immunization, the respiratory aerosol challenge of the Mycobacterium tuberculosis strain H37Rv was performed at the 12th week at a dose of 50-100 CFU / mouse, the lung and spleen tuberculosis loads were detected in the 10th week after the challenge.

Specific method: Aseptically take the lungs and spleen of mice, grind the organs in a mortar, add 2ml of PBS buffer, then dilute the liquid in five gradients, and inoculate 0.1ml of each gradient liquid into 7H11 on the culture medium. Cultured in a 37°C incubator for 18 days, and counted CFU. See Figure 12 for the results.

2.7 Detection of histopathological damage in mice after TB subunit vaccine LT70 enhanced BCG immunization and strain challenge

Observation of organ tissue slices in experimental mice: After the mice were dissected, the right lung tissue was taken, and paraffin sections were stained with hematoxylin-eosin (HE) staining to observe the pathological damage of the lungs; Stain to observe the number of Mycobacterium tuberculosis in the tissue. See Figure 13 and Figure 14 for the results: PBS group: the average area of tuberculous lesions accounted for 17% of the slice area; BCG group: the average area of tuberculous lesions accounted for 11% of the slice area; Lesions accounted for an average of 7% of the slice area.

Finally, it should be noted that: the above is only a preferred embodiment of the present invention, and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, for those skilled in the art, it still The technical solutions recorded in the foregoing embodiments may be modified, or some technical features thereof may be equivalently replaced. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

sequence listing

<110> Lanzhou University

<120> A Mycobacterium tuberculosis fusion protein and its preparation method and application

the

<210> 1

<211> 1980

<212> DNA

<213> Artificial sequence

the

<400> 1

atgacagagc agcagtggaa tttcgcgggt atcgaggccg cggcaagcgc aatccaggga 60

aatgtcacgt ccattcattc cctccttgac gaggggaagc agtccctgac caagctcgca 120

gcggcctggg gcggtagcgg ttcggaggcg taccagggtg tccagcaaaa atgggacgcc 180

acggctaccg agctgaacaa cgcgctgcag aacctggcgc ggacgatcag cgaagccggt 240

caggcaatgg cttcgaccga aggcaacgtc actgggatgt tcgcagaatt cttctcccgg 300

ccggggctgc cggtcgagta cctgcaggtg ccgtcgccgt cgatgggccg cgacatcaag 360

gttcagttcc agagcggtgg gaacaactca cctgcggttt atctgctcga cggcctgcgc 420

gcccaagacg actacaacgg ctggatatc aacacccccgg cgttcgagtg gtactaccag 480

tcgggactgt cgatagtcat gccggtcggc gggcagtcca gcttctacag cgactggtac 540

agcccggcct gcggtaaggc tggctgccag acttacaagt gggaaacctt cctgaccagc 600

gagctgccgc aatggttgtc cgccaacagg gccgtgaagc ccaccggcag cgctgcaatc 660

ggcttgtcga tggccggctc gtcggcaatg atcttggccg cctaccaccc ccagcagttc 720

atctacgccg gctcgctgtc ggccctgctg gacccctctc aggggatggg gcctagcctg 780

atcggcctcg cgatgggtga cgccggcggt tacaaggccg cagacatgtg gggtccctcg 840

agtgacccgg catgggagcg caacgaccct acgcagcaga tccccaagct ggtcgcaaac 900

aacacccggc tatgggttta ttgcgggaac ggcaccccga acgagttggg cggtgccaac 960

atacccgccg agttcttgga gaacttcgtt cgtagcagca acctgaagtt ccaggatgcg 1020

tacaacgccg cgggcgggca caacgccgtg ttcaacttcc cgcccaacgg cacgcacagc 1080

tgggagtact ggggcgctca gctcaacgcc atgaagggtg acctgcagag ttcgttaggc 1140

gccggcagat ctttcgcagt cacgaacgac ggggtgatta tgaggctgtc gttgaccgca 1200

ttgagcgccg gtgtaggcgc cgtggcaatg tcgttgaccg tcggggccgg ggtcgcctcc 1260

gcagatcccg tggacgcggt cattaacacc acctgcaatt acgggcaggt agtagctgcg 1320

ctcaacgcga cggatccggg ggctgccgca cagttcaacg cctcaccggt ggcgcagtcc 1380

tatttgcgca atttcctcgc cgcaccgcca cctcagcgcg ctgccatggc cgcgcaattg 1440

caagctgtgc cgggggcggc acagtacatc ggccttgtcg agtcggttgc cggctcctgc 1500

aacaactatg agctcatgac cacggcgcgt gatatcatga atgcgggtgt cacctgtgtt 1560

ggcgagcacg aaacgttgac cgcagcagca cagtacatgc gcgaacatga tatcggcgca 1620

ttgccgattt gcggcgacga tgatcgtctg cacggtatgc tgaccgaccg cgatatcgtt 1680

atcaagggtc tggccgcagg cttggacccg aacaccgcga ccgccggtga actggcacgt 1740

gacagcatct attacgtcga cgcgaacgcc agcattcaag agatgctgaa cgtgatggaa 1800

gagcatcagg tgcgtcgtgt cccggttatc agcgaacatc gtctggttgg tatcgttacc 1860

gaagccgaca tcgcacgtca cctgccggag cacgcgattg ttcagttcgt gaaagcgatt 1920

tgcagcccga tggcgttggc gtctcgtcaa aagggcgaca caaaatttat tctaaatgca 1980

<210> 2

<211> 660

<212> PRT

<213> artificial protein

<400> 2

Met Thr Glu Gln Gln Trp Asn Phe Ala Gly Ile Glu Ala Ala Ala Ser

1 5 10 15

Ala Ile Gln Gly Asn Val Thr Ser Ile His Ser Leu Leu Asp Glu Gly

20 25 30

Lys Gln Ser Leu Thr Lys Leu Ala Ala Ala Trp Gly Gly Ser Gly Ser

35 40 45 45

Glu Ala Tyr Gln Gly Val Gln Gln Lys Trp Asp Ala Thr Ala Thr Glu

50 55 60 60

Leu Asn Asn Ala Leu Gln Asn Leu Ala Arg Thr Ile Ser Glu Ala Gly

65 70 75 80

Gln Ala Met Ala Ser Thr Glu Gly Asn Val Thr Gly Met Phe Ala Glu

85 90 95

Phe Phe Ser Arg Pro Gly Leu Pro Val Glu Tyr Leu Gln Val Pro Ser

100 105 110

Pro Ser Met Gly Arg Asp Ile Lys Val Gln Phe Gln Ser Gly Gly Asn

115 120 125

Asn Ser Pro Ala Val Tyr Leu Leu Asp Gly Leu Arg Ala Gln Asp Asp

130 135 140

Tyr Asn Gly Trp Asp Ile Asn Thr Pro Ala Phe Glu Trp Tyr Tyr Gln

145 150 155 160

Ser Gly Leu Ser Ile Val Met Pro Val Gly Gly Gln Ser Ser Phe Tyr

165 170 175

Ser Asp Trp Tyr Ser Pro Ala Cys Gly Lys Ala Gly Cys Gln Thr Tyr

180 185 190

Lys Trp Glu Thr Phe Leu Thr Ser Glu Leu Pro Gln Trp Leu Ser Ala

195 200 205

Asn Arg Ala Val Lys Pro Thr Gly Ser Ala Ala Ile Gly Leu Ser Met

210 215 220

Ala Gly Ser Ser Ala Met Ile Leu Ala Ala Tyr His Pro Gln Gln Phe

225 230 235 240

Ile Tyr Ala Gly Ser Leu Ser Ala Leu Leu Asp Pro Ser Gln Gly Met

245 250 255

Gly Pro Ser Leu Ile Gly Leu Ala Met Gly Asp Ala Gly Gly Tyr Lys

260 265 270

Ala Ala Asp Met Trp Gly Pro Ser Ser Asp Pro Ala Trp Glu Arg Asn

275 280 285

Asp Pro Thr Gln Gln Ile Pro Lys Leu Val Ala Asn Asn Thr Arg Leu

290 295 300

Trp Val Tyr Cys Gly Asn Gly Thr Pro Asn Glu Leu Gly Gly Ala Asn

305 310 315 320

Ile Pro Ala Glu Phe Leu Glu Asn Phe Val Arg Ser Ser Asn Leu Lys

325 330 335

Phe Gln Asp Ala Tyr Asn Ala Ala Gly Gly His Asn Ala Val Phe Asn

340 345 350

Phe Pro Pro Asn Gly Thr His Ser Trp Glu Tyr Trp Gly Ala Gln Leu

355 360 365

Asn Ala Met Lys Gly Asp Leu Gln Ser Ser Leu Gly Ala Gly Arg Ser

370 375 380

Phe Ala Val Thr Asn Asp Gly Val Ile Met Arg Leu Ser Leu Thr Ala

385 390 395 400

Leu Ser Ala Gly Val Gly Ala Val Ala Met Ser Leu Thr Val Gly Ala

405 410 415

Gly Val Ala Ser Ala Asp Pro Val Asp Ala Val Ile Asn Thr Thr Cys

420 425 430

Asn Tyr Gly Gln Val Val Ala Ala Leu Asn Ala Thr Asp Pro Gly Ala

435 440 445

Ala Ala Gln Phe Asn Ala Ser Pro Val Ala Gln Ser Tyr Leu Arg Asn

450 455 460

Phe Leu Ala Ala Pro Pro Pro Gln Arg Ala Ala Met Ala Ala Gln Leu

465 470 475 480

Gln Ala Val Pro Gly Ala Ala Gln Tyr Ile Gly Leu Val Glu Ser Val

485 490 495

Ala Gly Ser Cys Asn Asn Tyr Glu Leu Met Thr Thr Ala Arg Asp Ile

500 505 510

Met Asn Ala Gly Val Thr Cys Val Gly Glu His Glu Thr Leu Thr Ala

515 520 525

Ala Ala Gln Tyr Met Arg Glu His Asp Ile Gly Ala Leu Pro Ile Cys

530 535 540

Gly Asp Asp Asp Arg Leu His Gly Met Leu Thr Asp Arg Asp Ile Val

545 550 555 560

Ile Lys Gly Leu Ala Ala Gly Leu Asp Pro Asn Thr Ala Thr Ala Gly

565 570 575

Glu Leu Ala Arg Asp Ser Ile Tyr Tyr Val Asp Ala Asn Ala Ser Ile

580 585 590

Gln Glu Met Leu Asn Val Met Glu Glu His Gln Val Arg Arg Val Pro

595 600 605

Val Ile Ser Glu His Arg Leu Val Gly Ile Val Thr Glu Ala Asp Ile

610 615 620

Ala Arg His Leu Pro Glu His Ala Ile Val Gln Phe Val Lys Ala Ile

625 630 635 640

Cys Ser Pro Met Ala Leu Ala Ser Arg Gln Lys Gly Asp Thr Lys Phe

645 650 655

Ile Leu Asn Ala

660

<210> 3

<211> 2070

<212> DNA

<213> Artificial sequence

<400> 3

atgacagagc agcagtggaa tttcgcgggt atcgaggccg cggcaagcgc aatccaggga 60

aatgtcacgt ccattcattc cctccttgac gaggggaagc agtccctgac caagctcgca 120

gcggcctggg gcggtagcgg ttcggaggcg taccagggtg tccagcaaaa atgggacgcc 180

acggctaccg agctgaacaa cgcgctgcag aacctggcgc ggacgatcag cgaagccggt 240

caggcaatgg cttcgaccga aggcaacgtc actgggatgt tcgcagaatt cggtggtggt 300

ggttctggtg gtggtggttc tggtggtggt ggttctttct cccggccggg gctgccggtc 360

gagtacctgc aggtgccgtc gccgtcgatg ggccgcgaca tcaaggttca gttccagagc 420

ggtgggaaca actcacctgc ggtttatctg ctcgacggcc tgcgcgccca agacgactac 480

aacggctggg atatcaacac cccggcgttc gagtggtact accagtcggg actgtcgata 540

gtcatgccgg tcggcgggca gtccagcttc tacagcgact ggtacagccc ggcctgcggt 600

aaggctggct gccagactta caagtgggaa accttcctga ccagcgagct gccgcaatgg 660

ttgtccgcca acagggccgt gaagcccacc ggcagcgctg caatcggctt gtcgatggcc 720

ggctcgtcgg caatgatctt ggccgcctac cacccccagc agttcatcta cgccggctcg 780

ctgtcggccc tgctggaccc ctctcagggg atggggccta gcctgatcgg cctcgcgatg 840

ggtgacgccg gcggttacaa ggccgcagac atgtggggtc cctcgagtga cccggcatgg 900

gagcgcaacg accctacgca gcagatcccc aagctggtcg caaacaacac ccggctatgg 960

gtttattgcg ggaacggcac cccgaacgag ttgggcggtg ccaacatacc cgccgagttc 1020

ttggagaact tcgttcgtag cagcaacctg aagttccagg atgcgtacaa cgccgcgggc 1080

gggcacaacg ccgtgttcaa cttcccgccc aacggcacgc acagctggga gtactggggc 1140

gctcagctca acgccatgaa gggtgacctg cagagttcgt taggcgccgg cggtggtggt 1200

ggttctggtg gtggtggttc tggtggtggt ggttctagat ctttcgcagt cacgaacgac 1260

ggggtgatta tgaggctgtc gttgaccgca ttgagcgccg gtgtaggcgc cgtggcaatg 1320

tcgttgaccg tcggggccgg ggtcgcctcc gcagatcccg tggacgcggt cattaacacc 1380

acctgcaatt acgggcaggt agtagctgcg ctcaacgcga cggatccggg ggctgccgca 1440

cagttcaacg cctcaccggt ggcgcagtcc tatttgcgca atttcctcgc cgcaccgcca 1500

cctcagcgcg ctgccatggc cgcgcaattg caagctgtgc cgggggcggc acagtacatc 1560

ggccttgtcg agtcggttgc cggctcctgc aacaactatg agctcatgac cacggcgcgt 1620

gatatcatga atgcgggtgt cacctgtgtt ggcgagcacg aaacgttgac cgcagcagca 1680

cagtacatgc gcgaacatga tatcggcgca ttgccgattt gcggcgacga tgatcgtctg 1740

cacggtatgc tgaccgaccg cgatatcgtt atcaagggtc tggccgcagg cttggacccg 1800

aacaccgcga ccgccggtga actggcacgt gacagcatct attacgtcga cgcgaacgcc 1860

agcattcaag agatgctgaa cgtgatggaa gagcatcagg tgcgtcgtgt cccggttatc 1920

agcgaacatc gtctggttgg tatcgttacc gaagccgaca tcgcacgtca cctgccggag 1980

cacgcgattg ttcagttcgt gaaagcgatt tgcagcccga tggcgttggc gtctcgtcaa 2040

aagggcgaca caaaatttat tctaaatgca 2070

<210> 4

<211> 690

<212> PRT

<213> artificial protein

<400> 4

Met Thr Glu Gln Gln Trp Asn Phe Ala Gly Ile Glu Ala Ala Ala Ser

1 5 10 15

Ala Ile Gln Gly Asn Val Thr Ser Ile His Ser Leu Leu Asp Glu Gly

20 25 30

Lys Gln Ser Leu Thr Lys Leu Ala Ala Ala Trp Gly Gly Ser Gly Ser

35 40 45 45

Glu Ala Tyr Gln Gly Val Gln Gln Lys Trp Asp Ala Thr Ala Thr Glu

50 55 60 60

Leu Asn Asn Ala Leu Gln Asn Leu Ala Arg Thr Ile Ser Glu Ala Gly

65 70 75 80

Gln Ala Met Ala Ser Thr Glu Gly Asn Val Thr Gly Met Phe Ala Glu

85 90 95

Phe Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

100 105 110

Phe Ser Arg Pro Gly Leu Pro Val Glu Tyr Leu Gln Val Pro Ser Pro

115 120 125

Ser Met Gly Arg Asp Ile Lys Val Gln Phe Gln Ser Gly Gly Asn Asn

130 135 140

Ser Pro Ala Val Tyr Leu Leu Asp Gly Leu Arg Ala Gln Asp Asp Tyr

145 150 155 160

Asn Gly Trp Asp Ile Asn Thr Pro Ala Phe Glu Trp Tyr Tyr Gln Ser

165 170 175

Gly Leu Ser Ile Val Met Pro Val Gly Gly Gln Ser Ser Phe Tyr Ser

180 185 190

Asp Trp Tyr Ser Pro Ala Cys Gly Lys Ala Gly Cys Gln Thr Tyr Lys

195 200 205

Trp Glu Thr Phe Leu Thr Ser Glu Leu Pro Gln Trp Leu Ser Ala Asn

210 215 220

Arg Ala Val Lys Pro Thr Gly Ser Ala Ala Ile Gly Leu Ser Met Ala

225 230 235 240

Gly Ser Ser Ala Met Ile Leu Ala Ala Tyr His Pro Gln Gln Phe Ile

245 250 255

Tyr Ala Gly Ser Leu Ser Ala Leu Leu Asp Pro Ser Gln Gly Met Gly

260 265 270

Pro Ser Leu Ile Gly Leu Ala Met Gly Asp Ala Gly Gly Tyr Lys Ala

275 280 285

Ala Asp Met Trp Gly Pro Ser Ser Asp Pro Ala Trp Glu Arg Asn Asp

290 295 300

Pro Thr Gln Gln Ile Pro Lys Leu Val Ala Asn Asn Thr Arg Leu Trp

305 310 315 320

Val Tyr Cys Gly Asn Gly Thr Pro Asn Glu Leu Gly Gly Ala Asn Ile

325 330 335

Pro Ala Glu Phe Leu Glu Asn Phe Val Arg Ser Ser Asn Leu Lys Phe

340 345 350

Gln Asp Ala Tyr Asn Ala Ala Gly Gly His Asn Ala Val Phe Asn Phe

355 360 365

Pro Pro Asn Gly Thr His Ser Trp Glu Tyr Trp Gly Ala Gln Leu Asn

370 375 380

Ala Met Lys Gly Asp Leu Gln Ser Ser Leu Gly Ala Gly Gly Gly Gly

385 390 395 400

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg Ser Phe Ala

405 410 415

Val Thr Asn Asp Gly Val Ile Met Arg Leu Ser Leu Thr Ala Leu Ser

420 425 430

Ala Gly Val Gly Ala Val Ala Met Ser Leu Thr Val Gly Ala Gly Val

435 440 445

Ala Ser Ala Asp Pro Val Asp Ala Val Ile Asn Thr Thr Cys Asn Tyr

450 455 460

Gly Gln Val Val Ala Ala Leu Asn Ala Thr Asp Pro Gly Ala Ala Ala

465 470 475 480

Gln Phe Asn Ala Ser Pro Val Ala Gln Ser Tyr Leu Arg Asn Phe Leu

485 490 495

Ala Ala Pro Pro Pro Gln Arg Ala Ala Met Ala Ala Gln Leu Gln Ala

500 505 510

Val Pro Gly Ala Ala Gln Tyr Ile Gly Leu Val Glu Ser Val Ala Gly

515 520 525

Ser Cys Asn Asn Tyr Glu Leu Met Thr Thr Ala Arg Asp Ile Met Asn

530 535 540

Ala Gly Val Thr Cys Val Gly Glu His Glu Thr Leu Thr Ala Ala Ala

545 550 555 560

Gln Tyr Met Arg Glu His Asp Ile Gly Ala Leu Pro Ile Cys Gly Asp

565 570 575

Asp Asp Arg Leu His Gly Met Leu Thr Asp Arg Asp Ile Val Ile Lys

580 585 590

Gly Leu Ala Ala Gly Leu Asp Pro Asn Thr Ala Thr Ala Gly Glu Leu

595 600 605

Ala Arg Asp Ser Ile Tyr Tyr Val Asp Ala Asn Ala Ser Ile Gln Glu

610 615 620

Met Leu Asn Val Met Glu Glu His Gln Val Arg Arg Val Pro Val Ile

625 630 635 640

Ser Glu His Arg Leu Val Gly Ile Val Thr Glu Ala Asp Ile Ala Arg

645 650 655

His Leu Pro Glu His Ala Ile Val Gln Phe Val Lys Ala Ile Cys Ser

660 665 670

Pro Met Ala Leu Ala Ser Arg Gln Lys Gly Asp Thr Lys Phe Ile Leu

675 680 685

Asn Ala

690

Claims (10)

1. A Mycobacterium tuberculosis fusion protein, which is the protein in sequence 2 and sequence 4 in the sequence listing. 2. the coding gene of the mycobacterium tuberculosis fusion protein described in claim 1. 3. The coding gene according to claim 2, characterized in that it is the gene described in 1) or 2) as follows: 1) its nucleotide sequence is sequence 1 or 3 in the sequence listing; 2) A nucleotide sequence complementary to sequence 1 or 3 in the sequence listing; 3) A DNA molecule that hybridizes to the DNA sequence defined in 1) under stringent conditions and encodes the fusion protein. 4. The recombinant expression vector containing the coding gene of claim 2 or 3. 5. A host cell containing the recombinant expression vector of claim 4. 6. the preparation method of the mycobacterium tuberculosis fusion protein described in claim 1, comprises the expression and the purification of fusion protein, it is characterized in that: the expression of described tuberculosis mycobacterium fusion protein is: the thalline containing fusion protein is added Shake culture in LB medium, add protein inducer IPTG at a final concentration of 0.5mmol/L, induce shaking culture for 4 hours at 27°C-31°C (29°C is optimal), and obtain supernatant after post-treatment as the expression product. 7. The application of the Mycobacterium tuberculosis fusion protein according to claim 1 in the preparation of tuberculosis subunit vaccine. 8. A tuberculosis subunit vaccine comprising the Mycobacterium tuberculosis fusion protein according to claim 1. 9. tuberculosis subunit vaccine according to claim 8, is characterized in that: described tuberculosis subunit vaccine is made up of mycobacterium tuberculosis fusion protein, DDA and PolyI:C according to claim 1; As preferably, The mass ratio of the Mycobacterium tuberculosis fusion protein, DDA and PolyI:C according to claim 1 is: 1:25:5. 10. the preparation method of tuberculosis subunit vaccine described in claim 8 is characterized in that: the preparation method of every 200 μ L tuberculosis subunit vaccine is as follows: fusion protein is diluted to 0.2mg/ml with PBS damping fluid; PolyI:C uses PBS The buffer solution was dissolved to 1mg/ml; DDA was prepared to 2.5mg/ml with PBS, cooled to room temperature after water bath; take 50 μl of PolyI:C solution and the same amount of fusion protein solution, mix well, and place at room temperature; add dropwise to the mixed solution 100 μL of DDA solution, and then fully emulsified to make the vaccine in a uniform creamy state to obtain the tuberculosis subunit vaccine.