CN108619499A - A kind of tuberculosis vaccine and its preparation process - Google Patents

Abstract

The invention discloses a tuberculosis vaccine and a preparation process thereof, comprising a recombinant lentivirus expression plasmid pLenti-spHIL47/pLenti-spHII1 and a lentivirus packaged with the recombinant lentivirus expression plasmid. It can significantly improve the ability of macrophages to present Mycobacterium tuberculosis antigens, and enhance the function of vaccines to stimulate the body to produce antigen-specific immune responses; it can effectively activate macrophages infected by Mycobacterium tuberculosis, and improve the autoimmunity of macrophages. phagocytic level, promoting macrophage killing and clearing the function of Mycobacterium tuberculosis; the present invention can effectively promote the removal of Mycobacterium tuberculosis infected in the body; the present invention can significantly shorten the cycle of tuberculosis treatment when used in combination with tuberculosis chemotherapy drugs; The combined use of the present invention and tuberculosis chemotherapy drugs can significantly delay the recurrence of tuberculosis after treatment; compared with other lentiviral vector vaccines, the present invention has good macrophage targeting and safety in use.

Description

A kind of tuberculosis vaccine and its preparation process

technical field

The invention belongs to the field of genetic engineering and the technical field of novel tuberculosis vaccines, and in particular relates to a tuberculosis vaccine and a preparation process thereof.

Background technique

Tuberculosis (TB) is a global health problem, generating 8 million new cases and 2 million deaths each year. The emergence of multi-drug and pan-drug resistant TB strains has only exacerbated this problem. The life cycle of Mtb has 3 phases. In the acute phase following initial infection, bacteria replicate in the host and express early secreted proteins that induce a host immune response. As the immune response begins to control the infection, Mtb enters a latent asymptomatic state in which the bacteria become non-replicating, parasitizing primarily within macrophages and encased in granulomas. The bacteria can persist in this latent state for many years in infected individuals, making diagnosis and treatment of the disease difficult. In some cases, the bacteria are reactivated and start replicating again, returning to the disease state. Reactivation can occur for many reasons, including immunosuppression caused by diseases such as HIV, treatments such as chemotherapy, or a weakened immune system due to aging. Worldwide, an estimated 2 billion people are latently infected with Mtb, and reactivation of latent Mtb is responsible for most new cases of active TB disease. Reactivation is associated with pulmonary inflammation, necrosis, and cavitation (a process that allows lesions to drain into the bronchi). Aerosols produced when individuals with bronchial lesions cough allow the Mtb organism to spread to uninfected susceptible persons, and thus maintain the cycle of shedding.

At present, the treatment of tuberculosis mainly relies on chemical drug treatment. A common problem is that the treatment cycle is very long. Even for infections caused by sensitive bacteria, the treatment cycle is at least 6 months, and the treatment cycle for drug-resistant tuberculosis is even longer. . Excessively long treatment cycles make patients' compliance with treatment not bad, often leading to irregular treatment, resulting in the emergence and prevalence of drug-resistant bacteria. On the other hand, the emergence of multidrug-resistant tuberculosis has made the traditional chemotherapy of tuberculosis face huge obstacles, and some extensively drug-resistant tuberculosis even faces the plight of no cure. Therefore, immune adjuvant therapy, including therapeutic vaccine, is considered as one of the effective ways to improve the efficacy of tuberculosis treatment.

At present, the tuberculosis vaccines in the prior art mainly exert anti-tuberculosis effects by activating lymphocytes, but tuberculosis mainly parasitizes in macrophages in the body. Although lymphocytes are considered to be the main effector cells of tuberculosis immunity, it is still necessary to It acts through macrophages, and a large number of studies have confirmed that Mycobacterium tuberculosis can inhibit the function of macrophages through a series of mechanisms, including sensitivity to external stimuli. Therefore, even if lymphocytes are well activated, it is still difficult to clear macrophages. The problem with intracellular tuberculosis.

The existing tuberculosis chemotherapeutic drugs have a good killing and clearing effect on the active infection of sensitive Mycobacterium tuberculosis, but the main problem they face is that they have poor killing effect on latent infection bacteria, which leads to the predicament that the current treatment course of tuberculosis is too long. At present, the stimulating antigen used in most tuberculosis vaccines is the immunodominant antigen secreted by Mycobacterium tuberculosis in the early stage. Although it can stimulate a strong immune response, it cannot recognize Mycobacterium tuberculosis in a state of latent infection, so it cannot kill and treat mycobacterium tuberculosis. Elimination of Mycobacterium tuberculosis in latent infection state is of little clinical help.

In recent years, there have also been a small number of reports on the use of latent infection-specific antigens for vaccine design. However, due to the existence of: (1) the activation of specific lymphocytes is also the main factor, which cannot kill and remove latent tuberculosis bacteria in macrophages; 2) Since Mycobacterium tuberculosis can inhibit the function of macrophages, the efficiency of vaccine antigen presentation to lymphocytes is poor, and it is difficult to obtain the desired effect of stimulating specific lymphocyte activation.

Contents of the invention

The purpose of the present invention is to provide a tuberculosis vaccine and its preparation process, which can significantly improve the ability of macrophages to present Mycobacterium tuberculosis antigens, enhance the function of the vaccine to stimulate the body to produce antigen-specific immune responses, and effectively activate the macrophages. Phage cells, enhance the ability of macrophages to kill and clear intracellular Mycobacterium tuberculosis. The invention discloses a tuberculosis vaccine, which comprises a recombinant lentivirus expression plasmid pLenti-spHL47/pLenti-spHI1 and a lentivirus packaged with the recombinant lentivirus expression plasmid.

Preferably, the recombinant lentiviral expression plasmid pLenti-spHL47/pLenti-spHI1 includes a lentiviral framework plasmid, a macrophage-specific promoter, an expressed gene, an IFN-γ-induced protein, and an internal ribosome binding sequence.

Preferably, in any of the above schemes, the lentiviral framework plasmid is CS-CDF-CG-PRE.

Preferably, in any of the above schemes, the macrophage-specific promoter is SP146.

Preferably, in any of the above schemes, the expressed gene is hspX gene.

Preferably, in any of the above schemes, the IFN-γ-induced protein is lrg47 and/or irgm1 gene.

Preferably, in any of the above schemes, the internal ribosome binding sequence is an IRES sequence.

The present invention also provides a preparation process for a tuberculosis vaccine. The gene Y and the lrg47/irgm1 gene specifically expressed during the latent infection period of Mycobacterium tuberculosis are connected through a ribosome binding sequence, cloned into a lentiviral expression vector, and macrophage-specific The expression of the gene is controlled by a sex promoter, and then the lentiviral expression vector is packaged into a lentivirus.

Preferably, the following steps are included: (1) replacing the CMV promoter in the lentiviral framework plasmid CS-CDF-CG-PRE with a macrophage-specific promoter X to obtain an empty expression vector for macrophage-specific expression of the lentivirus pLenti-X;

(4) Obtain the Y gene specifically expressed during the latent infection period of Mycobacterium tuberculosis, insert it into pLenti-X, obtain the recombinant lentiviral expression plasmid pLenti-Y, and clone the Y gene into the plasmid pIRES to obtain the recombinant plasmid pIRES- Y;

(5) Obtain the lrg47 and irgm1 genes, and clone them into the plasmid pIRES-Y, respectively, to construct the Y-IRES-lrg47 and Y-IRES-irgm1 sequences;

(4) Use the macrophage-specific promoter X to replace the CMV promoter in the lentiviral framework plasmid CS-CDF-CG-PRE to obtain the macrophage-specific expression lentiviral expression vector pLenti-X, and then Y-IRES -lrg47 and Y-IRES-irgm1 sequences were respectively cloned into pLenti-X to obtain recombinant lentiviral expression vectors pLenti-HIL47 and pLenti-HII1;

(5) Use lentiviral expression plasmids pLenti-X, pLenti-Y, pLenti-HIL47 or pLenti-HII1, and co-transfect 293T cells with lentiviral packaging plasmids pMD ₂ G and pSPAX ₂ to obtain recombinant lentiviral particles and concentrate lentiviral Particles, respectively obtain empty lentivirus (LV-X), Y recombinant lentivirus (LV-Y), therapeutic vaccine, that is, Y, LRG47 co-expression lentivirus LV-HIL47, Y and IRGM1 co-expression lentivirus LV-HII1 .

Preferably, in any of the above schemes, X in step (1) is at least SP146.

Preferably, in any of the above schemes, Y in step (2) is at least hspX.

Preferably, in any of the above schemes, identification of the recombinant plasmid is also included before step (6).

Preferably, in any of the above schemes, in step (2), insert the hspX gene into the downstream of the SP146 promoter in pLenti-SP146 to obtain the recombinant lentiviral expression plasmid pLenti-hspX, and at the same time clone the hspX gene into the upstream of the IRES in the plasmid pIRES In the multiple cloning restriction site, obtain the recombinant plasmid pIRES-hspX;

Preferably, in any of the above schemes, in step (3), the lrg47 and irgm1 genes are respectively cloned into the multiple cloning restriction site downstream of the IRES sequence in the plasmid pIRES-hspX to construct hspX-IRES-lrg47 and hspX-IRES-irgm1 sequence;

Preferably, in any of the above schemes, in step (4), the hspX-IRES-lrg47 and hspX-IRES-irgm1 sequences are respectively cloned downstream of the SP146 promoter in pLenti-SP146 to obtain recombinant lentiviral expression vectors pLenti-HIL47 and pLenti -HII1.

The tuberculosis vaccine of the present invention has the following advantages in the preparation process: (1) easy to prepare and use: the antigen HspX and IFN-γ-induced protein specifically expressed during the latent infection period of Mycobacterium tuberculosis can be co-expressed by a lentivirus LRG47/IRGM1; (2) Efficient: Lentivirus has the advantages of high infection efficiency and long-term expression of target molecules, especially for macrophages and other cells that are difficult to transfect by conventional methods, which can ensure the effectiveness of target molecules Enter macrophages for high-efficiency expression; (3) This vaccine uses the antigen specifically expressed in the latent infection stage of Mycobacterium tuberculosis, which can activate the tuberculosis bacteria in the latent infection stage; (4) induces the protein through IFN-γ LRG47/IRGM1 promotes macrophage autophagy. On the one hand, it can promote the fusion of Mycobacterium tuberculosis phagosomes and lysosomes in macrophages, and enhance the function of macrophages to kill and clear tuberculosis. The antigen presentation ability of the cells can promote the ability to present HspX, and activate the specific lymphocytes targeting Mycobacterium tuberculosis in the latent infection stage at a higher level; (5) In order to avoid off-target effects, this vaccine uses a macrophage-specific promoter The expression of hspX and lrg47/irgm1 is controlled so that they can only be expressed specifically in macrophages, thereby minimizing possible toxic and side effects during vaccine use.

Beneficial effect

The invention discloses a tuberculosis vaccine and a preparation process thereof, comprising a recombinant lentivirus expression plasmid pLenti-spHL47/pLenti-spHI1 and a lentivirus packaged with the recombinant lentivirus expression plasmid. have the following

Beneficial effect:

(1) It can significantly improve the ability of macrophages to present Mycobacterium tuberculosis antigens, and enhance the function of vaccines to stimulate the body to produce antigen-specific immune responses;

(2) It can effectively activate macrophages infected by Mycobacterium tuberculosis, improve the autophagy level of macrophages, and promote the function of macrophages to kill and remove Mycobacterium tuberculosis;

(3) The present invention can effectively promote the body to remove Mycobacterium tuberculosis infected in the body;

(4) The combination of the present invention and the tuberculosis chemotherapeutic drug can significantly shorten the cycle of tuberculosis treatment;

(5) The combination of the present invention and tuberculosis chemotherapeutic drugs can significantly delay the recurrence of tuberculosis after treatment;

(6) Compared with other lentiviral vector vaccines, the present invention has good macrophage targeting and safety in use.

Description of drawings

Figure 1 RT-PCR detection of recombinant gene mRNA transcription levels in RAW264.7 cells in each lentivirus infection group and control group (LV-sp146, LV-hspX, LV-HIL47 and uninfected control group; ^* P<0.05, ^** P <0.01);

Figure 2 RT-PCR detection of recombinant gene mRNA transcription levels in RAW264.7 cells in each lentivirus infection group and control group (LV-sp146, LV-hspX, LV-HII1 and uninfected control group; ^* P<0.05, ^** P <0.01);

Figure 3 Western-blot detection of recombinant protein expression levels in RAW264.7 cells in each lentivirus infection group and control group. Left: western-blot detection results, 1-4 are: uninfected control group (Control), LV-sp146 group, LV-hspX group, LV-HIL47 infected group; right: grayscale scanning results of LRG47 electrophoresis bands ( ^* P<0.05);

Figure 4 Western-blot detection of recombinant protein expression levels in RAW264.7 cells in each lentivirus infection group and control group. Left: western-blot detection results, 1-4 are: uninfected control group (Control), LV-sp146 group, LV-hspX group, LV-HII1 infected group; right: grayscale scanning results of IRGM1 electrophoresis band ( ^* P<0.05);

Figure 5 Western-blot method to detect the expression level of autophagy-related protein LC3 in each lentivirus infection group and control group RAW264.7 cells;

Figure 6 Flow cytometry detection of autophagy levels of RAW264.7 cells in each lentivirus infection group and control group ( ^* P<0.05);

Figure 7 The expression levels of MHC II, CD80 and CD86 on the surface antigen presentation of RAW264.7 cells in each lentivirus infection group and control group (* indicates P<0.05 compared with the uninfected control group, △ indicates the comparison with the LV-sp146 infected group Compare P<0.05);

Figure 8 The results of detection of lymphocyte activation-related molecules after mixed culture of RAW264.7 cells and HSPX-sensitized lymphocytes in each lentivirus infection group and control group. The left figure shows the expression of CD69 on the surface of CD3+T cells detected by flow cytometry, and the right figure shows the level of IFN-γ produced by CD3+T cells detected by flow cytometry (* indicates that compared with the uninfected control group, P <0.05, △ means P<0.05 compared with LV-sp146 infection group);

Fig. 9 Detection results of the activation of spleen lymphocytes in splenocytes of mice in each lentivirus infection group and control group after being stimulated by HSPX in vitro. A: The expression of CD69 on the surface of CD3+T cells detected by flow cytometry; B: The level of IFN-γ produced by CD3+T cells detected by flow cytometry; C: The use of CFSE fluorescent dye combined with flow cytometry The proliferation level of detected splenic lymphocytes (* indicates P<0.05 compared with LV-sp146 infection group);

Figure 10 Lentivirus-treated/treated mouse lung, spleen tissue load test results (* indicates P<0.05 compared with untreated/treated control group, △ indicates P<0.05 compared with LV-hspX treated/treated group);

Fig. 11 Pathological changes of lung tissue of mice treated with lentivirus/treatment (①～④ are respectively untreated group, LV-sp146 treated group, LV-hspX treated group and LV-HIL47 treated group; HE staining, 20×);

Figure 12 The effect of chemotherapeutic drugs combined with LV-HIL47 vaccine treatment on the bacterial load of mouse lung and spleen tissue (no Mycobacterium tuberculosis was cultured in the lung tissue of mice treated with chemotherapeutic drugs combined with LV-HIL47 vaccine; * indicates the same as untreated /P<0.05 compared with the treatment control group, △ means P<0.05 compared with the chemotherapy group alone);

Figure 13 Effects of chemotherapeutic drugs combined with LV-HIL47 vaccine treatment on the pathological changes of mouse lung tissue (①～④ are respectively untreated group, LV-HIL47 treatment group, INH+PZA treatment group, INH+PZA+LV-HIL47 treatment group ; HE staining, 20×);

Figure 14: After chemotherapy, LV-HIL47 vaccine was used to detect the bacterial load in lung tissue of mice (* indicates P<0.05 compared with untreated/treated control group, △ indicates P<0.05 compared with simple chemotherapy group);

Figure 15 Effect of LV-HIL47 vaccine treatment on pathological changes of mouse lung tissue after chemotherapy (①～③ are untreated group, INH+PZA treatment group and INH+PZA+LV-HIL47 treatment group respectively; HE staining, 20 ×);

Figure 16 Mycobacterium tuberculosis culture in the lung tissue of the tuberculosis mice of the chemotherapy group (left) and chemotherapy combined with LV-HIL47 vaccine group (the vertical axis is the amount of bacteria in each mouse, and the arrow shows that no tuberculosis has been cultured Mycobacterium mice);

Fig. 17 is a schematic diagram of the principle of the tuberculosis vaccine of the present invention.

Detailed ways

The following examples are a further description of the content of the present invention as an explanation of the technical content of the present invention, but the essential content of the present invention is not limited to the following examples, those of ordinary skill in the art can and should know any Simple changes or replacements of the essential spirit of the invention shall fall within the scope of protection required by the present invention.

Example 1

A tuberculosis vaccine, the design principle of which is shown in Figure 17, includes a recombinant lentiviral expression plasmid pLenti-spHL47/pLenti-spHI1 and lentiviral particles packaged with the recombinant lentiviral expression plasmid. The lentiviral particles are used to carry the above-mentioned lentiviral expression vectors, so that they can efficiently enter cells and express the above-mentioned recombinant target molecules for a long time.

Among them, pLenti-spHL47/pLenti-spHI1 includes the following main components:

(1) Lentiviral framework plasmid: CS-CDF-CG-PRE. Function: used to provide frame information of lentiviral expression vector.

(2) Macrophage specific promoter: SP146. Function: This is a macrophage-specific promoter, which is used to control the expression of hspX gene and lrg47/irgm1 gene, so that they can only be expressed in macrophages, but not in other cells.

(3) hspX gene: an expression gene that specifically expresses the antigen HspX during the latent infection period of Mycobacterium tuberculosis, and is used to express HspX to sensitize specific lymphocytes;

(4) lrg47 or irgm1 gene: lrg47 and irgm1 are highly conserved homologous genes with similar biological functions. They are called lrg47 in mouse genes and irgm1 in human genes. These two are IFN-γ-induced proteins, which can induce autophagy in cells and improve the antigen presentation ability of macrophages.

(5) IRES sequence: the internal ribosome binding sequence, which is used to connect the hspX gene and the lrg47/igrm1 gene so that they can be co-expressed under the control of the same promoter.

Example 2

The preparation technology of tuberculosis vaccine of the present invention comprises the following steps:

1. Using the plasmid pSP146-GFP as a template, the macrophage-specific promoter SP146 fragment was amplified by PCR technology (primers used: S1/S2, the sequence is shown in Table 1), and then molecular cloning technology (refer to Sambrook.J editor-in-chief The third edition of "Molecular Cloning Experiment Guide"), the SP146 promoter was cloned into the lentiviral framework plasmid CS-CDF-CG-PRE using restriction enzymes EcoRI and AgeI to replace the CMV promoter in the original plasmid, Obtain macrophage-specific expression lentiviral expression vector pLenti-SP146.

2. Use the PCR method (refer to the third edition of "Molecular Cloning Experiment Guide" edited by Sambrook.J) to amplify and obtain the hspX gene specifically expressed during the latent infection period of Mycobacterium tuberculosis (primers used: H1/H2, the sequence is shown in Table 1 ), which was inserted between the AgeI and BsrGI restriction sites downstream of the SP146 promoter in pLenti-SP146 to obtain the recombinant lentiviral expression plasmid pLenti-hspX. At the same time, the hspX gene was cloned into the plasmid pIRES between the NheI and MluI restriction sites upstream of the IRES to obtain the recombinant plasmid pIRES-hspX.

3. Obtain the lrg47 and irgm1 genes (primer sequences are L1/L2 and I1/I2 respectively, see Table 1 for the sequences) by reverse transcription PCR method (with reference to the third edition of "Molecular Cloning Experiment Guide" edited by Sambrook.J), and respectively Cloned into the plasmid pIRES-hspX between the BamHI and NotI restriction sites downstream of the IRES sequence, and constructed recombinant plasmids phspX-IRES-lrg47 and phspX-IRES-irgm1 containing the sequences hspX-IRES-lrg47 and hspX-IRES-irgm1 respectively .

4. Using the PCR method to amplify and obtain hspX-IRES-lrg47 and hspX-IRES-irgm1 gene fragments from plasmids phspX-IRES-lrg47 and phspX-IRES-irgm1 respectively (primers used: HIL1/HIL2, the sequences are shown in Table 1), And they were respectively cloned into the downstream of the SP146 promoter in pLenti-SP146 by restriction endonucleases AgeI and BsrGI to obtain recombinant lentiviral expression vectors pLenti-HIL47 and pLenti-HII1.

5. Recombinant plasmids were identified by restriction endonuclease separation and sequencing.

6. Use the above-identified correct lentiviral expression plasmids pLenti-SP146, pLenti-hspX, pLenti-HIL47 or pLenti-HII1 to co-transfect 293T cells with lentiviral packaging plasmids pMD2G and pSPAX2, and collect the cells for culture at 72 hours after transfection Clear, obtain recombinant lentivirus particles, concentrate lentivirus particles by ultracentrifugation, and obtain empty-loaded lentivirus (LV-sp146), hspX recombinant lentivirus (LV-hspX), and therapeutic vaccine, that is, co-expression of HSP X and LRG47 Lentivirus LV-HIL47 co-expressed lentivirus LV-HII1 with HSP X and IRGM1.

Primers used in the present invention in table 1

Example 3

The identification of the tuberculosis vaccine of the present invention specifically includes the following aspects:

1. Expression identification: LV-sp146, LV-hspX, LV-HIL47, and LV-HII1 were used to infect macrophage RAW264.7 cells, and cells not infected with lentivirus were set as controls. The cells in each group were collected 72 hours after infection, and the total RNA and protein of the cells were extracted. The expression levels of hspX and lrg47/irgm1 were detected by RT-PCR technology, and the expression levels of HSP X and LRG47/IRGM1 proteins were detected by Western blotting. The results showed that , after LV-hspX, LV-HIL47 and LV-HII1 infected RAW264.7 cells, they could all effectively express the corresponding recombinant proteins in the cells, as shown in Figures 1-4. Figure 1 is RT-PCR detection of recombinant gene mRNA transcription levels in each lentivirus infection group and control group RAW264.7 cells; Figure 2 is RT-PCR detection of recombinant gene levels in each lentivirus infection group and control group RAW264.7 cells mRNA transcription level; Figure 3 is Western-blot detection of recombinant protein expression levels in each lentivirus infection group and control group RAW264.7 cells; Figure 4 is Western-blot detection of each lentivirus infection group and control group RAW264.7 Expression levels of recombinant proteins in cells.

2. Functional identification of promoting autophagy: LV-sp146, LV-hspX, and LV-HIL47 were used to infect macrophage RAW264.7 cells, and cells not infected with lentivirus were set as controls. The cells in each group were collected 72 hours after infection, and then Western blotting was used to detect the expression levels of autophagy-related molecules LC 3 Ⅰ and LC 3 Ⅱ. Autophagy levels, identifying the function of therapeutic vaccine-expressed LRG47/IRGM1 in modulating autophagy. Western-blot results showed that compared with the control group, the expression level of LC3II in LV-HIL47-infected macrophages was significantly up-regulated, as shown in Figure 5. The results of flow cytometry based on Cyto-IDAutophagy Detection also showed that the autophagy level of macrophages in the LV-HIL47 infection group was significantly up-regulated, as shown in Figure 6.

3. Identification of therapeutic vaccines to enhance the antigen presentation function of macrophages

(1) LV-sp146, LV-hspX, and LV-HIL47 were used to infect macrophage RAW264.7 cells, and cells not infected with lentivirus were set as controls. The cells in each group were collected 72 hours after infection, and the expression levels of CD80, CD86 and MHC II on the surface of macrophages related to antigen presentation were detected by flow cytometry. The results showed that, compared with the uninfected control group, the expression levels of MHC II and CD80 in each virus-infected group were significantly up-regulated, and the expression level of CD86 had no significant difference; compared with the empty lentivirus LV-sp146-infected group, LV-HIL47 The expression levels of CD80, CD86 and MHC II in the macrophages of the group were significantly up-regulated, as shown in Figure 7, indicating that the expression of LRG47 can significantly up-regulate the autophagy level of macrophages.

(2) C57BL/c mice were immunized by subcutaneous inoculation with recombinant HSP X protein, and mouse splenocytes were extracted after 6 weeks for use; mouse peritoneal macrophages were isolated and cultured, and LV-sp146, LV-hspX and LV -HIL47 infection, 72h later, these macrophages were used for mixed culture with the mouse splenocytes prepared above, and the proliferation level of splenocytes, the expression level of spleen lymphocyte activation-related molecule CD69, and the IFN- in the culture supernatant were respectively detected. The level of γ. The results showed that the mixed culture of macrophages in the LV-HIL47 infection group and HSP X-sensitized lymphocytes could significantly enhance the activation level of CD3+ T cells, which was reflected in: the expression of CD69 was significantly up-regulated in the lymphatic details activation specimen; the secretion of IFN- The level of γ was significantly up-regulated, as shown in Figure 8. These results suggest that LV-HIL47-infected macrophages can more effectively present HSPX antigen to lymphocytes.

(3) C57BL/c mice were infected with LV-sp146, LV-hspX, and LV-HIL47 respectively. After 2 weeks of infection, splenocytes were isolated from the mice, stimulated with recombinant HSP X protein, and lymphocyte activation-related molecules were detected 48 hours later. The expression level of CD69 and the level of IFN-γ in the culture supernatant, and the proliferation level of splenocytes were detected after 72 hours. The results showed that compared with other control groups, splenocytes of LV-HIL47-infected mice could activate spleen lymphocytes more effectively after HSPX rechallenge in vitro, which was manifested in (1) activation of spleen lymphocyte surface after HSPX protein stimulation The expression of molecule CD69 was significantly up-regulated, as shown in Figure 9A; (2) the level of IFN-γ produced by splenic lymphocytes was significantly up-regulated after HSPX protein stimulation, as shown in Figure 9B; (3) the proliferation of splenic lymphocytes after HSPX protein stimulation levels increased significantly, as shown in Figure 9A.

Example 4

Detection of the effect of the tuberculosis vaccine LV-HIL47 prepared by the invention in treating tuberculosis in mice

Mycobacterium tuberculosis H37Rv was used to establish a tuberculosis mouse model by tail vein injection, and the mice were randomly divided into 4 groups after 4 weeks of infection, namely: no treatment control group (no treatment), empty lentivirus treatment Control group (with LV-sp146 treatment), hspX recombinant lentivirus treatment group (with LV-hspX treatment), HSP X and LRG47 co-expression lentivirus treatment group (with LV-HIL47 treatment). At the same time, C57BL/c mice that were not infected with the device were used as healthy controls. The lentiviral treatment was administered by nasal drip method, the virus titer was 5×10 ⁷ TU/ml, 100ul/time, once every 2 weeks, a total of 3 times. Mice in each group were sacrificed 10 weeks after infection, and the bacteria load in lung and spleen tissue was detected by inoculating M7H10 plate culture method after tissue homogenization, and the lung tissue was observed by paraffin embedding, ultrathin section and HE staining. Pathological changes, and evaluate the effect of LV-HIL47 on the treatment of tuberculosis in mice. The results showed that compared with other control groups, the bacterial load in lung and spleen tissue of LV-HIL47 treated mice was significantly reduced, as shown in Figure 10, and the pathological changes in lung tissue were relatively milder, as shown in Figure 11.

Example 5

Effect detection of tuberculosis vaccine LV-HIL47 of the present invention as an adjuvant chemotherapy drug in treating tuberculosis in mice

1. Adopt Mycobacterium tuberculosis H37Rv to establish the tuberculosis mouse model through the tail vein injection method. After 4 weeks of infection, the mice are randomly divided into 4 groups, which are respectively: the control group (not receiving any treatment), the vaccine treatment group (only received LV-HIL47 treatment), chemotherapy group (received INH+PZA chemotherapy for 4 weeks), combined treatment group (received INH+PZA chemotherapy and LV-HIL47 treatment at the same time). Chemotherapy regimen: INH (0.1g/L)+PZA (8g/L), administered by intragastric administration, once a day, 100 μl each time. Lentiviral treatment plan: virus titer 5×10 ⁷ TU/ml, administered by nasal drip, 100ul/time, once every 2 weeks, a total of 3 times. Mice in each group were sacrificed 10 weeks after infection, and the bacteria load in lung and spleen tissue was detected by inoculating M7H10 plate culture method after tissue homogenization, and the lung tissue was observed by paraffin embedding, ultrathin section and HE staining. Pathological changes, to evaluate the effect of LV-HIL47 adjuvant chemotherapy drugs in the treatment of tuberculosis in mice. The results showed that, compared with the chemotherapy alone group, the chemotherapy combined with vaccine treatment group could further reduce the bacterial load in lung and spleen tissues of mice, as shown in Figure 12 . It is particularly worth mentioning that, 2 weeks after the completion of the treatment, no mycobacterium tuberculosis was cultured in the lung tissues of the mice in the LV-HIL47 vaccine combined with chemotherapy group, suggesting that LV-HIL47 can greatly improve the elimination of tuberculosis by chemotherapy drugs. Competence of Mycobacterium tuberculosis in mice. The results of histopathological examination also showed that the histopathological changes of the lungs in the combined treatment group were milder than those in the chemotherapy alone group, as shown in Figure 13.

2. Detection of the effect of the therapeutic vaccine LV-HIL47 on killing persistent bacteria:

Mycobacterium tuberculosis H37Rv was used to establish the tuberculosis mouse model by tail vein injection, and the mice were randomly divided into three groups after 4 weeks of infection: control group (no treatment), chemotherapy group (received INH+PZA chemotherapy, lasted for 4 weeks), the combined treatment group (first received 4 weeks of INH+PZA chemotherapy as in the chemotherapy group, and then received LV-HIL47 once). Chemotherapy regimen: INH (0.1g/L)+PZA (8g/L), dissolved in drinking water, and fed by mice freely. Lentivirus treatment plan: virus titer 5×10 ⁷ TU/ml, administered by nasal drip, 100ul/time. Mice in each group were sacrificed 14 weeks after infection, and the bacterial load of lung tissue was detected by inoculating M7H10 plate culture method after tissue homogenization, and the pathological changes of lung tissue were observed by paraffin embedding, ultrathin section and HE staining of lung tissue To evaluate the effect of LV-HIL47 on killing the residual bacteria after chemotherapy. The results showed that compared with the untreated control group, the amount of bacterial load in the lung tissue of the mice in the chemotherapy group was significantly reduced, but the combined treatment group could further reduce the load of bacteria in the lung tissue on this basis, as shown in Figure 14, but the lung tissue There was no significant difference in histopathological changes, as shown in Figure 15, suggesting that LV-HIL47 vaccine treatment can effectively enhance the ability of mice to kill and clear residual bacteria in lung tissue after chemotherapy.

3. Detection of the effect of therapeutic vaccine LV-HIL47 on shortening the treatment course of tuberculosis:

Mycobacterium tuberculosis H37Rv was used to establish the tuberculosis mouse model by tail vein injection, and the mice were randomly divided into two groups after 4 weeks of infection: chemotherapy group (received INH+PZA chemotherapy for 8 weeks), combined treatment group ( They received chemotherapy drugs as in the chemotherapy group, and at the same time received lentiviral LV-HIL47 treatment from the 4th week after infection). Chemotherapy regimen: INH (0.1g/L)+PZA (8g/L), dissolved in drinking water, and fed by mice freely. Lentiviral treatment plan: virus titer 5×10 ⁷ TU/ml, administered by nasal drip, 100ul/time, once every 2 weeks, a total of 3 times. The mice in each group were sacrificed in batches from 4 weeks after infection, with an interval of 4 weeks between each batch, and 3 mice in each group were sacrificed for the detection of bacterial load, a total of 7 batches. After the mice in each group were sacrificed, the lung tissues were taken out, homogenized and inoculated with M7H10 plate culture method to detect the bacterial load of the lung tissues, and the effect of LV-HIL47 adjuvant chemotherapy drugs on shortening the treatment course of tuberculosis in mice was evaluated. The results showed that no bacteria could be cultured in the lungs of all the mice in the combination treatment group of chemotherapy drugs and vaccines at 8 weeks after treatment, and it was maintained until 24 weeks. By the end of the experiment, that is, at 28 weeks after treatment, only one mouse Mycobacterium tuberculosis was cultured from the lung tissues of the mice, while Mycobacterium tuberculosis could be cultured from the lung tissues of all the mice in the chemotherapy group after 8 weeks of treatment, and no tuberculosis isolates could be cultured from all the mice by 12 weeks after treatment. Mycobacterium tuberculosis, but this sterile state was only maintained for 8 weeks. Mycobacterium tuberculosis was cultured in 1 mouse at 20 weeks, and bacteria were cultured in the lung tissues of all mice at 24 weeks. As shown in Figure 16. The above results show that compared with chemotherapy alone, chemotherapy combined with vaccine treatment can clear the infected Mycobacterium tuberculosis in mice faster and maintain a longer sterility time, indicating that it can effectively delay the recurrence of tuberculosis mice after treatment .

Claims (10)

1. A tuberculosis vaccine, characterized in that it comprises a recombinant lentiviral expression plasmid pLenti-spHL47/pLenti-spHI1 and a lentivirus packaged with the recombinant lentiviral expression plasmid. 2. A tuberculosis vaccine according to claim 1, wherein the recombinant lentiviral expression plasmid pLenti-spHL47/pLenti-spHI1 comprises a lentiviral framework plasmid, a macrophage specific promoter, an expression gene, IFN-γ Induced protein and internal ribosome binding sequences. 3. A tuberculosis vaccine according to claim 2, characterized in that the lentiviral framework plasmid is CS-CDF-CG-PRE. 4. The tuberculosis vaccine according to claim 2, wherein the macrophage-specific promoter is SP146. 5. The tuberculosis vaccine according to claim 2, characterized in that the expressed gene is hspX gene. 6. A tuberculosis vaccine according to claim 2, characterized in that the IFN-γ-induced protein is lrg47 and/or irgm1 gene. 7. according to the preparation technology of tuberculosis vaccine described in any one of the above-mentioned claims, it is characterized in that, the gene Y and lrg47/irgm1 gene specifically expressed in latent infection period of mycobacterium tuberculosis are connected by ribosome binding sequence, and it is cloned into In the lentiviral expression vector, a macrophage-specific promoter is used to control gene expression, and then the lentiviral expression vector is packaged into a lentivirus. 8. The preparation process of a kind of tuberculosis vaccine according to claim 7, is characterized in that, comprises the following steps: (1) adopt macrophage specific promoter X to replace in lentiviral framework plasmid CS-CDF-CG-PRE The CMV promoter was used to obtain macrophage-specific expression lentiviral empty expression vector pLenti-X; (2) Obtain the Y gene specifically expressed during the latent infection period of Mycobacterium tuberculosis, insert it into pLenti-X, obtain the recombinant lentiviral expression plasmid pLenti-Y, and clone the Y gene into the plasmid pIRES to obtain the recombinant plasmid pIRES- Y; (3) Obtain the lrg47 and irgm1 genes, and clone them into the plasmid pIRES-Y, respectively, to construct the Y-IRES-lrg47 and Y-IRES-irgm1 sequences; (4) Use the macrophage-specific promoter X to replace the CMV promoter in the lentiviral framework plasmid CS-CDF-CG-PRE to obtain the macrophage-specific expression lentiviral expression vector pLenti-X, and then Y-IRES -lrg47 and Y-IRES-irgm1 sequences were respectively cloned into pLenti-X to obtain recombinant lentiviral expression vectors pLenti-HIL47 and pLenti-HII1; (5) Use lentiviral expression plasmids pLenti-X, pLenti-Y, pLenti-HIL47 or pLenti-HII1, and co-transfect 293T cells with lentiviral packaging plasmids pMD ₂ G and pSPAX ₂ to obtain recombinant lentiviral particles and concentrate lentiviral Particles, respectively obtain empty lentivirus (LV-X), Y recombinant lentivirus (LV-Y), therapeutic vaccine, that is, Y, LRG47 co-expression lentivirus LV-HIL47, Y and IRGM1 co-expression lentivirus LV-HII1 . 9. A tuberculosis vaccine according to claim 8, characterized in that X in the step (1) is at least SP146. 10. A tuberculosis vaccine according to claim 8, characterized in that Y in step (2) is at least hspX.