CN107217026B - Recombinant mycobacterium smegmatis strain with c-di-AMP catabolic enzyme knocked out and application thereof - Google Patents

Abstract

The invention relates to a recombinant Mycobacterium smegmatis strain with a c-di-AMP catabolic enzyme knocked out. Respectively connecting the upstream and downstream homologous arms of a mycobacterium smegmatis c-di-AMP degrading enzyme gene MSMEG _2630 into a pMSG360 vector, transforming a mycobacterium smegmatis competent cell containing recombinant enzymes gp60 and gp61pJV53 plasmids by using the recombinant plasmids, screening and obtaining a recombinant mycobacterium smegmatis strain (rMS-delta CnpB) with a c-di-AMP degrading enzyme knocked out, wherein the preservation number of the recombinant mycobacterium smegmatis strain is as follows: CCTCC M2016336. rMS-delta CnpB bacterial strain immune mouse can regulate mouse body fluid and cell immune response, especially Th1 type cell immune response after infecting mycobacterium tuberculosis and raise the protection of body against mycobacterium infection, so that it may be used in developing vaccine and preparation for preventing and treating tuberculosis.

Description

Recombinant mycobacterium smegmatis strain with c-di-AMP catabolic enzyme knocked out and application thereof

Technical Field

The invention belongs to the field of tuberculosis vaccines and immunotherapy. The invention relates to a recombinant Mycobacterium smegmatis strain with a c-di-AMP catabolic enzyme knocked out. The invention also relates to the application of the recombinant mycobacterium smegmatis strain in vaccines and preparations for preventing and/or treating tuberculosis.

Background

Current status of tuberculosis prevalence

Tuberculosis (TB) is a chronic infectious disease that seriously harms human health worldwide. Mycobacterium tuberculosis (Mtb) is the causative agent of TB, and according to WHO report, 1040 ten thousand of new cases are estimated globally in 2015, 120 ten thousand (11%) of HIV patients die of TB in total 140 ten thousand. TB is still ranked in the first ten causes of death. Six countries, indian, indonesia, china, nigeria, pakistan and south africa account for 60% of the total number of new cases. Active TB control studies and measures have avoided 4900 million TB deaths in 2000 to 2015, but the current prevalence and risk of TB indicate that there is a significant gap in TB prevention, diagnosis and treatment.

Current situation of tuberculosis prevention and cure

Bacillus Calmette-guerin vaccine (BCG) is the only vaccine currently used for tuberculosis prevention. BCG is mainly used as a preventive vaccine before Mtb infection, and can not prevent and control the relapse of Mtb in a latent infection state aiming at the early infection of Mtb, but has the defects of short protection period, weak protective immune response and the like, and is used as an attenuated live vaccine and can not be used for the vaccination of people with low immunity, so that the development of a safe and reliable novel TB vaccine is urgent.

In the aspect of TB treatment, as the course of TB chemotherapy is longer than 6 months, patients cannot keep on taking the medicine and have poor compliance, and often cause drug resistance, especially the occurrence of extensive tuberculosis drug resistance and super drug resistant tubercle bacillus. With the progressive understanding of the nature of Mtb, the Th1 type cellular immune response of TB patients is proved to be low, so that the therapeutic vaccine capable of improving the immune function of organisms, particularly the Th1 type response has better application prospect. The vaccine is mainly used for asymptomatic carriers infected with Mtb or patients suffering from diseases, selectively induces specific and non-specific immune response of human bodies, and finally achieves the purpose of treatment (the

MI, et al, therapeutic vaccines for tubericulosis-a systematic review. vaccine 2014,32(26): 3162-8). The novel TB vaccines currently under development mainly comprise subunit vaccines, gene vaccines, recombinant BCG vaccines, attenuated or enhanced whole-cell live vaccines, auxotrophic live vaccines, vaccines combined with DC and the like, but no novel vaccine can replace the traditional BCG or be used for the immunotherapy of the TB.

Study of recombinant mycobacterial vaccines

The construction of recombinant live vaccines by introducing exogenous genes into mycobacteria is one of the important research directions in this field. BCG is a live vaccine vector for mycobacteria which is researched more, but has the problems of slow growth, low transformation efficiency, complex genetic operation and the like, and BCG is an attenuated strain and cannot be used for inoculation of people with low immunity.

Mycobacterium smegmatis (Ms) is a non-pathogenic Mycobacterium with fast growth and high transformation efficiency and a strong cellular immune adjuvant, and has immunological characteristics similar to BCG. rMS expressing IL12 and GLS fusion genes is constructed, such as Zhu DY, et al (Recombinant M.modematis vaccine targeted delivery IL-12/GLS expression genes canonic specific cellular antigen M.tuberculosis in BALB/c microorganism, 2007,25(4):638-648.) and the like, and the vaccine can stimulate T lymphocyte proliferation, start Th1 cell immune response, promote cytokine secretion and improve immune clearance of organisms to mycobacteria. Tullius MV et al (High cellular levels of mycobacterial proteins glutamine synthesis and superoxide dismutase in active growth processes products to High expression and extracellular catalytic promoter which is a protein-specific expression. immunological Cell biol.2003,87(7):209-215.) transferred Mtb protein Ms into expression, which revealed that the biochemical and immunological properties of the recombinant protein and the native protein were almost identical. In addition, Ms grows at a fast rate, Goldstone RM et al (A new)

vector and Expression protocol for fast and effective recombinant protein Expression in Mycobacterium smegmatis. protein Expression and Purification,2008,57(1):81-87.) establishes a method for soluble Expression of Mtb protein in Ms strain, and researches show that the Ms Expression exogenous protein has high yield which is 5-10 times of BCG. Bashiri et al use Ms as a recombinant protein expressed by host bacteria, and are useful for structural and functional studies (Bashiri G, Baker EN. production of recombinant proteins in Mycobacterium for structural and functional students. protein Sci.2015,24(1): 1-10.). Thus, Ms is an excellent carrier for live mycobacterial vaccines.

Recombinant live Mycobacterium smegmatis (rMs) vaccine for the treatment of Mtb infectionRestoring protective immunity and promoting mononuclear macrophage to generate more H₂O₂And NO, effectively eliminating Mtb, enhancing the immune response capability of the organism (slow vaccine, etc. the effect of the Mycobacterium smegmatis vaccine on the generation of nitric oxide on mouse macrophage, proceedings of Chinese academy of medical sciences, 2009, (4) 410-; it also can change the Th1/Th2 pattern of immune response, shift the immune response from Th2 to Th1, thus helping the body immune cells kill Mtb persisting bacteria and drug-resistant bacteria, and shorten the course of chemotherapy (slow vaccine, etc. effect of Mycobacterium smegmatis vaccine on mouse cytokine production and Th1/Th2 response, Chinese tuberculosis and J.Resinatum, 2005, (11): 49-52.). Ms is nontoxic mycobacterium, the safety of the M.smegmatis vaccine is proved by a plurality of experiments, and the safety of the M.smegmatis vaccine is proved in China (long-term toxicity research of the cell-free M.smegmatis vaccine to guinea pigs, China New medicine journal, 2006, (19):1640 and 1643.) that the cell-free Ms vaccine has no obvious toxic effect on the guinea pigs after long-term use, and the cell-free Ms prepared by the Ms vaccine can be used for immunotherapy of TB, so that a better effect is achieved (research of M.smegmatis as an immunomodulator, China microbiology and immunology journal, 2005, (9):752 and 755.). Therefore, the rMS has the remarkable advantages of high efficiency, good safety and the like, and is an important tool for novel preventive and therapeutic vaccines of TB.

The selection of a suitable target antigen can significantly improve the protective efficiency of the vaccine. The study of Mtb protective antigens has been continued for many years, mainly with secreted proteins such as ESAT-6, CFP10, Ag85, TB10.4, Mtb39, Mtb32, etc., antigens of the heat shock family such as Hsp65, Hsp70, HspX, etc., and the fusion protein Mtb72f, etc. However, no recombinant vaccine for prevention and treatment is available at present, which indicates that a new Mtb antigen needs to be screened widely to obtain a recombinant mycobacterium with better immune effect for preventing and treating TB.

The second signal molecule c-di-AMP becomes a new target for bacterial vaccine research

In 2008, a bacterial signaling molecule, Cyclic di-adenosine (c-di-AMP) (Witte, G., et al., structural biological chemistry of bacterial protein variants activity regulated by DNA recognition) was newly discovered 2008.30(2): 167-78.). c-di-AMP is involved in various physiological processes such as cell wall synthesis, pressure induction, biofilm formation, ion metabolism of bacteria such as Staphylococcus aureus, Streptococcus, Bacillus subtilis, and Listeria pseudomonads (Krasteva PV et al Versatile models of cellular regulation via cyclic peptides. Nat Chem biol.2017,13(4): 350-. Studies have shown that c-di-AMP regulates Mtb cell length, cell wall formation, lipid metabolism, and pathogenicity. Studies have also shown that c-di-AMP can directly activate STING, induce innate immune responses characteristic of Host cell type I Interferon release (Andrade WA, equivalent. group B Streptococcus degradation cycles-di-AMP to Modulate STING-dependent type I Interferon production. cell Host Microbe.2016,20(1):49-59.), and that c-di-AMP alone or in combination with antigenic immunity can Modulate Th1/Th2/Th17 adaptive immune responses, with significant immune adjuvant effects (Ebensen, T, et al bis- (3',5') -Cyclic dimeric adenosine monophosphorus: strong Th 1/2/17 promoter Th co assay. vaccine, Vaccine, 5229 (32): 201110-201120). c-di-AMP is only present in bacteria, but not in eukaryotic cells, and thus, c-di-AMP is a new target for bacterial vaccine and drug research.

c-di-AMP is synthesized from two molecules of ATP catalyzed by a Diadenylate cyclase (DacA). Rv3586 is the DAC enzyme in Mtb that synthesizes c-di-AMP. Jun Yang et al (Deletion of the cyclic di-Amphodiolesterase gene (cnpB) in Mycobacterium tuberculosis leads to reduced viral gene in a motor model of infection.2014,93(1):65-79.) study demonstrated that Rv2837c, which contains the DHH domain, is a c-di-AMP degrading enzyme of Mtb and was named CnpB. Manikandan K et al (Two-stepsynthesis and hydrolosis of cyclic di-AMP in Mycobacterium tuberculosis. PLoSOne.201423; 9(1): e86096.) found that Rv2837c can degrade c-di-AMP first to pApApApApApApApApA and further to 5' -AMP. It is presumed that, in the bacterial pathogenic process, Rv2837c has an important regulatory effect, as found by Postic G et al (propagation of Nrn A homology from Mycobacterium tuberculosis and Mycoplasma pneumoniae.201218 (1): 155-65.). In a further study by Jun Yang et al, the level of c-di-AMP in the mutant Rv2837c was increased, the mutant infected STING-/-deficient mice M phi, and IL-1 beta release was significantly increased. Animal experiments show that the strain promotes host immunity to eliminate Mtb, and animal survival time is prolonged. Dey RJ et al (Inhibition of immune cell metabolism by m. tuberculosis pathology homology. nat. Chem biol.2017,13(2): 210. cona 217.) found that the toxicity of Mtb can be reduced by the mutant c-di-AMP catabolic enzyme Rv2837c, while the use of Rv2837c inhibitors can change the outcome of Mtb infection by activation of the cytoplasmic cGAS-STING pathway. In conclusion, the Rv2837c protein containing the DHH structural domain has the enzymatic activity of degrading c-di-AMP, the enzyme is mutated in Mtb, the virulence of the strain is reduced, the c-di-AMP level of bacteria can be obviously increased, the secreted c-di-AMP can activate the inherent immune response mediated by a cytoplasmic STING pathway, and finally the immune clearance of intracellular bacteria is promoted, so the c-di-AMP degrading enzyme of the DHH structural domain becomes a new target point for vaccine and drug research.

In Ms, mseg _2630 encodes a DHH domain protein similar to Rv2837c, with 82% homology. The MSMEG _2630 gene is 1023 bp, and the molecular weight of the encoded protein is about 35.5 kDa. Tang Q et al (Functional Analysis of a c-di-AMP-specific phosphorus pesticide MsPDE from Mycobacterium smegmatis. int J Biol Sci.2015,11(7):813-24) at national Huazhong agriculture university adopt sacB-lacZ auxotrophy screening system to construct MSMEG _2630 gene mutant. Phenotypic studies have shown that MSMEG _2630 has phosphodiesterase activity similar to that of Rv2837c, hydrolyzing c-di-AMP. The MSMEG-2630 gene is knocked out, the colony morphology, lipid metabolism and ion metabolism of the Ms strain are changed, and the c-di-AMP level of the strain is obviously increased. Therefore, MSMEG-2630 is the c-di-AMP degrading enzyme of Ms, the gene is knocked out, the level of c-di-AMP produced by bacteria is obviously increased, and the secreted c-di-AMP promotes innate immune response after the bacterial strain is immunized, thereby being beneficial to host immunity to eliminate intracellular bacteria.

Disclosure of Invention

The invention aims to obtain a recombinant Mycobacterium smegmatis strain with a c-di-AMP catabolic enzyme knocked out. The strain can be used for the development of tuberculosis prevention and therapeutic vaccines.

The invention is realized by the following steps:

the genome of Ms is used as a template, the upstream and downstream homologous arms of the c-di-AMP degrading enzyme gene MSMEG _2630 are amplified by PCR and are respectively connected into a pMSG360 vector, and the positive cloning plasmid is named as PW 75. An Ms competent cell is prepared, a pJV53 plasmid containing recombinase gp60 and gp61 genes is transformed, and a positive clone is named as Ms-pJV 53. Preparing competent cells of Ms-pJV53 strain, electrically transforming recombinant plasmid PW75, screening positive clone by bleomycin resistance plate, identifying target gene knockout condition by PCR and Westren-blot, and naming the positive clone as recombinant smegmatis ramaria cladribium strain (rMS-delta CnpB) with knockout of c-di-AMP catabolic enzyme, which is delivered to China center for type culture collection and has the collection number of: CCTCC M2016336.

The recombinant rMS-delta CnpB is applied to a vaccine and a preparation for preventing and treating tuberculosis.

After the strain rMS-delta CnpB is subjected to amplification culture, 10 is taken⁷Mice were immunized subcutaneously, the immunization was performed 2 times at 2-week intervals, and the humoral immune response level and the cellular immune response level (splenic lymphocyte proliferation, cytokine transcription and secretion level) were measured 6 weeks after completion of the immunization; the infected immunized mice were challenged with Mtb H37Ra strain 6 weeks after completion of immunization, and after 6 weeks, the humoral immune response level and cellular immune response level (splenic lymphocyte proliferation, cytokine transcription and secretion level) were measured, and the number of major organ-borne bacteria was counted. The result shows that the obtained rMS-delta CnpB strain can induce the humoral immune response reaction of the mouse when the mouse is immunized, but the overall level is not high; in cellular immunity, the spleen lymphocyte proliferation can be induced remarkably, the IFN-gamma level is increased but is lower than Ms, and the Th2 type cytokine IL-10 level is not increased remarkably; after infection with H37Ra, the Th1 type cytokines IFN-gamma and IL-2 levels are obviously increased, while the IL-10 level is still kept at a relatively low level, which indicates that moderate humoral and cellular immune responses can be induced after rMS-delta CnpB immunization, and after infection, the cellular immune response level is rapidly increased, particularly Th1 type response, is beneficial to host anti-infection immunity. Both Ms and rMS-. DELTA.CnpB immunizations were resistant to a number of H37Ra challenge infections. Both rMS-delta CnpB strain and Ms strain have the function of inducing host immune response, and rMS-delta CnpB strain is simple in immunity, low in IFN-gamma induction level, and after infection, it can induce host immune responseThe Th1 type cell factor is obviously increased, and the IL-10 level is still kept at a lower level, which shows that the regulation of the response of Th1/Th2 is obviously better than Ms, thus having better application prospect.

Drawings

FIG. 1: the MSMEG _2630 upstream and downstream homology arms are cloned into a pMSG360 vector enzyme digestion identification result diagram.

FIG. 2: rMS-delta CnpB recombinant bacterium genotype identification PCR result chart.

FIG. 3: western-blot result chart for phenotypic identification of rMS-delta CnpB recombinant bacteria.

FIG. 4: rMS-delta CnpB immune mouse serum antibody detection result chart.

FIG. 5: spleen lymphocyte proliferation result chart of rMS-delta CnpB immunized mice.

FIG. 6: RT-PCR detection result chart of spleen lymphokine transcription level of rMS-delta CnpB immune mice.

FIG. 7: ELISA detection result chart of spleen lymphokine secretion level of rMS-delta CnpB immune mice.

FIG. 8: RT-PCR detection result chart of spleen lymphokine secretion level after rMS-delta CnpB immune mouse H37Ra infection.

FIG. 9: ELISA detection result chart of spleen lymphokine secretion level after rMS-delta CnpB immune mouse H37Ra infection.

FIG. 10: results of the number of the organs and the number of the bacteria in the lotus after the rMS-delta CnpB immune mice H37Ra are infected. Proof of deposit

Categorical naming

Mycobacterium smegmatis rMS-delta CnpB

Latin character name

Mycobacterium smegmatis rMS-ΔCnpB

Name of depository

China center for type culture Collection

Address: eight-way Lojia mountain in Wuchang region of Wuhan city of Hubei province

Date of storage

2016 (6 months) and 20 days

Accession number

CCTCC M 2016336

Detailed Description

Construction of MSMEG-2630 gene homologous recombination knockout plasmid

According to Ms MC²Primers are designed for upstream and downstream sequences of genome MSMEG _2630 gene of 155 strains:

primers were synthesized by Shanghai Biotech, Inc. With Ms MC²155 strains of genome as template, PCR amplification of target gene, reaction parameters of pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30s, renaturation at 57 ℃ for 30s, extension at 72 ℃ for 30s, 30 cycles, extension at 72 ℃ for 5 min.1% agarose gel electrophoresis analysis after the last cycle shows that 456bp and 468bp target bands are respectively amplified, a gel recovery kit recovers target fragments, the fragments are respectively cut by enzyme, are connected with pMSG360 plasmid which is cut by enzyme and recovered in sequence, and transform E.coli DH5 α competent cells, bleomycin resistance plate screening is used, positive clones are picked and inoculated in LB liquid culture medium for shaking culture at 37 ℃ overnight, the kit extracts plasmid DNA, the cut by enzyme identifies positive plasmid to carry out sequence determination (figure 1), the sequence determination result shows that the positive plasmid and Ms MC are subjected to sequence determination²The published sequences of the MSMEG _2630 gene of the 155 strains of genome are completely consistent, which indicates that the MSMEG _2630 gene homologous recombination knockout plasmid is successfully constructed and is named as PW75, and the sequences of the MSMEG _2630 gene inserted into the vector are respectively shown as SEQ ID NO:1 and NO: 2.

Construction of MSMEG-2630 Gene knockout Strain (rMS-. DELTA.CnpB)

Preparation of Ms competent cells

Ms is inoculated in 3mL of 7H9 culture medium, cultured at 37 ℃ for 48H with shaking at 100rpm, transferred to 50mL of culture medium according to the proportion of 1:100, and cultured continuously until OD is reached₆₀₀0.8-1.0, and carrying out ice bath for 1 h. Washing with pre-cooled 10% glycerol solution for three times, packaging (100 μ l/tube), and storing at-80 deg.C.

Construction of Ms competent cells containing recombinases gp60 and gp61

pJV53 was amplified in E.coli, plasmids were purified by the kit, 45. mu.l of Ms competent cells were thawed on ice, 5. mu.L of pJV53 plasmid was added, mixed and left on ice for 10min, 50. mu.L of the mixture was transferred to a 0.1cm electric rotating cup (Biorad), parameters for electric transformation: 1.25kv, 25 muF, 1000 omega, left on ice for 10min, added with 1mL of 7H9 for 2H, spread antibiotic plates, and cultured at 37 ℃ for 3-4d until colony formation. Positive clones were identified by PCR. Designated Ms-pJV 53.

Construction of MSMEG _2630 knock-out Strain (rMS- Δ CnpB)

Preparing competent cells and an electrotransformation method according to the method, preparing the Ms-pJV53 strain into the competent cells, electrotransforming PW75, screening and cloning a bleomycin resistant plate, selecting clone PCR identification, amplifying the strain which can not amplify target genes by using an MSMEG-2630 primer as a knockout strain (figure 2), selecting positive clone in a 7H9 culture medium, shaking and culturing at 37 ℃ for 3-4d, preparing a total bacterial protein sample, transferring the sample to a PVDF membrane after 12 percent SDS-PAGE electrophoresis, taking polyclonal mouse serum immunized by using MSMEG-2630 protein as a primary antibody, Westren-blot identification result shows that the target protein at rMs strain bacterial protein of 36kDa is deleted, showing that the target genes are successfully knocked out (figure 3), knocking out the positive clone of a recombinant trichome ramification strain (rMS-delta Cnpb) named as c-di-AMP resolvase, and delivering the recombinant trichome ramification strain (rMS-delta Cnpb) to China center for type culture collection, the preservation number is as follows: CCTCC M2016336.

rMS-delta CnpB immune mouse response level detection

Grouping and immunizing experimental animals: female BALB/c mice at 6 weeks of age were randomized into 4 groups (n ═ 6), normal control (Naive), non-immunized (UN), Ms-immunized and rrms- Δ CnpB-immunized. Ms and rMs group 10⁷CFU is immunized subcutaneously in groin for 2 times at 2 weeks intervals; at 6w after completion of immunization, mice were sacrificed and the humoral and cellular immune levels were examined.

1. Detection of humoral immunity level of immunized mice

The specific antibodies in the serum of each group of immunized mice are detected by an indirect method by taking Ms bacterial protein as an antigen coating ELISA plate, and the results show that the Ms immune group and the rMS-delta CnpB immune group can detect the antibodies with lower concentration (figure 4).

2. Immune mouse cell immune level detection

(1) Proliferation of splenic lymphocytes

Immunization in mice6w after completion, splenic lymphocytes from each group of mice were isolated aseptically and inoculated with 1 × 10 per well in a 96-well plate⁵After the cells were cultured for 72 hours under stimulation with Ms somatic protein at a final concentration of 25mg/L, 20. mu.L/well of MTS (5mg/mL) was added thereto, and the cells were further cultured for 4 hours, and finally 10% SDS was added thereto at 25. mu.L/well to measure the A490 value. The results are expressed as the proliferation index SI: SI ═ a490 (experimental-blank)/a 490 (negative-blank). The results showed that SI of Ms immune group and rMS- Δ CnpB immune group showed an increasing trend (P) with Ms mycoprotein as stimulating protein<0.05) but no significant difference (P) between the two groups>0.05) (fig. 5).

(2) Spleen lymphokine transcription level

6w after the completion of immunization, mouse spleen tissues are taken aseptically, the total RNA of the tissues is extracted by the kit, the transcription level of the cell factors is detected by a qRT-PCR relative quantitative method, and the results show that: the Ms and rMS- Δ CnpB immunization groups showed a significant increase in IFN- γ, IL-2 and IL-10 transcript levels (P <0.001) with no significant difference between the two groups (P >0.05) (fig. 6).

(3) Level of spleen lymphokine secretion

Collecting the spleen lymphocyte stimulation culture supernatant, and detecting the levels of cytokines IFN-gamma, IL-2 and IL-10 in the supernatant by ELISA. The results show that: the Ms and rMS- Δ CnpB immunization groups had significantly increased levels of IFN- γ, IL-2, and IL-10 transcripts on average (P <0.001), while the rMS- Δ CnpB immunization group had significantly lower levels of IFN- γ secretion than the Ms group (P <0.05), indicating that rMS- Δ CnpB immunization may modulate host cell immunity such that the level of IFN- γ responses decreased after immunization against Ms (fig. 7).

Protective Effect of rMS- Δ CnpB immunized mice on H37Ra infection

6w after completion of the immunization of the mice in the above groups, Mtb H37Ra 10⁴CFU/mice immunized by tail vein challenge infection alone, 6w post infection, mice were sacrificed and tested as follows.

1. Spleen lymphokine transcription level after H37Ra infection in immunized mice

Mouse lung and spleen tissues are taken aseptically, total RNA of the tissues is extracted by a kit, and spleen lymphokine transcription level is detected by a qRT-PCR absolute quantitative method, so that the results show that the transcription levels of IFN-gamma, IL-2 and IL-10 of an Ms immune group and an rMS-delta CnpB immune group are obviously increased on average, the transcription levels of Th1 type cytokines IFN-gamma and IL-2 of the rMS-delta CnpB immune group are obviously higher than that of an Ms group (P <0.05 and P <0.01), and the transcription level of Th2 type cytokine IL-10 is equivalent to that of the Ms group (P >0.05) (figure 8).

2. Spleen lymphokine secretion level after H37Ra infection of immunized mice

Separating mouse spleen lymphocytes by the same method, stimulating and culturing by antigen, detecting the cytokine level in cell culture supernatant by an ELISA method, and showing that the secretion levels of IFN-gamma and IL-2 in the UN group, the Ms immune group and the rMS-delta CnpB immune group are obviously increased, and the secretion levels of IFN-gamma and IL-2 in the rMS-delta CnpB immune group are obviously higher than that in the Ms immune group (P <0.01 and P < 0.05); the IL-10 secretion level of each group was significantly increased, while the IL-10 level of the rMS- Δ CnpB immune group was significantly increased at a level lower than those of the UN and Ms groups (P <0.01) (FIG. 9).

3. Major viscera and Hejun cell number after H37Ra infection of immunized mice

After 6w of infection, the spleen and the lung are taken aseptically, 5ml of 1640 culture solution is added, the mixture is fully ground on a 200-mesh screen, 100 mu L of suspension is taken for multiple dilution, and the number of the viscera and the lotus bacteria is counted. The results show that both Ms and rrms- Δ CnpB immunized mice were effective against Mtb H37Ra infection (P <0.01, P <0.001), and there was no difference between Ms and rrms- Δ CnpB immunized groups (P >0.05) (fig. 10).

SEQUENCE LISTING

<110> the fourth military medical university of the Chinese people liberation army

<120> c-di-AMP lyase-knocked-out recombinant Mycobacterium smegmatis strain and application thereof

<160>2

<210>1

<211>456

<212>DNA

<213> Mycobacterium smegmatis (Mycobacterium smegmatis)

<400>1

gatatcggcc atcgagtacg agatcaagga tccgcgcctg gcgggcgtga cgatcaccga 60

cgcgaaggtg tccggggatc tgcacgacgc cacgctgtac tacaccgtgc tgggggcttc 120

gctcgacgag gacccggatt acgagggtgc ggcagcggcg ctggagaagg ccaaaggtgt 180

gctgcgcacc aaggtgggcg ccgggaccgg ggtgaggttc accccgaccc tggcgttcgt 240

ccgggacacc gtgcccgacg cggcgcaccg gatggaggag ctgctggcac gggcgcgggc 300

cgcggacgag gatctggcga gagttcggga gggcgccaag cacgcaggcg atcccgaccc 360

gtaccgtgtt ggcggggcgg aggacacaga cggggatacc gacggggacg aacgctgagg 420

atgccggtga cgacaaccga tcccaagacc aagctt 456

<210>2

<211>468

<212>DNA

<213> Mycobacterium smegmatis (Mycobacterium smegmatis)

<400>2

tctagagcgg gatactccgc gaccggttcg gccgacgacg tcgtacaggc gctcgcacgg 60

gcccttggct gatgcggcgg gcgacctgcc cggcggtccc gacgacgacg cccagggcgc 120

cgcactgacg caggcgacgg gccggcgcat cgccaaactc gcgtttcccg cgctcggcgt 180

ccttgccgca gaacccatct acctgctttt cgacctcgcg atcgtcgggc gcctcggcgc 240

ggtgagcctc gccggcctgg cgatcggcgg gctggtcctc ggactcgtca actcccaggg 300

cacgttcctg tcctacggca ccacggcccg ctcggcgcgg ttctacgggg cgggggaccg 360

gacctcggcg gtggccgagg gtgtgcaggc cacctggctc gcgctcggtc tcggactgct 420

gatcatcgcc gtggtggaag ccgttgcggt gccgatgctt tcggtacc 468

Claims (1)

1. A recombinant Mycobacterium smegmatis strain with c-di-AMP catabolic enzyme knocked out is used for preparing vaccines and preparations for preventing and/or treating tuberculosis, and is characterized in that: the recombinant Mycobacterium smegmatis strain with the c-di-AMP resolvase knocked out is prepared by knocking out an MSMEG _2630 gene of c-di-AMP resolvase in a Mycobacterium smegmatis genome by a homologous recombination method by adopting pJV53 plasmid containing recombinase gp60 and gp61 genes, and screening to obtain the recombinant Mycobacterium smegmatis strain which is abbreviated as rMS-delta CnpB and has the preservation number of: CCTCC M2016336.

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