CN107217026A - A kind of recombinant mycobacterium smegmatis strain of knockout c di AMP catabolic enzymes and its application - Google Patents

Abstract

The present invention relates to a kind of recombinant mycobacterium smegmatis strain of knockout c di AMP catabolic enzymes.Mycobacterium smegmatis c di AMP degrading enzyme genes MSMEG_2630 upstream and downstream homology arm is connected into pMSG360 carriers respectively, recombinant plasmid transformed contains the mycobacterium smegmatis competent cell of recombinase gp60 and gp61pJV53 plasmid, the recombinant mycobacterium smegmatis strain (rMS Δ CnpB) of one plant of knockout c di AMP catabolic enzyme is screened and obtains, its deposit number is：CCTCC M 2016336.Mouse is immunized in rMS Δ CnpB bacterial strains; adjustable mouse body fluid and cellullar immunologic response; Th1 type cellullar immunologic responses especially after infection mycobacterium tuberculosis; and improve the protection of the anti-mycobacterial infections of body; thus available for the development for preventing and/or treating vaccine lungy and preparation, with relatively good application prospect.

Description

A kind of recombinant mycobacterium smegmatis strain of knockout c-di-AMP catabolic enzymes and its application

Technical field

The invention belongs to tuberculosis vaccine and immunotherapy field.The present invention relates to a kind of knockout c-di-AMP catabolic enzymes Recombinant mycobacterium smegmatis strain.It is used to prevent the invention further relates to above-mentioned recombinant mycobacterium smegmatis strain and/or treats tuberculosis Vaccine and preparation in application.

Background technology

TB endemic present situation

Tuberculosis (Tuberculosis, TB) is the chronic infectious disease that human health is seriously endangered in global range.Tuberculosis Mycobacteria (Mycobacterium tuberculosis, Mtb) is TB pathogen, and according to WHO report, 2015, the whole world was estimated Counting in new cases 1 040 ten thousand, new cases has 1,200,000 (11%) HIV patients, and 1,400,000 people die from TB altogether.TB is dead former Top ten is still come because in.This six countries of India, Indonesia, China, Nigeria, Pakistan and South Africa account for newly Send out the 60% of total cases.Positive TB study on prevention and measure avoided 49,000,000 TB death at 2000 to 2015, but Current TB epidemic status and the extent of injury show that the breach in terms of TB preventions, diagnosis and treatment is also very big.

Tuberculosis prevention and treatment present situation

BCG vaccine (Bacillus Calmette-Gu é rin vaccine, BCG) be currently used for tuberculosis prophylaxis only One vaccine.BCG is directed to Mtb early infection mainly as the preventative vaccine before Mtb infection, it is impossible to which prevention and control are latent Infection Status Mtb recurrence is lied prostrate, but there is the shortcomings of protection period is short, protective immune response is not strong, and as a kind of Attenuated live vaccine, it is impossible to be used in the inoculation of immunocompromised crowd, therefore develop safe and reliable novel TB vaccines and compel in eyebrow Eyelash.

In terms of TB treatment, because TB chemotherapy treatments are up to more than 6 months, patient is caused to adhere to taking medicine and complying with Property is poor, and often causes resistance, extensive Drug Resistance for Tuberculosis and super Resistance Mycobacterium Tuberculosis particularly occurs.Recognize with to Mtb essence Progressively deeply, it is relatively low to have proven to TB patient's Th1 type cell immune responses, therefore can improve body's immunity, especially Th1 The therapeutic vaccine of type reaction has preferable application prospect.Such vaccine be mainly used in oneself infection Mtb Asymptomatic Carriers or Be oneself morbidity patient, selectively induce the specificity and nonspecific immune response of human body, be finally reached treatment purpose (MI,et al.Therapeutic vaccines for tuberculosis--a systematic review.Vaccine.2014,32(26):3162-8).The TB new generation vaccines being currently being deployed mainly include subunit's epidemic disease Seedling, gene vaccine, recombinant BCG vaccine, attenuation or enhanced whole cell live vaccine, auxotroph live vaccine, the epidemic disease with reference to DC Seedling etc., but can substitute traditional BCG or immunization therapy for TB there is presently no a kind of new generation vaccine.

The research of recombinant mycobacterium vaccine

It is one of the area research important directions that allogenic gene importing mycobacteria is built into live recombined vaccines.BCG It is the more mycobacteria live vaccine vectors of research, but there are problems that slow-growing, transformation efficiency is low, genetic manipulation, And BCG sheets are as attenuated strain, it is impossible to be used in immunocompromised crowd is inoculated with.

Mycobacterium smegmatis (Mycobacterium smegmatis, Ms) is that a kind of growth is fast, the high non-cause of transformation efficiency Germ mycobacteria and strong cellular immunity adjuvant, with the immunological characteristic similar to BCG.Such as Zhu DY, et al. (Recombinant M.smegmatis vaccine targeted delivering IL-12/GLS into macrophages can induce specific cellular immunity against M.tuberculosis in BALB/c mice.Vaccine,2007,25(4):The rMS of expression IL12 and GLS fusions 638-648.) etc. is constructed, should Vaccine can stimulate T lymphopoiesis, start Th1 cellullar immunologic responses, promote cytokine secretion, improve body to branch The immune clearance of bacillus.(the High extracellular levels of mycobacterium such as Tullius MV tuberculosis glutamine synthetase and superoxide dismutase in actively growing cultures are due to high expression and extracellular stability rather than to a protein-specific export mechanism.Immunol Cell Biol.2003,87 (7):Mtb albumen 209-215.) is transferred to Ms expression, as a result shows that the biochemistry and immunological characteristic of recombinant protein and native protein are several It is identical.In addition, Ms fast growths, (the A new such as Goldstone RMvector and expression protocol for fast and efficient recombinant protein expression in Mycobacterium smegmatis.Protein Expression and Purification,2008,57(1):81- 87.) method for establishing the soluble-expression Mtb albumen in Ms bacterial strains, its research shows, Ms expression foreign protein yield it is high and its Yield is 5~10 times of BCG.The recombinant protein that Bashiri etc. is expressed with Ms as Host Strains, available for 26S Proteasome Structure and Function research (Bashiri G,Baker EN.Production of recombinant proteins in Mycobacterium smegmatis for structural and functional studies.Protein Sci.2015,24(1):1- 10.).Therefore, Ms is the excellent carrier of mycobacteria live vaccine.

Recombinant Mycobacterium smegmatis (rMs) live vaccine be used for Mtb infect treatment, can by recovering protective immunity, Mononuclear macrophage is promoted to produce more H₂O₂And NO, Mtb is effectively removed, (Xu Miao waits shame to enhancing immune response ability Dirty mycobacterial vaccine produces nitric oxide production influence Chinese Academy of Medical Sciences journal, 2009, (4) to mouse macrophage: 410-412.)；The Th1/Th2 patterns of immune response can also be changed, immune response is offset from Th2 types to Th1 types, so as to help Mtb is killed in immune cell and withholds bacterium and drug-fast bacteria, and shortening chemotherapy treatment, (the Mycobacterium smegmatis vaccines such as Xu Miao are to small The Chinese tuberculosis of influence and breathing magazine of the generation of mouse cell factor and Th1/Th2 responses, 2005, (11):49-52.).Ms is nothing The mycobacteria of poison, many experiments have proven to its security, (the acellular Mycobacterium smegmatis vaccine such as domestic Xu Miao, kingdom control Chinese Journal of New Drugs, 2006, (19) are studied to the long term toxicity of cavy:1640-1643.) research shows, be used for a long time without thin Born of the same parents Ms vaccine in guinea pigs is without notable toxic action, and the acellular Ms bacterium that they prepare are used for TB immunization therapy, achieve Relatively good effect (mycobacterium smegmatis as immunomodulator the Chinese microbiologies of research and Journal of Immunology, 2005, (9):752-755.).Therefore, rMS have efficiently, the significant advantage such as security is good, be the new prevention and treatment vaccines of TB Important tool.

Suitable target antigen is selected to be remarkably improved the protective efficacy of vaccine.Research for Mtb protective antigens has been held Continue for many years, mainly have secretory protein such as ESAT-6, CFP10, Ag85, TB10.4, MTB39 and MTB32 etc., heat shock family is such as Hsp65, Hsp70, HspX etc., and the antigen such as fusion protein MTB72f.But, do not obtain yet available for prevention and treatment at present Recombinant vaccine, shows to still need to screen new Mtb antigens extensively, with the more preferable recombinant mycobacterium of adaptive immune effect, for TB Preventing and treating.

Secondary signal molecule c-di-AMP turns into bacterial vaccine and studies novel targets

, a kind of Bacterial signal molecules of new discovery in 2008 --- the adenosine of ring two (Cyclic di-adenosine Monophosphate, c-di-AMP) (Witte, G., et al.Structural biochemistry of a bacterial checkpoint protein reveals diadenylate cyclase activity regulated by DNA recombination intermediates.Mol Cell,2008.30(2):167-78.).C-di-AMP participates in golden yellow Portugal Cell wall synthesis, pressure sensitive, the biofilm of the bacteriums such as grape coccus, streptococcus, Bacillus subtillis, Liszt pseudomonad The various physiological processes such as formation, ion metabolism (Krasteva PV et al.Versatile modes of cellular regulation via cyclic dinucleotides.Nat Chem Biol.2017,13(4):350-359).Study table It is bright, c-di-AMP regulations Mtb thalline length, cell membrane formation, lipid metaboli and pathogenic.Research is it is also shown that c-di-AMP can Directly activate STING, innate immune response (Andrade WA, et that the release of inducing host cell I types interferon is characterized al.Group B Streptococcus Degrades Cyclic-di-AMP to Modulate STING-Dependent Type I Interferon Production.Cell Host Microbe.2016,20(1):49-59.), and c-di- AMP antigen immunes alone or in combination, can adjust Th1/Th2/Th17 adaptive immune responses, with significant immunoadjuvant function (Ebensen,T,et al.Bis-(3',5')-cyclic dimeric adenosine monophosphate:strong Th1/Th2/Th17 promoting mucosal adjuvant.Vaccine,2011,29(32):5210-20.)。c-di- AMP is existed only in bacterium, and is not had in eukaryotic, therefore c-di-AMP turns into the new target of bacterial vaccine and drug research Point.

C-di-AMP is catalyzed through two adenyl cyclases (Diadenylate cyclase A, DacA) by two molecule ATP and closed Into.Rv3586 is the DAC enzymes of synthesis c-di-AMP in Mtb.(the Deletion of the cyclic di-AMP such as Jun Yang phosphodiesterase gene(cnpB)in Mycobacterium tuberculosis leads to reduced virulence in a mouse model of infection.2014,93(1):65-79.) research has shown that, contains DHH knots The Rv2837c in structure domain is Mtb c-di-AMP digestive enzymes, and is named as CnpB.(the Two-step such as Manikandan K synthesis and hydrolysis of cyclic di-AMP in Mycobacterium tuberculosis.PLoS One.2014 23；9(1):E86096.) find that c-di-AMP first can be degraded to pApA by Rv2837c, be further degraded to 5'- AMP.(the Characterization of NrnA homologs from Mycobacterium such as Postic G tuberculosis and Mycoplasma pneumoniae.2012 18(1):155-65.) find that Rv2837c has RNA Enzymatic activity, thus it is speculated that it has important adjustment effect in bacterium pathogenic course.Jun Yang etc. further study show that, Rv2837c mutant strain c-di-AMP levels are raised, the mutant infection STING-/notable increasing of-deficient mice M φ, IL-1 β releases Plus.Zoopery shows that the bacterial strain promotes host immune to remove Mtb, animal survival time extension.Dey RJ etc. (Inhibition of innate immune cytosolic surveillance by an M.tuberculosis phosphodiesterase.Nat Chem Biol.2017,13(2):210-217.) research is found, mutation c-di-AMP is decomposed Enzyme Rv2837c can reduce Mtb toxicity, and the use of Rv2837c inhibitor, can pass through swashing for endochylema cGAS-STING paths It is living, change the final result of Mtb infection.In summary, the Rv2837c albumen containing DHH domains has degraded c-di-AMP enzyme Activity, the enzyme is mutated in Mtb, not only declines Strain Virulence, bacterium c-di-AMP levels is significantly increased, secretion C-di-AMP can activate the innate immune response of endochylema STING paths mediation, finally promote the immune clearance of intracellular bacterium, therefore, Also the c-di-AMP digestive enzymes of DHH domains turn into the novel targets of vaccine and drug research.

In Ms, MSMEG_2630 encodes the DHH domain protein similar to Rv2837c, and both homologys are 82%. The 023bp of MSMEG_2630 genes 1, encoding proteins molecular weight is about 35.5kDa.Domestic Hua Zhong Agriculture University Tang Q etc. (Functional Analysis of a c-di-AMP-specific Phosphodiesterase MsPDE from Mycobacterium smegmatis.Int J Biol Sci.2015,11(7):813-24) lacked using sacB-lacZ nutrition Swaged screening system, constructs Ms MSMEG_2630 gene mutations strain.Phenotype research shows, MSMEG_2630 have with Phosphodiesterase activity similar Rv2837c, hydrolyzable c-di-AMP.Knockout MSMEG_2630 genes, Ms bacterial strains colonial morphology, Lipid metaboli and ion metabolism change, and the c-di-AMP levels of bacterial strain significantly rise.Therefore, MSMEG_2630 is Ms c- Di-AMP digestive enzymes, knock out the gene, and bacterium produces c-di-AMP levels and dramatically increased, after the bacterial strain is immune, its c- secreted Di-AMP will promote innate immune response, be conducive to host immune to remove intracellular bacterium.

The content of the invention

The purpose of the present invention is to obtain a kind of recombinant mycobacterium smegmatis strain of knockout c-di-AMP catabolic enzymes.The bacterial strain Development available for tuberculosis prophylaxis and therapeutic vaccine.

What the present invention was realized in：

Using Ms genome as template, PCR amplification c-di-AMP degrading enzyme genes MSMEG_2630 upstream and downstream homology arm, It is connected into pMSG360 carriers respectively, positive colony plasmid is named as PW75.Ms competent cells are prepared, conversion contains restructuring The pJV53 plasmids of enzyme gp60 and gp61 gene, positive colony is named as Ms-pJV53.Prepare Ms-pJV53 bacterial strain competence thin Born of the same parents, electricity conversion recombinant plasmid PW75, blasticidin resistance plate screening positive colony, PCR and Westren-blot identification purposes Gene knockout situation, positive colony is named as recombinant mycobacterium smegmatis strain (the rMS- Δs for knocking out c-di-AMP catabolic enzymes CnpB), China typical culture collection center preservation has been sent, its deposit number is：CCTCC M 2016336.

Described restructuring rMS- Δs CnpB is used to prevent and treat the application in tuberculosis vaccine and preparation.

Described bacterial strain rMS- Δs CnpB expands after culture, takes 10⁷Subcutaneous inoculation mouse, exempts from interval 2 weeks, is immunized 2 times, exempts from 6 weeks after the completion of epidemic disease, (spleen lymphocyte proliferation, cell factor turn for detection humoral immune response level and cellullar immunologic response level Record and secretion level)；6 weeks after the completion of immune, mouse is immunized with H37Ra plants of challenge infections of Mtb, after 6 weeks, humoral immunity is detected Response level and cellullar immunologic response level (spleen lymphocyte proliferation, cell factor transcription and secretion level), and count main Internal organs lotus bacterium number.As a result show, mouse is immunized in the rMS- Δ CnpB bacterial strains of acquisition, can induce mouse humoral immune responsing reaction, But integral level is not high；In cellular immunity, significant spleen lymphocyte proliferation, the rise of IFN-γ level are can induce, but level is low In Ms, and Th2 cytokines IL-10 levels are not raised significantly；After H37Ra challenge infections, Th1 cytokines IFN- γ and IL-2 levels are significantly raised, and IL-10 levels are remained at than relatively low level, can after showing that rMS- Δs CnpB is immune The humoral and cellular immune response response of appropriateness is induced, and after infecting, cellullar immunologic response level rises rapidly, and especially Th1 types are anti- Should, contribute to host's anti-infectious immunity.Ms and rMS- Δs CnpB is immunized, and can resist a certain amount of H37Ra challenge infections.rMS- Δ CnpB bacterial strains and Ms bacterial strains have induction host immune response, and rMS- Δ CnpB bacterial strains are immunized merely, induce IFN-γ level is not high, and after infecting, it induces Th1 cytokines significantly to raise, and IL-10 levels are remained at than relatively low Level, show to be significantly better than Ms to the regulation that Th1/Th2 reacts, thus with relatively good application prospect.

Brief description of the drawings

Fig. 1：MSMEG_2630 upstream and downstream homology arms are cloned into pMSG360 carrier digestion qualification result figures.

Fig. 2：RMS- Δ CnpB recombinant bacterium genotype identification PCR result figures.

Fig. 3：RMS- Δ CnpB recombinant bacterium phenotypic evaluation Western-blot result figures.

Fig. 4：RMS- Δ CnpB immune serum antibody test result figures.

Fig. 5：Mice spleen lymphocytes proliferation result figure is immunized in rMS- Δs CnpB.

Fig. 6：The horizontal RT-PCR testing result figures of mouse spleen lymphocyte factor transcription are immunized in rMS- Δs CnpB.

Fig. 7：The horizontal ELISA testing result figures of mouse spleen lymphocyte cytokine secretion are immunized in rMS- Δs CnpB.

Fig. 8：The horizontal RT-PCR testing results of splenic lymphocytes cytokine secretion after mouse H37Ra infection are immunized in rMS- Δs CnpB Figure.

Fig. 9：The horizontal ELISA testing results of splenic lymphocytes cytokine secretion after mouse H37Ra infection are immunized in rMS- Δs CnpB Figure.

Figure 10：Internal organs lotus bacterium number result figure after mouse H37Ra infection is immunized in rMS- Δs CnpB.Preservation is proved

Classification And Nomenclature

Mycobacterium smegmatis rMS- Δs CnpB

Latin name

Mycobacterium smegmatis rMS-ΔCnpB

Depositary institution's title

China typical culture collection center

Address：Wuhan City, Hubei Province Wuchang District Bayi Road Luo Jia Shan

Preservation date

On June 20th, 2016

Preserving number

CCTCC M 2016336

Embodiment

MSMEG_2630 homologous recombinations knock out the structure of plasmid

According to Ms MC²155 plants of genome MSMEG_2630 gene upstream and downstream primers：

By Shanghai Sheng Gong bio tech ltd synthetic primer.With Ms MC²155 plants of genome is template, and PCR expands Increase target gene, response parameter：95 DEG C of pre-degenerations 5min, 95 DEG C of denaturation 30s, 57 DEG C of renaturation 30s, 72 DEG C of extension 30s, 30 are followed Ring, 72 DEG C of extension 5min after last circulation.1% agarose gel electrophoresis analysis shows, amplify 456bp and 468bp mesh respectively Band.Gel reclaims kit reclaims purpose fragment, digestion respectively, successively with the pMSG360 plasmids of same digestion and recovery Connection, and Transformed E .coli DH5 α competent cells, use bleomycin resistance plate screening.Picking positive colony is inoculated in LB 37 DEG C of shaken cultivations of fluid nutrient medium are stayed overnight, and kit extracts DNA, and digestion identification positive plasmid carries out sequencing (figure 1).Sequencing results are shown, with Ms MC²155 pnca gene group MSMEG_2630 genes upstream and downstream announcement sequence is completely the same, Show that successfully building MSMEG_2630 homologous recombinations knocks out plasmid, is named as PW75, the MSMEG_ inserted in the carrier 2630 gene upstream and downstream sequences are respectively such as SEQ ID NO:1 and NO:Shown in 2.

The structure of MSMEG_2630 gene knockouts strain (rMS- Δ CnpB)

It is prepared by Ms competent cells

Ms is inoculated in 3mL7H9 culture mediums, 37 DEG C of 100rpm shaken cultivation 48h, by 1:100 ratio is transferred in 50mL trainings Base is supported, continues to cultivate to OD₆₀₀=0.8~1.0, ice bath 1h.10% glycerite of precooling is washed three times, dispenses (100 μ l/ Pipe), -80 DEG C of preservations.

Build the Ms competent cells containing recombinase gp60 and gp61

PJV53 is expanded in Escherichia coli, kits plasmid melts the μ l of Ms competent cells 45, added on ice The μ L of pJV53 plasmids 5, place 10min on ice after mixing, in the electric revolving cup (Biorad) that 50 μ L mixed liquors are moved to 0.1cm, and electricity turns Change parameter：1.25kv, 25 μ F, 1000 Ω, place 10min on ice, add 1mL7H9 culture 2h, are coated with antibiotic flat board, 37 DEG C 3-4d is cultivated to bacterium colony generation.PCR identifies positive colony.It is named as Ms-pJV53.

Build MSMEG_2630 and knock out strain (rMS- Δ CnpB)

Competent cell and electric method for transformation are prepared according to the method described above, take Ms-pJV53 bacterial strains to prepare competent cell, Electricity conversion PW75, blasticidin resistance plate screening clone, picked clones PCR identifications are expanded with MSMEG_2630 primers, it is impossible to The bacterial strain of target gene is amplified to knock out strain (Fig. 2), picking positive colony is in 7H9 culture mediums, 37 DEG C of concussion and cultivate 3-4d, system After standby bacterial protein sample, 12%SDS-PAGE electrophoresis, pvdf membrane is transferred to, with many grams after MSMEG_2630 protein immunizations Grand mice serum is primary antibody, and Westren-blot qualification results are shown, destination protein is lacked at rMs plants of mycoprotein 36kDa, table Bright target gene successful knockout (Fig. 3), positive colony is named as the recombinant mycobacterium smegmatis strain for knocking out c-di-AMP catabolic enzymes (rMS- Δ CnpB), has sent China typical culture collection center preservation, and its deposit number is：CCTCC M 2016336.

The detection of mouse response level is immunized in rMS- Δs CnpB

Experimental animal is grouped and immune：6 week old female BAl BIcs/c mouse are taken to be randomly divided into 4 groups (n=6), Normal group (Naive), non-immune group (UN), Ms immune groups and rMS- Δ CnpB immune groups.Ms and rMs groups are with 10⁷CFU/ is only through groin Subcutaneous immunizations, are spaced 2 weeks, are immunized 2 times；6w after the completion of immune, puts to death mouse, detects humoral and cellular immune response level.

1st, the detection of mouse humoral immune level is immunized

Using Ms mycoproteins as antigen coat elisa plate, specific antibody in indirect method detection each group immune serum, As a result show, the antibody (Fig. 4) of Ms immune groups and the detectable low concentration of rMS- Δ CnpB immune groups.

2nd, the detection of mouse cell immune level is immunized

(1) spleen lymphocyte proliferation

The 6w after the completion of mouse immune, sterile separation each group mouse spleen lymphocyte, the inoculation 1 × 10 per hole of 96 orifice plates⁵It is individual Cell, adding final concentration of 25mg/L Ms mycoproteins stimulates after culture 72h, adds the MTS (5mg/mL) in 20 μ L/ holes, after Continuous culture 4h, is eventually adding the μ L/ holes of 10%SDS 25, determines A490 values.As a result represented with proliferation index SI：SI=A490 is (real Test group-blank control)/A490 (negative control-blank control).As a result show, using Ms mycoproteins as stimulates the protein, Ms exempts from Epidemic disease group and rMS- Δ CnpB immune groups SI are in rise trend (P<0.05) without significant difference (P, but between two groups>0.05) (Fig. 5).

(2) splenic lymphocytes factor transcription level

6w after the completion of immune, sterile to take mouse boosting tissue, kit extracts total tissue RNA, the inspection of qRT-PCR relative quantifications method Cell factor transcriptional level is surveyed, is as a result shown：Ms immune groups and rMS- Δ CnpB immune groups IFN-γ, IL-2 and IL-10 transcriptions Level significantly rises (P<0.001), and without significant difference (P between two groups>0.05) (Fig. 6).

(3) splenic lymphocytes cytokine secretion level

Collect above-mentioned splenic lymphocytes stimulate cell factor IFN-γ in culture supernatant, ELISA detection supernatants, IL-2 and IL-10 level.As a result show：Ms immune groups and rMS- Δ CnpB immune groups IFN-γ, IL-2 and IL-10 transcriptional levels show Write and rise (P<0.001), and rMS- Δ CnpB immune group IFN-γ secretion levels are substantially less than Ms groups (P<0.05) rMS-, is shown The immune host cells that may adjust of Δ CnpB are immunized, and IFN-γ reaction level declines (Fig. 7) after making it immune to Ms.

The protective effect that mouse is infected H37Ra is immunized in rMS- Δs CnpB

6w after the completion of above-mentioned each group mouse immune, with Mtb H37Ra 10⁴What CFU/ was only immunized through tail vein challenge infection 6w after mouse, infection, puts to death mouse, is detected as follows.

1st, splenic lymphocytes factor transcription level after mouse H37Ra infection is immunized

Sterile to take mouse lung, spleen tissue, kit extracts total tissue RNA, and qRT-PCR absolute quantitations method detection spleen lymph is thin Intracellular cytokine transcriptional level, as a result shows, Ms immune groups and rMS- Δ CnpB immune groups IFN-γ, IL-2 and IL-10 transcriptional levels Significantly rise, rMS- Δ CnpB immune group Th1 cytokines IFN-γ, IL-2 transcriptional levels are significantly higher than Ms groups (P< 0.05, P<0.01), Th2 cytokines IL-10 transcriptional levels (P suitable with Ms groups>0.05) (Fig. 8).

2nd, splenic lymphocytes cytokine secretion level after mouse H37Ra infection is immunized

Ibid method separating mouse splenic lymphocytes, antigenic stimulus and after cultivating, in ELISA method detection cells and supernatant Cytokine levels, as a result show, UN groups, Ms immune groups and rMS- Δ CnpB immune groups IFN-γ, IL-2 secretion levels are equal Significantly rise, rMS- Δ CnpB immune groups IFN-γ, IL-2 secretion levels are significantly higher than Ms immune groups (P<0.01, P<0.05)； Each group IL-10 secretion levels significantly rise, and rMS- Δ CnpB immune group IL-10 levels increase amplitude be substantially less than UN groups and Ms groups (P<0.01, P<0.01) (Fig. 9).

3rd, main organs lotus bacterium number after mouse H37Ra infection is immunized

Infect after 6w, it is sterile to take spleen, lungs, 5ml 1640 culture mediums are added, after being fully ground on 200 eye mesh screens, are taken After 100 μ L suspension doubling dilutions, internal organs lotus bacterium number is counted.As a result show, Ms immune groups and rMS- Δ CnpB immune group mouse are equal It is effective against Mtb H37Ra infection (P<0.01, P<0.001), it is not present between Ms immune groups and rMS- Δ CnpB immune groups Difference (P>0.05) (Figure 10).

SEQUENCE LISTING

<110>The Fourth Military Medical University of P.L.A

<120>A kind of recombinant mycobacterium smegmatis strain of knockout c-di-AMP catabolic enzymes and its application

<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 (2)

1. a kind of recombinant mycobacterium smegmatis strain of knockout c-di-AMP catabolic enzymes, using containing recombinase gp60 and gp61 gene PJV53 plasmids, by methods of homologous recombination will in mycobacterium smegmatis genome encode c-di-AMP catabolic enzymes MSMEG_ 2630 gene knockouts, the recombinant mycobacterium smegmatis strain that screening is obtained is referred to as rMS- Δ CnpB, and its deposit number is：CCTCC M 2016336。

2. it is used to prevent by a kind of recombinant mycobacterium smegmatis strain of knockout c-di-AMP catabolic enzymes described in claim 1 And/or the application in treatment tuberculosis vaccine and preparation.