CN113322221B - Recombinant mycobacterium smegmatis strain knocking out virulence related genes and application thereof - Google Patents

Abstract

The invention relates to a recombinant mycobacterium smegmatis strain for knocking out a virulence related gene Ms esat-6, which is rMs-delta E6 for short and has the preservation number of: cctccc M2021462. The invention also relates to application of the recombinant mycobacterium smegmatis rMs-delta E6 in vaccines and preparations for preventing and/or treating tuberculosis. The rMs- ΔE6 strain has reduced survival in macrophages and rMs- ΔE6 induced immune responses have a higher protective effect on Mtb infection than Ms. Therefore, rMs- Δe6 can be used not only as a safer vaccine carrier, but also as a carrier for prophylactic or therapeutic vaccines; the anti-Mtb protective immune response inducing agent also has the characteristic of inducing the anti-Mtb protective immune response, can be used for developing an immune therapeutic agent, and has good application prospect.

Description

Recombinant mycobacterium smegmatis strain knocking out virulence related genes and application thereof

Technical Field

The present invention belongs to the field of tuberculosis vaccine and immunotherapy. In particular to a recombinant mycobacterium smegmatis strain knocking out a virulence related gene Ms esat-6and application thereof.

Background

Current situation of tuberculosis epidemic

Tuberculosis (TB) caused by mycobacterium Tuberculosis (Mycobacterium Tuberculosis, mtb) infection is still a serious infectious disease worldwide. The WHO estimated that the global new onset of TB was 1,000 tens of thousands in 2019, with about 140 tens of thousands dying from TB. Currently, the worldwide population with latent tuberculosis infection (latent tuberculosis infection, LTBI) is approaching 20 hundred million, and 5% -10% of latent infected people can be converted into active TB, which is the main source of adult TB. The new onset of TB in our country is second only to india, the second world (who. Global numeric report, 2020.). TB is one of the major diseases that is important to control in China.

Current situation of tuberculosis vaccine research

BCG vaccine (Bacillus Calmette-Guerin vaccinee, BCG) is an attenuated live vaccine obtained by inoculating mycobacterium bovis (Mycobacterium bovis) into an artificial culture medium for 13 years and 230 passages continuously, and is the only vaccine currently used for TB prevention. BCG is effective in preventing severe TB in children, but the overall protective efficiency for adult TB varies from 0% to 80%. BCG has good safety but cannot be used as an attenuated live vaccine for vaccination of immunocompromised individuals (fanma, s., et al Tuberculosis vaccine: A journey from BCG to present. Life Sci, 2020.252:p.117594.). Studies have shown that BCG has reduced immunoprotection due to the loss of some strain-specific antigen during passage, which results in imperfect immunoprotection. In addition, the overall protective effect of BCG is not ideal due to various factors such as differences in vaccine strains in different regions, genetic diversity of individuals in the population, etc., which are affected by mycobacteria in the environment (Schrager, L.K., et al, the status of tuberculosis vaccine development. Lancet effect Dis,2020.20 (3): p.e28-e 37). Thus, the development of new vaccines is critical for the control of TB epidemic.

In recent years, there has been much research into novel TB vaccines, including the screening of a range of candidate vaccines and different vaccine immunization strategies. There are 14 total TB vaccines reported by WHO to have entered clinical trials in 2020, including viral vector vaccines, protein/adjuvant subunit vaccines, mycobacterial lysates, and live mycobacterial vaccines. The improvement of Mtb or BCG etc. by genetic engineering methods to reduce virulence of strains, or to increase immunoprotection efficacy of attenuated/non-virulent strains of mycobacteria, is one of the directions in the construction and research strategies of new vaccines for TB (Schrager, l.k., et al, the status of tuberculosis vaccine development.lancet effect Dis,2020.20 (3): p.e28-e 37).

Recombinant mycobacterium live vaccine is one of the important directions for research of novel TB vaccine. The current TB vaccine is more studied for mycobacterial vectors, including attenuated Mtb, BCG, mycobacterium vaccae and Mycobacterium smegmatis (Mycobacterium smegmatis, ms), etc. Compared with BCG, ms is a non-pathogenic mycobacterium, contains a type vii secretion system similar to Mtb, and has a growth rate 10 times faster than BCG, expresses foreign proteins at high yields, and expresses Mtb-related proteins nearly completely identical to the biochemical and immunological properties of native proteins in Ms (Anew Gateway vector and expression protocol for fast and efficient recombinant protein expression in Mycobacterium smegmatis. Protein Expression and Purification,2008,57 (1): 81-87.). Ms is therefore an excellent TB vaccine vector. Ms has been successfully used as a vaccine adjuvant in the united States for the prevention and treatment of TB (Mycobacterium semgatis vaccinee. Recombinant Mycobacterium smegmatis (Recombinant Ms, rMs) live vaccines can be used in combination with drugs for the treatment of Mtb infection (Yang Chun, he Yonglin, xu Lei, zhang Li, izhengjun, li Na, wang Yuwei, zhu Daoyin. Comparison of Recombinant Mycobacterium smegmatis with microcard in murine tuberculosis immunotherapy J.Immunol, 2009,25 (06): 685-688). Previous studies of the subject group found that Ms and rMs (rMs-AEL, patent number: ZL 201010189471.3) overexpressing Ag85B and ESAT-6 fusion proteins had protective effects against Mtb and prevented to some extent the recurrence of latent infection (Wang P, wang L, zhang W, bai Y, kang J, hao Y, luo T, shi C, xu Z.immunology efficacy of recombinant Mycobacterium smegmatis expressing Ag B-ESAT6 fusion protein against persistent tuberculosis infection in micro vaccine immunother.2014;10 (1): 150-158.) in addition, this rMs infection induced increased secretion of TNF- α, induced enhanced phagocytic function in cells, contributing to the enhancement of host anti-infective innate immune response (Hao Yanfei, luo Tailai, wang Ping, et al, recombinant Mycobacterium smegmatis Ag85B-ESAT6-rMs had an effect on mouse macrophage function. J.cell and molecular immunology 2012.28 (04): 361). The study including the subject group at home and abroad proves that Ms has safety and reliability as live vector vaccine and immunomodulator. Thus, ms can be used in the development of prophylactic and therapeutic vaccines and immunotherapeutic agents.

Ms is mainly parasitic bacteria of putrescence, and can also be planted in certain parts of human body, so that the pathogenicity is weak. However, under certain conditions Ms may also cause diseases including: lymphadenitis, osteomyelitis, cellulitis, wound infection, suppurative granulomatosis inflammation, etc. (Brown-Elliott BA, phililey jv. Rapid growth mycobacteria. Microbiol spectra, 2017,5 (1): 1-19; tian Jinyi, ma Xuzhu, li Ni. Mycobacterium smegmatis skin soft tissue infection 1. Journal of chinese infection and chemotherapy, 2020,20 (06): 681-682.). Therefore, the pathogenicity of Ms is further reduced, the safety of Ms is improved, and the application of Ms in TB prevention and treatment vaccines and immunotherapeutic agents is facilitated.

Mycobacterium tuberculosis ESAT-6 research progress

The full length of the 6kDa early secretion target antigen (6kDa early secretory antigenic target,ESAT-6) coding gene is 288bp, and the protein is composed of 95 amino acids. ESAT-6 is present only in Mtb and mycobacterium bovis strains, whereas it is deleted in the BCG genome (Groschel, m.i., et al Recombinant BCG Expressing ESX-1 of Mycobacterium marinum Combines Low Virulence with Cytosolic Immune Signaling and Improved TB Protection.Cell Rep,2017.18 (11): p.2752-2765). Studies have shown that ESAT-6 can form pores directly in the membrane of the vacuoles containing mycobacteria, promote the escape of mycobacteria from the vacuoles and spread among cells, provide a physiological basis for intracellular colonization, replication of virulent mycobacteria, and are believed to be associated with the virulence of Mtb (Smith, J., J.Manoranjan, M.Pan, et al, evidence for pore formation in host cell membranes by ESX-1-encrypted ESAT-6and its role in Mycobacterium marinum escape from the vacuole.Infect Immun,2008.76 (12): 5478-87.).

The study of the mechanism of ESAT-6 to promote bacterial intracellular survival has found that ESAT-6 induces an increase in the level of the autophagy marker molecule LC3 II, while inhibiting autophagy flow by activating mTOR (Dong, h., et al, ESAT6 inhibits autophagy flux and promotes BCG proliferation through mTOR. Biochem Biophys Res Commun,2016.477 (2): p.195-201). Earlier studies found that ESAT-6-CFP10 fusion proteins were able to inhibit the formation of autophagosomes induced by Mtb infection, promoting intracellular survival of Mtb (Shi Changhong, mao Fengfeng, zhao Yong, et al, experimental studies of ESAT 6and CFP10 fusion proteins inhibiting macrophage autophagosome formation. Chinese zoonotic journal 2011.27 (05): 414-417.). In addition, recombinant ESAT-6 proteins inhibit autophagosome formation by down-regulating microRNA-30a, resulting in increased intracellular viability of mycobacteria (Behura, A., A.Mishra, S.Chugh, et al, ESAT-6 modulates Calcimycin-induced autophagy through microRNA-30a in mycobacteria infected macrophages.J Infect,2019.79 (2): 139-152.). The above study demonstrates that ESAT-6 promotes survival of mycobacteria within macrophages by interfering with the different stages of autophagy. Thus ESAT-6 is considered to be a virulence factor for Mtb.

The amino acid sequence of ESAT-6 in different mycobacteria is studied, and the amino acid sequence of ESAT-6 between pathogenic mycobacteria (Mtb, mycobacterium bovis) is found to be highly homologous, and the similarity is as high as 90%. Ms esat-6 is a homologous gene to Mtb esat-6, and the amino acid sequence similarity of the two encoded proteins is 72% (Peng, X., G.Jiang, W.Liu., et al, characterization of differential pore-forming activities of ESAT-6 proteins from Mycobacterium tuberculosis and Mycobacterium smegmatis.FEBS Lett,2016.590 (4): 509-19.). Thus, by blocking the strategy of Ms esat-6, a more safe rMs can be obtained for the development of vaccine vectors or immunomodulators.

CRISPR/Cas system for mycobacterial gene knockout

CRISPR (clustered regularly interspaced short palindromic repeats and CRISPR-associated protein), a regularly clustered, short palindromic repeat of intervals and CRISPR-related proteins, is widely present in the genome of bacteria and archaea as an acquired immune system that helps bacteria/archaea to resist interference by phage and foreign DNA. The CRISPR/Cas9 system plays a role in gene editing, and the process comprises the following steps: after the guide RNA (sgRNA) binds to the Cas9 post-protein, the Cas9 protein begins to search on the genome for the protospacer adjacent motif (protospacer adjacent motif, PAM); the Cas9-sgRNA locally breaks the DNA double strand from the PAM end, so that the sgRNA enters into an RNA-DNA heteroduplex structure formed by complementation with the DNA; then Cas9 protein cleaves DNA double strand, initiating DNA double strand breaks; homologous recombination or nonhomologous end joining repair is initiated. Non-homologous end joining is an error-prone repair mechanism that typically introduces insertions or deletions at the DNA breaks, resulting in gene knockouts, enabling directed editing of the gene (Pickar-Oliver, A.and C.A.Gersbach, the next generation of CRISPR-Cas technologies and applications. Nat Rev Mol Cell Biol,2019.20 (8): 490-507.). CRISPR/Cas systems have been successfully used at present for the construction of various mycobacterial knockout strains such as Mtb, ms (yanMY, li SS, ding XY, guo XP, jin Q, sun YC.A CRISPR-Assisted Nonhomologous End-Joining Strategy for Efficient Genome Editing in Mycobacterium turbosis.mBio, 2020;11 (1): e 02364-19.).

According to literature reports, gene editing efficiency is related to PAM sequences (Rock, J.M., F.F.Hopkins, A.Chavez, et al Programmable transcriptional repression in mycobacteria using an orthogonal CRISPR interference platform. Nat Microbiol, 2017.2:16274). There is no report on the applicability of Ms esat-6 knockout strain.

Disclosure of Invention

The invention provides a recombinant mycobacterium smegmatis strain for knocking out virulence related genes and application thereof.

The invention is realized by the following technical scheme:

the invention is based on Ms MC ² 155 strain Ms esat-6 gene sequence, design and synthesize sgRNA as SEQ ID NO.1 and SEQ ID NO.2 sequenceThe sequence is used for constructing a plasmid vector, the Ms esat-6 gene is knocked out from a Ms genome by a CRISPR/Cas9 technology, and the strain rMs-delta E6 is obtained through screening and identification. The recombinant rMs-delta E6 is delivered to China center for type culture Collection with the accession number: cctccc M2021462. With Ms MC ² The survival rate of strain rMs- ΔE6 in macrophages was reduced compared to strain 155, and the protective effect of the induced immune response on Mtb infection was higher than Ms.

Use of recombinant mycobacterium smegmatis strain rMs- Δe6 for the preparation of a vaccine and/or formulation for the prevention and/or treatment of tuberculosis.

According to the invention, sgRNA target sequences of SEQ ID NO.1 and SEQ ID NO.2 of a sequence table are designed on a Ms esat-6 gene sequence according to a PAM site (ATGGAAT), and the Ms esat-6 is successfully knocked out by a CRISPR/Cas9 system, so that the preservation number is obtained: rMs- ΔE6 of CCTCC M2021462. rMs- ΔE6 on the one hand has reduced viability in the cells and further reduced virulence; on the other hand, the protective power of the immune response induced by the vaccine against Mtb infection is higher than that of Ms. Therefore, the strain can be used as a safer vaccine carrier for preventing or treating vaccine; can induce higher anti-Mtb protective immune response, can be used for developing immunotherapeutic agents, and has good application prospect.

Drawings

FIG. 1 is a schematic diagram showing the results of rMs-. DELTA.E6 sequencing.

FIG. 2 is a schematic representation of rMs- ΔE6 survival in macrophages.

FIG. 3 is a graph showing the number of visceral organs charged after Mtb infection in rMs-. DELTA.E6 immunized mice.

Preservation certificate

Classification naming

Mycobacterium smegmatis rMs-DeltaE 6

Latin Wen Xueming

Mycobacteria smegmatis rMs-ΔE6

China center for type culture Collection

Address:

eight paths of Lopa nationality mountain in Wuhan city of Hubei province in Wuchang district

E-mail:cctcc@whu.edu.cn

Telephone: 027-6875 4052

Date of preservation

2021, 4, 27

Deposit number

CCTCC M 2021462

Detailed Description

Construction and identification of rMs- ΔE6 Strain

(1) Construction of sgRNA expression vector and sgRNA target sequence design

Ms esat-6 (MSMEG_0066) gene sequences were obtained in the Mycobacterium genome database (https:// mycobrowse. Epfl. Ch /), and PAM sequences were searched for in this gene sequence according to literature (Rock, J.M., F.F.Hopkins, A.Chavez, et al Programmable transcriptional repression in mycobacteria using an orthogonal CRISPR interference platform. Nat Microbiol, 2017.2:16274.). PAM site "ATGGAAT" was selected to design sgrnas. And Bbs I enzyme cutting sites are added at the 5' ends of Forward (Forward) and Reverse (Reverse) sequences of the sgRNA target DNA sequences respectively to synthesize single-stranded Oligo DNA sequences of the sgRNA Forward and the sgRNA Reverse, wherein the sequences are shown as SEQ ID NO.1 and SEQ ID NO. 2.

The single-stranded Oligo DNA is annealed to form a double-stranded DNA with cohesive ends. The annealing system was Oligo Forward (100. Mu.M) 1. Mu.L, oligo Reverse (100. Mu.M) 1. Mu.L, T4 PNK (NEB) 0.5. Mu.L, 10 XT 4 Ligation Buffer 1. Mu.L, ddH2O 6.5. Mu.L, and the total volume was 10. Mu.L. The annealing procedure of the PCR instrument is 30min at 37 ℃;95 ℃ for 5min;95 ℃ for 30min, minus 1 ℃ per cycle, ramp 0.1 ℃/s, +60s per cycle;65 ℃ for 40min, minus 1 ℃ per cycle, ramp 0.1 ℃/s, +60s per cycle;20 ℃ is infinity.

The PW267 plasmid tangential by Bbs I enzyme was ligated with annealed double-stranded Oligo DNA, and the ligation product was transformed into E.coli (E.coli) DH 5. Alpha. Competent cells, and selected with kanamycin resistance plates. Clones identified as positive by PCR were picked up and inoculated in LB liquid medium, cultured overnight at 37℃with shaking, and plasmid DNA was extracted. Positive recombinants were sequenced. Sequencing results showed that the target DNA sequence had been successfully cloned into the PW267 plasmid, indicating successful construction of the sgRNA expression vector, designated PW267-sgRNA.

(2) Construction of helper plasmid-containing Strain Ms-PW268

The steps for preparing Ms competent cells were as follows: the frozen Ms were inoculated in 5mL 7H9 medium and incubated at 37℃until saturated. The saturated culture was transferred to a 250mL Erlenmeyer flask containing 50mL 7H9 complete medium and shake cultured at 80rpm to OD ₆₀₀ And approximately 0.8. Ice-bath the bacterial liquid for 1h; centrifuging at 8 000rpm at 4deg.C for 10min to collect thallus, and discarding supernatant; the bacterial pellet was resuspended and washed with 1/2, 1/4, 1/8 of the culture volume of pre-chilled 10% glycerol, respectively, and finally the bacterial pellet was resuspended with 1/100 of the culture volume of 10% glycerol solution and stored at-70℃until use.

Electrically transforming the PW268 plasmid into Ms competent cells: taking 50 mu L of Ms competent cells, and melting on ice; adding 5 mu L PW268 plasmid, mixing, transferring into a precooled electrorotating cup with the height of 0.1cm, and ice-bathing for 5min; the electrical conversion parameters were: voltage 1.25kV, capacitance 25 muF, resistance 1 000Ω; transferring the mixture into 1mL 7H9 complete culture medium without antibiotics, and culturing at 37 ℃ for 2H; the bacterial pellet was collected and plated onto 7H10 solid plates containing 25. Mu.g/mL kanamycin; inverted culturing at 37 ℃ for 3d until colony formation. The kana resistance gene of PW268 plasmid was amplified by PCR. Agarose gel electrophoresis showed successful amplification of the specific band, indicating that PW268 successfully transformed the Ms strain, which was designated Ms-PW268, expanded and maintained.

(3) Construction and identification of Ms esat-6 knockout mutants

Construction and identification of mutants: the procedure for the preparation of Ms-PW268 competent cells was as described in (2). 200 mu L of Ms-PW268 competent cells are taken, 500ng of sgRNA expression plasmid PW267-sgRNA is added, after uniform mixing, the mixture is transferred into a precooled electrorotating cup with the thickness of 0.2cm gap, and the mixture is subjected to ice bath for 5min, and PW268 empty plasmid with equal quality is transformed to be used as a control; the electrical conversion parameters were: voltage 2.5kV, capacitance 25 muF, resistance 1 000 omega; adding 1mL 7H9 complete culture medium without antibiotics, and resuscitating at 37 ℃ for 6H; the bacterial suspension was plated onto a plate of three antibodies 7H10 containing simultaneously 25. Mu.g/mL kanamycin (Kan), 100. Mu.g/mL bleomycin (Zeo), 50ng/mL tetracycline (ATc), and incubated in reverse at 37℃until visible colonies appeared. And (3) picking colonies to separate monoclonal, amplifying target gene sequences by PCR, and sequencing and analyzing nucleotide sequences of amplified fragments. As a result of sequence alignment analysis, as shown in FIG. 1, it was found that the 4 th to 11 th nucleotides of the target gene open reading frame of the 2-1# transformant were deleted by 8 consecutive bases. Sequencing showed that the target gene Ms esat-6 was successfully knocked out.

Obtaining of non-resistant mutant strains: 2-1# clone is selected and inoculated into 3mL of 7H9 complete culture medium, and Kan with the final concentration of 5 mug/mL is added; after the broth had been saturated, 1:100 was transferred to 3mL of 7H9 complete medium without antibiotics and incubated for an additional 5 days. Sucrose is reversely screened to lose auxiliary plasmid, after clone is grown, kan plate, zeo plate and non-resistance plate are sequentially marked, and whether resistance is lost is confirmed; the Ms esat-6 mutant that successfully lost resistance was designated rMs-. DELTA.E6. The obtained recombinant rMs-delta E6 is sent to China center for type culture Collection with the preservation number: cctccc M2021462.

Survival of rMs- ΔE6 in macrophages

Culturing mouse macrophage line RAW264.7, preparing single cell suspension, inoculating 2×10 in 24-hole cell culture plate ⁵ Culturing the individual cells overnight; the fresh complete medium without the diabody was replaced, and the concentration of Ms and rMs-. DELTA.E6 strain was adjusted to 2X 10 with the complete medium without the diabody, respectively ⁷ CFU/mL, 100. Mu.L of bacterial suspension was added to give an MOI of 10; adding the control group (NT) to the complete medium; after 2h infection, the cell supernatant was discarded and the cells were washed 3 times with pre-chilled PBS; adding a complete culture medium containing 200 mug/mL gentamicin (gentamicin), and continuously culturing for 2 hours to kill extracellular bacteria; the complete medium without the double antibody was replaced, which was then recorded as 0h after infection; 1mL of 0.025% SDS was added to lyse the cells at room temperature for 2min after the culture medium was discarded at 0h, 6h, 24h and 48h, respectively; diluting cell lysate with sterile PBS at 10-fold gradient to obtain 10 ^-1 、10 ^-2 100. Mu.L of each dilution was spread on a 7H10 plate, cultured upside down for 3 days, and the number of colonies on the plate was counted, and the result was expressed as Log ₁₀ CFU display.

As shown in FIG. 2, it was found that after rMs- ΔE6 infection of RAW264.7 cells for 6 hours, the intracellular bacterial count was reduced by 0.17Log compared with Ms ₁₀ CFU(P<0.05 A) is provided; rMs- ΔE6 is similar to the number of bacteria charged to Ms 24h after infection; when infection is prolonged to 48hBoth strains showed increased intracellular replication, but rMs-. DELTA.E6 was reduced by 0.18Log compared to Ms ₁₀ CFU(P<0.05). Knocking out esat-6 was shown to decrease the replication capacity of Ms in macrophages, indicating a further decrease in virulence of the rMs- Δe6 strain.

Protective force of the immune response induced by rMs- ΔE6 immunized mice against Mtb infection

Based on the viable count of Ms and rMs-. DELTA.E6, the strain was diluted to 1X 10 with sterile PBS ⁸ CFU/mL. 100. Mu.L of 1X 10 is taken respectively ⁷ Ms and rMs- ΔE6 of CFU were immunized subcutaneously on the back with 6-8 week old female BALB/c mice, two weeks apart, three times. 6 weeks after immunization was completed, 2X 10 was used ⁵ CFU Mtb H37Ra infects mice via respiratory tract challenge. 4 weeks after infection, spleen and lung of mice were aseptically separated, 4mL of RPMI 1640 culture solution was added, and after sufficient grinding on a 40 μm screen, the mixture was diluted with the grinding solution, and the mixture was applied to a 7H10 plate to count the number of bacteria in the organs, and the result was expressed as Log ₁₀ CFU display.

The number of the visceral bacteria is shown in FIG. 3. The lung lotus count result shows that the Ms immunized mice are reduced by 0.76Log compared with the non-immunized (UN) mice ₁₀ CFU, rMs- ΔE6 immunized mice reduced 2.68Log compared to non-immunized mice ₁₀ CFU(P<0.05 Lower 1.92Log than Ms immunized group ₁₀ CFU. In the rMs- ΔE6 immunized group, 1 mouse had lung harbor numbers reduced to undetectable. The result of the splenic lotus bacteria number shows that the rMs-delta E6 immunized mice have the splenic lotus bacteria number reduced by 0.48Log compared with the non-immunized mice ₁₀ CFU, 1.37Log lower than Ms immunization group ₁₀ CFU. Out of 6 mice in the rMs- ΔE6 immunized group, the number of splenic lotus in 3 (50%) mice was reduced to undetectable. The immune response induced by rMs-delta E6 immunized mice is shown to have protection against Mtb infection and is more protective than Ms.

The above results show that rMs- ΔE6 on the one hand has reduced viability in cells and further reduced virulence; on the other hand, the protective power of the immune response induced by the vaccine against Mtb infection is higher than that of Ms. Therefore, the strain can be used as a safer vaccine carrier for preventing or treating vaccine; can induce higher anti-Mtb protective immune response, can be used for developing immunotherapeutic agents, and has good application prospect.

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Sequence listing

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<120> recombinant Mycobacterium smegmatis strain for knocking out virulence-related gene and application thereof

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Claims (1)

1. The application of recombinant mycobacterium smegmatis strain with knock-out virulence related genes in preparing tuberculosis prevention vaccine and preparation is characterized in that: the recombinant mycobacterium smegmatis strain adopts sgRNA shown as SEQ ID NO.1 and SEQ ID NO.2 sequences to obtain a recombinant mycobacterium smegmatis strain knocking out virulence related gene Ms esat-6 by CRISPR/Cas9 technology, wherein the recombinant mycobacterium smegmatis strain is rMs-delta E6 for short, and the preservation number is: cctccc M2021462.

Images