CN113322221A - Recombinant mycobacterium smegmatis strain with virulence related gene knocked out and application thereof - Google Patents

Abstract

The invention relates to a recombinant mycobacterium smegmatis strain with a virulence related gene Ms esat-6 knocked out, which is rMs-delta E6 for short, and the preservation number is as follows: CCTCC 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- Δ E6 strain had decreased survival in macrophages, and the immune response induced by rMs- Δ E6 was more protective against Mtb infection than Ms. Therefore, rMs- Δ E6 can be used as a safer vaccine carrier and a carrier for a preventive or therapeutic vaccine; also has the characteristic of inducing protective immune response against Mtb, can be used for the development of immunotherapeutic agents, and thus has good application prospect.

Description

Recombinant mycobacterium smegmatis strain with virulence related gene knocked out and application thereof

Technical Field

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

Background

Current status of tuberculosis prevalence

Tuberculosis (TB) caused by Mycobacterium Tuberculosis (Mtb) infection remains a serious infectious disease worldwide. According to the WHO estimation, 1000 thousands of new TB people are sent globally in 2019, and about 140 thousands of new TB people die from TB. Currently, the population worldwide with latent tuberculosis infection (LTBI) is nearly 20 billion, and 5% -10% of latent infected people can be transformed into active TB, which is the main source of adult TB. The number of new TB people in China is only inferior to that in India and second place around the world (WHO. Global tuberculosis report.2020.). TB is one of the major diseases which are mainly controlled in China.

Current research situation for tuberculosis vaccine

BCG (Bacillus Calmette-Guerin vaccine) is an attenuated live vaccine obtained by inoculating Mycobacterium bovis (Mycobacterium bovis) into an artificial culture medium for 13 years and continuously passaging for 230 generations, and is the only vaccine currently used for preventing TB. BCG can effectively prevent serious TB of children, but the overall protection efficiency on adult TB is 0-80%. BCG has good safety, but as an attenuated live vaccine, it cannot be used for vaccination of people with low immunity (Fatima, S., et al, Tuberculosis vaccine: A journey from BCG to present. Life Sci,2020.252: p.117594.). Studies have shown that BCG immunoprotection is reduced because it loses some strain-specific antigens during passage, which results in imperfect immunoprotection. In addition, The overall protection effect of BCG is not ideal due to various factors such as The influence of mycobacteria in The environment, The difference of vaccine strains in different areas, The genetic diversity of individuals in The population and The like (Schrager, L.K., et al, The status of The tuberculosis vaccine maintenance. Lancet infection Dis,2020.20(3): p.e28-e 37). Therefore, the development of new vaccines is crucial for the prevention and control of TB epidemic.

In recent years, there has been much research on new TB vaccines, including screening of a range of candidate vaccines and different vaccine immunization strategies. There are 14 TB vaccines reported by WHO that have entered clinical trials in 2020, including viral vector vaccines, protein/adjuvant subunit vaccines, mycobacterial lysates, and live mycobacterial vaccines. Improvement of Mtb or BCG and The like by genetic engineering methods to reduce The virulence of strains, or to improve The immunoprotection efficacy of attenuated/avirulent strains of mycobacteria is one of The approaches in The construction and research strategies of new TB vaccines (Schrager, l.k., et al, The status of vaccine maintenance. lancet infection Dis,2020.20(3): p.e28-e 37).

Recombinant live mycobacterial vaccines are one of the important directions for the research of novel TB vaccines. Currently, most mycobacterial vectors for TB vaccine research include attenuated Mtb, BCG, Mycobacterium vaccae, Mycobacterium smegmatis (Ms), and the like. Compared with BCG, Ms is non-pathogenic mycobacteria, contains VII-type secretion system similar to Mtb, and has 10 times faster growth rate than BCG, high yield of expressed foreign protein, and Mtb-related protein expressed in Ms is almost completely consistent with the biochemical and immunological properties of natural protein (New Gateway vector and Expression protocol for the disease and Expression vector Expression in Mycobacterium smegmatis. protein Expression and Purification,2008,57(1): 81-87.). Thus, Ms is an excellent TB vaccine vector. Ms has been successfully used in the united States as a vaccine adjuvant for the prevention and treatment of TB (Mycobacterium tuberculosis vaccine. united States Patent 20060029620.). The Recombinant Mycobacterium smegmatis (Recombinant Ms, rMs) live vaccine can be used in combination with drugs for the treatment of Mtb infection (Populus spring, Hoyonglin, Xulei, Zhangli, Yizhengjun, Lina, Wangcongwei, Vermilion.) comparison of Recombinant Mycobacterium smegmatis and microcard in the immunotherapy of tuberculosis in mice. journal of immunology, 2009,25(06): 685-688). Preliminary studies on subjects found that Ms and rMs (rMs-AEL, patent No. ZL 201010189471.3) overexpressing the fusion proteins Ag85B and ESAT-6 have a protective effect against Mtb and prevent the recurrence of latent infection to some extent (Wang P, Wang L, Zhang W, Bai Y, Kang J, Hao Y, Luo T, Shi C, Xu Z. immunological infection efficacy of recombinant Mycobacterium smegmatis expressing Ag85B-ES 6 fusion protein and expression of tissue fusion protein. hum vaccine. 2014; 10(1): 150. Bu 158), the rMs-infected macrophages induce increased TNF-alpha secretion, induce enhanced phagocytic function in cells, and contribute to an increase in host anti-infectious innate immune response (Yanbifha, rotifer, Wangping, et al., Effect of recombinant Mycobacterium smegmatis Ag85B-ESAT6-rMs on macrophage function in mice. J. cell & molecular immunology 2012.28(04): 361-. The study at home and abroad including the subject group proves that Ms as a live vector vaccine and an immunomodulator has safety and reliability. Thus, Ms can be used in the development of prophylactic and therapeutic vaccines and immunotherapeutics.

Ms is mainly parasitic bacteria of sapropel and can also be planted in some parts of human body, and the pathogenicity is weak. However, under certain conditions Ms may also cause disease including: lymphadenitis, osteomyelitis, cellulitis, wound infection, pyogenic granulomatous inflammation, etc. (Brown-Elliott BA, Phlley JV, Rapid growing mycobacteriosis, Microbiol Spectr,2017,5(1): 1-19; Tianjin next, Ma prefab, Leni. Mycobacterium smegmatis skin soft tissue infection 1. J. infection and chemotherapy in China, 2020,20(06): 681) 682.). Therefore, the pathogenicity of the Ms is further reduced, the safety of the Ms is improved, and the application of the Ms in TB prevention and treatment vaccines and immunotherapeutics is facilitated.

Mycobacterium tuberculosis ESAT-6 research progress

The total length of the coding gene of the 6kDa early secretory target antigen (ESAT-6) is 288bp, and the coding gene is a protein consisting of 95 amino acids. ESAT-6 is present only in Mtb and M.bovis strains, but is absent from the BCG genome (Groscel, M.I., et al, Recombinant BCG Expressing ESX-1 of Mycobacterium marinum combinations Low Virus with cytolytic Immune signalling and Improved TB protection. cell Rep,2017.18(11): p.2752-2765.). Research shows that ESAT-6 can directly form holes on vacuole membranes containing mycobacteria, promote the escape of the mycobacteria from vacuoles and the diffusion of the mycobacteria among cells, provide physiological basis for intracellular colonization and replication of toxic mycobacteria, and is considered to be related to the virulence of Mtb (Smith, J.J., J.Manoranjan, M.Pan, et al, Evidence for pore formation in host cells, by ESX-1-segmented ESAT-6and bits roll in Mycobacterium marinum vaccine, 2008.76(12): 5478-87.).

In the mechanism study of ESAT-6 promoting the intracellular survival of bacteria, it was found that ESAT-6 induces the increase of the level of autophagy marker molecule LC3 II and simultaneously inhibits autophagy flow by activating mTOR (Dong, H., et al, ESAT6 inhibitors autophagy flux and proteins BCG promotion through MTOR. biochem Biophys Res Commun,2016.477(2): p.195-201.). Early studies found that the ESAT-6-CFP10 fusion protein can inhibit the formation of autophagosome induced by Mtb infection, and promote the intracellular survival of Mtb (Shichang hong, Maofeng peak, Zhaoyong, et al., Experimental study that ESAT 6and CFP10 fusion proteins inhibit the formation of macrophage autophagosome. China zoonosis report. 2011.27(05): 414-417.). In addition, recombinant ESAT-6 proteins inhibit the formation of autophagosome by down-regulating microRNA-30a, resulting in increased intracellular viability of mycobacteria (Behura, A. Mishra, S. Chugh, et al., ESAT-6 modulated calcium-induced autophagy microRNA-30a in mycobacteria induced macrophages. J Infect,2019.79(2): 139-152.). The above studies indicate that ESAT-6 promotes survival of mycobacteria within macrophages by interfering with different stages of autophagy. ESAT-6 is therefore considered to be a virulence factor for Mtb.

The amino acid sequence research of ESAT-6 in different mycobacteria finds that the amino acid sequence of ESAT-6 is highly homologous among pathogenic mycobacteria (Mtb, mycobacterium bovis) and the similarity is as high as 90%. Ms ESAT-6 is a homologous gene of Mtb ESAT-6, and the amino acid sequence similarity of the two encoded proteins is 72% (Peng, X., G.Jiang, W.Liu, et al, mutation of differential pore-forming activities of ESAT-6 proteins from Mycobacterium tuberculosis, FEBS Lett,2016.590(4): 509-19.). Therefore, by blocking the strategy of Ms esat-6, rMs with higher safety 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 proteins are regularly clustered, widely exist in genomes of bacteria and archaea, and serve as an acquired immune system to help bacteria/archaea resist interference of bacteriophage and exogenous DNA. The CRISPR/Cas9 system plays a role in gene editing by the following steps: after the guide RNA (sgRNA) binds to Cas9, the Cas9 protein begins to search for a pro-spacer adjacent motif (PAM) on the genome; cas9-sgRNA partially untwists a DNA double strand from a PAM end, so that the sgRNA enters into a RNA-DNA heteroduplex structure which is complementary with the DNA; then the Cas9 protein cleaves the DNA double strand, initiating a DNA double strand break; initiate homologous recombination or non-homologous end-joining repair. Non-homologous end joining is an error-prone repair mechanism, usually involving insertions or deletions at DNA breaks, resulting in gene knock-out, enabling targeted editing of genes (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). CRISPR/Cas systems have been successfully used for the construction of various mycobacterial knockout strains such as Mtb and Ms (Yan MY, Li SS, Ding XY, Guo XP, Jin Q, Sun YC.A CRISPR-amplified nonhomologus End-Joining strand for Efficient Genome Editing in Mycobacterium tuberculosis. 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 response in mycobacterial use of an orthogonal CRISPR interference plant, 2017.2: 16274.). At present, no Ms esat-6 knockout strain and relevant reports of the application of the strain exist.

Disclosure of Invention

The invention provides a recombinant mycobacterium smegmatis strain with a knockout virulence related gene and application thereof.

The invention is realized by the following technical scheme:

the invention is based on Ms MC²Ms esat-6 gene sequence of 155 strain, designing and synthesizing sgRNA sequences as SEQ ID NO.1 and SEQ ID NO.2, constructing a plasmid vector by using the sequences, knocking out Ms esat-6 gene from Ms genome by CRISPR/Cas9 technology, and obtaining rMs-delta E6 through screening and identification. The recombinant rMs-delta E6 is sent to China center for type culture Collection with the collection number as follows: CCTCC M2021462. And Ms MC²155 strain, rMs- Δ E6 showed a decreased survival rate in macrophages and induced immune responses that were more protective against Mtb infection than Ms.

The recombinant mycobacterium smegmatis strain rMs-delta E6 is used for preparing vaccines and preparations for preventing and/or treating tuberculosis.

The sgRNA target sequences of sequence tables SEQ ID NO.1 and SEQ ID NO.2 are designed on the Ms esat-6 gene sequence according to PAM locus (ATGGAAT), Ms esat-6 is successfully knocked out by a CRISPR/Cas9 system, and the obtained collection number is as follows: rMs- Δ E6 of CCTCC M2021462. rMs- Δ E6 on the one hand reduced viability in the cell, further attenuated virulence; on the other hand, it induced immune responses with greater protection against Mtb infection than Ms. Therefore, the strain can be used as a safer vaccine carrier for a preventive or therapeutic vaccine; can also induce the characteristic of higher Mtb-resistant protective immune response, can be used for the development of immunotherapeutic agents, and thus has good application prospect.

Drawings

FIG. 1 is a diagram showing the determination results of rMs- Δ E6 sequence.

FIG. 2 is a schematic representation of the survival of rMs- Δ E6 within macrophages.

FIG. 3 is a graph showing the number of visceral lotus bacteria in mice immunized with rMs- Δ E6 after Mtb infection.

Proof of deposit

Categorical naming

Mycobacterium smegmatis rMs-DELTA E6

Latin character name

Mycobacteria smegmatis rMs-ΔE6

China center for type culture Collection under the name of Collection entity

Address:

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

E-mail:cctcc@whu.edu.cn

Telephone: 027-68754052

Date of storage

2021, 4 months and 27 days

Accession number

CCTCC M 2021462

Detailed Description

Construction and identification of strain rMs-delta E6

(1) sgRNA target sequence design and sgRNA expression vector construction

The Ms esat-6(MSMEG _0066) gene sequence was obtained in the mycobacterial genome database (https:// mycobrown. Epfl. ch. /), and the PAM sequence was looked up in this gene sequence according to the literature (Rock, J.M., F.F.Hopkins, A.Chavez, et al., Programmmable transgenic expression in mycobacterial use an orthogonal CRISPR internal reference plant. Nat. Microbiol., 2017.2: 16274.). A PAM locus 'ATGGAAT' is selected to design sgRNA. Bbs I enzyme cutting sites are added at the 5' ends of the Forward (Forward) sequence and the Reverse (Reverse) sequence of the sgRNA target DNA sequence respectively to synthesize single-chain Oligo DNA sequences sgRNA Forward and sgRNA Reverse respectively, and 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 having a cohesive end. The annealing system is 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 6.5.5. mu.L, and the total volume is 10. mu.L. The annealing program of the PCR instrument is 30min at 37 ℃; 5min at 95 ℃; 30min at 95 ℃, per cycle at-1 ℃, ramp 0.1 ℃/s, +60s per cycle; 40min at 65 ℃, per cycle at-1 ℃, ramp 0.1 ℃/s, +60s per cycle; infinity at 20 ℃.

The linearized PW267 plasmid was digested with Bbs I and ligated to the annealed double-stranded Oligo DNA, and the ligation product was transformed into E.coli (E.coli) DH 5. alpha. competent cells and screened with kanamycin-resistant plates. And selecting the clone identified as positive by PCR, inoculating the clone to an LB liquid culture medium, carrying out shaking culture at 37 ℃ overnight, and extracting plasmid DNA. Positive recombinants were sequenced. Sequencing results show that a target DNA sequence is successfully cloned into a PW267 plasmid, and the successful construction of an sgRNA expression vector is shown and is named as PW 267-sgRNA.

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

The Ms competent cells were prepared by the following steps: frozen Ms were inoculated in 5mL of 7H9 medium and incubated to saturation at 37 ℃. The saturated culture was transferred into a 250mL Erlenmeyer flask containing 50mL of 7H9 complete medium and cultured with shaking at 80rpm to OD₆₀₀0.8. Carrying out ice bath on the bacterial liquid for 1 h; centrifuging at 8000 rpm at 4 deg.C for 10min, collecting thallus, and removing supernatant; resuspend and wash with pre-cooled 10% glycerol in culture volumes of 1/2, 1/4, 1/8, respectively, and finally resuspend the bacterial pellet with 1/100 culture volume 10% glycerol solution and store at-70 ℃ until use.

Electrically transforming Ms competent cells with PW268 plasmid: taking 50 mu L of Ms competent cells, and thawing on ice; adding 5 μ L PW268 plasmid, mixing, transferring into 0.1cm gap precooled electric rotary cup, and ice-cooling for 5 min; the electrical conversion parameters were: the voltage is 1.25kV, the capacitance is 25 muF, and the resistance is 1000 omega; transferring the mixture into 1mL of 7H9 complete medium without antibiotics, and culturing at 37 ℃ for 2H; collecting the bacterial precipitation, and coating the bacterial precipitation on a 7H10 solid culture plate containing 25 mu g/mL kanamycin; and (4) carrying out inverted culture at 37 ℃ for 3d until colonies are generated. The kanamycin resistance gene of the PW268 plasmid was PCR amplified. Agarose gel electrophoresis shows that a specific band is successfully amplified, which indicates that the Ms strain is successfully transformed by the PW268, is named as Ms-PW268, and is subjected to amplification culture and seed preservation.

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

Construction and identification of mutant strains: the preparation of Ms-PW268 competent cells was performed as described in (2). Taking 200 mu L Ms-PW268 competent cells, adding 500ng of sgRNA expression plasmid PW267-sgRNA, uniformly mixing, transferring to a precooled electric transfer cup with 0.2cm gap, carrying out ice bath for 5min, and converting PW268 empty plasmid with equal mass as a reference; the electrical conversion parameters were: the voltage is 2.5kV, the capacitance is 25 muF, and the resistance is 1000 omega; adding 1mL of 7H9 complete culture medium without antibiotics, and recovering for 6H at 37 ℃; the bacterial suspension was spread on a 7H10 triple-resistant plate containing 25. mu.g/mL kanamycin (Kan), 100. mu.g/mL bleomycin (Zeo) and 50ng/mL tetracycline (ATc), and the plate was cultured in an inverted state at 37 ℃ until a visible colony appeared. Selecting colony to separate single clone, PCR amplifying target gene sequence, sequencing and analyzing the nucleotide sequence of amplified segment. The results of the sequence alignment analysis are shown in FIG. 1, and it is found that the 4 th to 11 th nucleotides of the target gene open reading frame of the 2-1# transformant are subjected to continuous 8-base deletion. Sequence determination shows that the target gene Ms esat-6 is successfully knocked out.

Obtaining of non-resistant mutants: 2-1# clone was picked and inoculated into 3mL of 7H9 complete medium, Kan was added to a final concentration of 5. mu.g/mL; after the bacterial liquid is cultured to saturation, the bacterial liquid is transferred to 3mL of 7H9 complete medium culture medium without antibiotics in a ratio of 1:100, and the culture is carried out for 5 days. Reversely screening lost helper plasmids by using sucrose, sequentially marking a Kan plate, a Zeo plate and a non-resistance plate after growing clones, and determining whether resistance is lost; the Ms esat-6 mutant, which succeeded in losing resistance, was named rMs- Δ E6. The obtained recombinant rMs-delta E6 is sent to China center for type culture Collection with the collection number as follows: CCTCC M2021462.

Survival of rMs- Δ E6 in macrophages

Culturing mouse macrophage line RAW264.7, preparing single cell suspension, inoculating 2 × 10 in 24-well cell culture plate⁵Culturing the cells overnight; the medium was replaced with fresh complete medium containing no diabody, and the concentrations of Ms and rMs-. DELTA.E 6 strain were adjusted to 2X 10, respectively, using complete medium containing no diabody⁷CFU/mL, 100. mu.L of bacterial suspension was added to give an MOI of 10; control (NT) was added to complete medium; after 2h of infection, the cell supernatant was discarded and the cells were washed 3 times with pre-cooled PBS; adding complete culture containing 200 ug/mL gentamicin (gentamicin)Culturing the medium for 2h to kill extracellular bacteria; replacing the complete culture medium without the double antibody, and recording as 0h after infection; discarding the culture medium at 0h, 6h, 24h and 48h after infection, adding 1mL of 0.025% SDS, and lysing the cells at room temperature for 2 min; the cell lysate was diluted 10-fold in sterile PBS and 10 samples were taken^-1、10^-2Each 100. mu.L of the dilution was applied to a 7H10 plate, cultured for 3 days in an inverted state, and the number of colonies on the plate was counted to obtain the Log₁₀And (5) displaying by a CFU.

As shown in FIG. 2, the intracellular survival results show that the number of intracellular loaded bacteria is reduced by 0.17Log compared with Ms after rMs-delta E6 infects RAW264.7 cells for 6h₁₀CFU(P<0.05); rMs-delta E6 were similar in number of charged bacteria to Ms 24h after infection; when infection was prolonged to 48h, both strains increased intracellular replication, but rMs- Δ E6 decreased Ms by 0.18Log₁₀CFU(P<0.05). Knock-out of esat-6 was shown to decrease the ability of Ms to replicate within macrophages, indicating a further decrease in virulence of the rMs- Δ E6 strain.

Protection of immune responses induced by mice immunized with rMs- Δ E6 against Mtb infection

According to the results of viable count of Ms and rMs- Δ E6, the strains were diluted to 1 × 10 with sterile PBS⁸CFU/mL. Respectively taking 100 mu L of the extract, namely 1 multiplied by 10⁷Ms and rMs-delta E6 from CFU were immunized subcutaneously in the back of 6-8 week-old female BALB/c mice, two weeks apart, three times. 6 weeks after completion of immunization, 2X 10⁵CFU of Mtb H37Ra infected mice by respiratory tract challenge. 4 weeks after infection, the spleen and lung of the mouse were aseptically separated, 4mL of RPMI 1640 culture medium was added, the mixture was sufficiently ground on a 40 μm mesh screen, the ground solution was diluted in multiple proportions, the mixture was spread on a 7H10 plate, and the number of the live organs was counted, and the Log of the results was obtained₁₀And (5) displaying by a CFU.

The number of organs and their parasites is shown in FIG. 3. The number of pulmonary lotus bacteria results show that the Ms immunized mice are reduced by 0.76Log compared with the UN-immunized (UN) mice₁₀CFU, rMs-delta E6 immunized mice reduced 2.68Log compared to non-immunized mice₁₀CFU(P<0.05), 1.92Log is reduced compared with Ms immune group₁₀And (4) CFU. rMs- Δ E6 in the immunized group, the number of pneumococci in 1 mouse decreased to no level. The results of the number of splenic lotus bacteria show that the number of splenic lotus bacteria of rMs-delta E6 immunized mice is reduced by 0.48 compared with that of non-immunized miceLog₁₀CFU, 1.37Log lower than Ms immune group₁₀And (4) CFU. rMs- Δ E6 of 6 mice in the immunized group, 3 (50%) had a reduction in spleen lotus count to no detection. It was shown that rMs- Δ E6 immunized mice induced an immune response with protection against Mtb infection that was superior to Ms in protective effect.

The above results show that rMs- Δ E6, on the one hand, has reduced viability in cells and further attenuated virulence; on the other hand, it induced immune responses with greater protection against Mtb infection than Ms. Therefore, the strain can be used as a safer vaccine carrier for a preventive or therapeutic vaccine; can also induce the characteristic of higher Mtb-resistant protective immune response, can be used for the development of immunotherapeutic agents, and thus has good application prospect.

Sequence listing

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Claims (2)

1. A recombinant Mycobacterium smegmatis strain with a virulence related gene Ms esat-6 knocked out is called rMs-delta E6, and the preservation number is as follows: CCTCC M2021462.

2. Use of the recombinant mycobacterium smegmatis strain rMs- Δ E6 of claim 1 for the preparation of vaccines and formulations for the prevention and/or treatment of tuberculosis.

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