Controllable self-destruction engineering bacterium and preparation method thereof

Abstract

The invention provides a controllable self-destruction engineering bacterium and a preparation method thereof, attenuated salmonella typhimurium is taken as a carrier, plasmid containing PD-1 gene is introduced into the body of the bacterium, and the engineering bacterium VNP-mPD-1 is obtained; the preparation method comprises the following steps: preparing engineering bacteria VNP-mPD-1-N3, enabling the surface of the engineering bacteria to contain an azide group through a glycometabolism method to obtain the engineering bacteria VNP-mPD-1-N3 with the surface containing the azide group, and covalently linking a DBCO modified photosensitizer with the azide group on the surface of the engineering bacteria through a click chemistry method to prepare the controllable self-destruction engineering bacteria VNP-mPD-1-(at) IN. The controllable self-destruction engineering bacterium prepared by the invention is good in biological safety, can generate active oxygen under laser irradiation, and has anti-tumor performance.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and particularly relates to a controllable self-destructing engineered bacterium and a preparation method thereof. Background Art

[0002] The current anti-tumor treatment methods face many challenges, such as insufficient targeting accuracy, limited treatment range, and easy recurrence. Therefore, it is particularly urgent to develop new and highly targeted anti-tumor methods.

[0003] Photodynamic therapy (PDT) activates photosensitizers by using light of a specific wavelength, and then induces the generation of cytotoxic reactive oxygen species in the lesion area to precisely kill tumor cells. This method has significant advantages such as minimally invasive, highly controllable, low toxic side effects, and not easy to produce drug resistance. The core elements of PDT include photosensitizer (PS), oxygen, and light source. After being irradiated by light, the photosensitizer absorbs photons and enters the electronically excited state. The photosensitizer in this excited state undergoes intersystem crossing to form a long-lived excited triplet state, and may release energy through fluorescence, heat release, or other photophysical processes. Subsequently, the photosensitizer in the excited triplet state promotes the generation of reactive oxygen species (ROS) through two main mechanisms: in the type I reaction, it participates in electron transfer to generate free radicals and radical ions; in the type II reaction, it transfers energy to the ground state triplet molecular oxygen ( 3 O 2 ), generating highly reactive singlet oxygen ( 1 O 2 ). Photodynamic therapy is a treatment method that induces cell necrosis or apoptosis through a direct light damage mechanism. Specifically, when photosensitizers accumulate in organelles such as mitochondria and endoplasmic reticulum, they can induce oxidative stress reactions under specific light conditions, which further promotes the apoptotic pathway of cells. In addition to direct damage, PDT-induced apoptosis can further release tumor-associated antigens (TAA), promote dendritic cell maturation and activate T cells, thereby triggering an anti-tumor immune response, which is expected to promote cancer treatment. However, how the photosensitizer reaches the tumor tissue is a key issue, and most nanoparticles cannot deliver drugs deep into the tumor tissue.

[0004] Studies have found that some tumor cells express Programmed Cell Death-Ligand 1 (PD-L1) on their surface. After binding to Programmed Death 1 (PD-1) on the surface of T cells, it can activate the PD-1 / PD-L1 immune checkpoint signaling pathway, resulting in the inhibition of T cell function (such as the loss of cytotoxicity), thereby weakening the body's immune clearance ability against tumors and ultimately promoting tumor immune escape. Related studies have shown that specific types of Salmonella (such as VNP20009) tend to accumulate in tumor tissues, with a concentration far exceeding that of normal organ tissues, up to more than 1000 times. Although the exact mechanism by which Salmonella targets tumors has not been fully elucidated, this property may be closely related to tissue blood flow velocity, the hypoxic state of the tumor microenvironment, and the chemotactic properties of Salmonella itself. Salmonella typhimurium mainly exerts its anti-tumor activity by directly inducing apoptosis of tumor cells and activating the body's immune response. However, Salmonella has been halted in phase I clinical trials due to dose toxicity issues.

[0005] Therefore, it is necessary to develop an engineered bacterium that can highly target tumor tissues, generate reactive oxygen species at the tumor tissues, thereby killing tumor cells, and has a certain degree of biological safety. Summary of the Invention

[0006] In view of the disadvantages and deficiencies of the prior art, the present invention provides a controllable self-destructing engineered bacterium and its preparation method. The controllable self-destructing engineered bacterium has strong tumor tissue targeting ability, not only has strong killing ability against tumor tissues through its own toxicity and its PDT effect, but also has good controllable self-destructing ability due to the generation of ROS, improving its biological safety in vivo.

[0007] The technical solution of the present invention is as follows: A controllable self-destructing engineered bacterium, wherein the engineered bacterium is an attenuated Salmonella typhimurium carrying a plasmid and having an azide group on its surface, the plasmid is a plasmid containing the PD-1 (Programmed Death Protein 1) gene, and the azide group is covalently linked to a photosensitizer.

[0008] The attenuated Salmonella typhimurium is attenuated Salmonella typhimurium VNP20009.

[0009] Preferably, the plasmid containing the PD-1 gene is the pCMV-Pdcd1(mouse)-3×FLAG-Neo plasmid (abbreviated as the PD-1 plasmid).

[0010] Preferably, the source of the azide group is a substance having an azide group, and the substance having an azide group is D-alanine azide. Preferably, the photosensitizer is a dibenzocyclooctyne (DBCO)-modified photosensitizer, and its structural formula is as follows: .

[0011] The preparation method of the DBCO-modified photosensitizer comprises the following steps: S1. React compound 1 with compound 2 under reflux in the presence of piperidine and p-toluenesulfonic acid overnight to obtain product 1; S2. Under ice bath conditions, uniformly mix product 1 with tetrahydrofuran and LiOH, stir and react for 40 - 80 min, acidify the obtained substance to pH 3.0 to obtain product 2; S3. Uniformly mix product 2 with bis(pyrrolidino)carbonyliminium hexafluorophosphate and N,N-diisopropylethylamine, stir and react for 20 - 60 min, then add taurine and dibenzocyclooctyne-amine (DBCO-NH 2 ), stir and react in the dark for 8 - 20 h, precipitate the obtained product in diethyl ether, and then purify to obtain the DBCO-modified photosensitizer (abbreviated as photosensitizer IN).

[0012] In step S1, the molar ratio of compound 1 to compound 2 is 1:(3 - 6), the added mass of p-toluenesulfonic acid is 1 - 3% of the total mass of compound 1 and compound 2, and the added volume ratio of piperidine to the total mass of compound 1 and compound 2 is (0.8 - 2) mL / g. Preferably, the molar ratio of compound 1 to compound 2 is 1:5.2, the added mass of p-toluenesulfonic acid is 2.87% of the total mass of compound 1 and compound 2, and the added volume ratio of piperidine to the total mass of compound 1 and compound 2 is 1.15 mL / g.

[0013] In step S2, the molar ratio of product 1 to LiOH is 1:(8 - 12), and the added volume ratio of tetrahydrofuran to the mass of product 1 is (20 - 30) mL / g. Preferably, the molar ratio of product 1 to LiOH is 1:10.2, and the added volume ratio of tetrahydrofuran to the mass of product 1 is 25 mL / g.

[0014] In step S3, the molar ratio of product 2, bis(pyrrolidino)carbonyliminium hexafluorophosphate, N,N-diisopropylethylamine, taurine, and DBCO-NH 2 is 1:(4 - 8):(4 - 8):(3 - 7):(0.8 - 1.5). Preferably, the molar ratio of product 2, bis(pyrrolidino)carbonyliminium hexafluorophosphate, N,N-diisopropylethylamine, taurine, and DBCO-NH 2 is 1:6:6:5:1.

[0015] Among them, the structural formula of Compound 1 is: ; the structural formula of Compound 2 is: .

[0016] The present invention also provides a method for preparing the controllable self-destructing engineered bacteria, comprising the following steps: (1) Preparing engineered bacteria: Using attenuated Salmonella typhimurium as a vector, introducing a plasmid containing the PD-1 gene into attenuated Salmonella typhimurium by electroporation to obtain engineered bacteria; (2) Preparing engineered bacteria with azide groups on the surface: Then co-incubating and culturing the engineered bacteria with a substance having azide groups by a method of sugar metabolism to obtain engineered bacteria with azide groups on the surface; (3) Synthesis of controllable self-destructing engineered bacteria: Covalently connecting a photosensitizer with the engineered bacteria having azide groups on the surface by a method of click chemistry to obtain the controllable self-destructing engineered bacteria.

[0017] Preferably, in step (1), the conditions of the electroporation method are: voltage 2 - 3 KV, discharge time 3 - 6 ms. More preferably, the voltage is 2 KV, 2.5 KV or 3 KV, and the discharge time is 3 ms, 4 ms, 5 ms or 6 ms.

[0018] Preferably, step (1) specifically includes: inoculating attenuated Salmonella typhimurium into an LB liquid medium for culture, centrifuging, washing, and resuspending in sterile glycerol to obtain a suspension of competent bacteria, the viable cell count in the suspension of competent bacteria is 3×10 8 ~ 8×10 8 CFU / mL, adding 0.005 - 0.05 mg of the plasmid containing the PD-1 gene to each milliliter of the suspension of competent bacteria, mixing evenly, and performing electroporation under the conditions of 2 - 3 KV and 3 - 6 ms to obtain the engineered bacteria.

[0019] Preferably, the viable cell count in the suspension of competent bacteria is 3×10 8 ~4×10 8 CFU / mL, 4.1×10 8 ~4.9×10 8 CFU / mL, 5×10 8 ~6×10 8 CFU / mL, 6.1×10 8 ~7×10 8 CFU / mL or 7.1×10 8 ~ 8×10 8 CFU / mL.

[0020] Preferably, the addition amount of the plasmid containing the PD-1 gene is 0.01 - 0.03 mg. More preferably, the addition amount of the plasmid containing the PD-1 gene is 0.01 mg, 0.02 mg or 0.03 mg.

[0021] Preferably, step (2) specifically includes: culturing the engineered bacteria obtained in step (1) to a viable bacteria count of 0.5×10 6 ~5.5×10 6 CFU / mL of the engineered bacteria solution, adding a culture medium containing a substance with an azide group to the engineered bacteria solution, and culturing to obtain engineered bacteria with an azide group on the surface.

[0022] Preferably, the viable bacteria count in the engineered bacteria solution is 0.5×10 6 ~1.5×10 6 CFU / mL, 2×10 6 ~2.5×10 6 CFU / mL, 3×10 6 ~3.5×10 6 CFU / mL or 4×10 6 ~5.5×10 6 CFU / mL.

[0023] Preferably, in step (2), the concentration of the substance with an azide group in the culture medium is 2 - 10 mM, more preferably 3 - 9 mM, and even more preferably 4 mM, 5 mM, 6 mM, 7 mM or 8 mM.

[0024] Preferably, in step (2), the volume ratio of the engineered bacteria solution to the culture medium containing the substance with an azide group is 1:10 - 1:50, more preferably 1:15 - 1:45, and even more preferably 1:20, 1:25, 1:30, 1:35 or 1:40.

[0025] Preferably, in step (2), the culture temperature is 30 - 37°C, more preferably 32 - 37°C, and even more preferably 33°C, 34°C, 35°C, 36°C, 36.5°C or 37°C.

[0026] Preferably, in step (3), the culture time is 4 - 20 h, more preferably 5 - 18 h, and even more preferably 6 h, 8 h, 10 h, 12 h, 14 h or 16 h.

[0027] Preferably, step (3) specifically includes: adding 0.1 - 0.5 mg of the DBCO-modified photosensitizer to each milliliter of the engineered bacteria with an azide group on the surface, and reacting at 30 - 37°C for 2 - 16 h to obtain the controllable self-destructing engineered bacteria.

[0028] Preferably, in step (3), 0.2 - 0.4 mg of the photosensitizer modified with DBCO is added to each milliliter of the engineered bacteria with azide groups on the surface. More preferably, the addition amount of the photosensitizer modified with DBCO is 0.25 mg, 0.3 mg or 0.35 mg.

[0029] Preferably, in step (3), the reaction temperature is 33 - 37 °C. More preferably, the reaction temperature is 34 °C, 35 °C, 36 °C, 36.5 °C or 37 °C.

[0030] Preferably, in step (3), the reaction time is 2 - 16 h, more preferably 4 - 14 h, and even more preferably 5 h, 6 h, 8 h, 10 h, 12 h or 13 h.

[0031] The present invention also provides a pharmaceutical preparation, comprising the controllable self - destructing engineered bacteria.

[0032] The present invention also provides an application of the controllable self - destructing engineered bacteria in the preparation of anti - tumor drugs.

[0033] The beneficial effects of the present invention are as follows: 1. For the controllable self - destructing engineered bacteria prepared by the present invention, the plasmid containing the PD - 1 gene is transferred into attenuated Salmonella typhimurium by electroporation. Due to the hypoxic characteristics of tumor tissues, attenuated Salmonella typhimurium will target deep into the tumor tissues, and the number of bacteria at the tumor tissues is thousands of times that in normal tissues. In addition, the plasmid released by the engineered bacteria will be re - encoded into PD - 1 protein at the tumor cells, blocking the binding of PD - L1 protein on the surface of tumor cells to PD - 1 protein on the surface of T cells and preventing tumor cells from escaping.

[0034] 2. For the controllable self - destructing engineered bacteria prepared by the present invention, azide groups are made to be contained on the surface of bacteria by means of sugar metabolism, and then the photosensitizer modified with DBCO is covalently linked to the azide groups on the surface of bacteria by click chemistry. The bacteria highly target tumor tissues and can induce apoptosis of tumor cells. In addition, under laser irradiation, the controllable self - destructing engineered bacteria will generate reactive oxygen species, thus achieving the effect of killing tumor tissues. And because reactive oxygen species can also kill bacteria, the bacteria themselves will be eliminated and die due to reactive oxygen species, achieving good biosafety. Description of the Drawings

[0035] Figure 1 Photographs of attenuated Salmonella typhimurium VNP20009 (abbreviated as VNP) and engineered bacteria VNP - mPD - 1 cultured on solid LB agar medium containing ampicillin; Figure 2 Near - infrared fluorescence imaging analysis spectrum of the controllable self - destructing engineered bacteria; Figure 3Fluorescence microscopic imaging analysis atlas of controllable self-destructing engineered bacteria; Figure 4 Cell viability and death staining analysis atlas of controllable self-destructing engineered bacteria; Figure 5 CCK8 cell activity analysis results of controllable self-destructing engineered bacteria; Figure 6 1H NMR spectrum of photosensitizer. Specific implementation manners

[0036] Next, the technical solutions in the embodiments of the present invention will be described clearly and completely. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0037] Unless otherwise specified, the reagents and bacteria involved in the embodiments of the present invention are all commercially available products and can be obtained through commercial channels.

[0038] It should be noted that the attenuated Salmonella typhimurium VNP20009 used in the present invention was purchased from the American Type Culture Collection (ATCC), with the strain number YS1646 and the ATCC number 202165.

[0039] The pCMV-Pdcd1(mouse)-3×FLAG-Neo plasmid (abbreviated as PD-1 plasmid) was purchased from Wuhan Miaoling Biotechnology Co., Ltd., and the product number is P50504. Example 1

[0040] This example provides a preparation method of controllable self-destructing engineered bacteria, including the following steps: 1. Preparation of engineered bacteria VNP-mPD-1 First, pick a single colony from the LB agar plate containing attenuated Salmonella typhimurium VNP20009, inoculate it into 8 mL of LB liquid medium, and then place it in a shaker at 37 °C for overnight culture. Inoculate the overnight culture broth into 50 mL of fresh LB liquid medium at a ratio of 1:100, and culture it in a shaker at 37 °C for 3 h to make its OD 600 value reach 0.5 - 0.6. Ice-bath the broth for 30 min, centrifuge at 4 °C and 5500 rpm for 10 min, and discard the supernatant. Resuspend and wash the precipitate twice with pre-cooled nuclease-free water (centrifuge under the same conditions). Repeat the washing twice with pre-cooled 30% sterile glycerol. Finally, resuspend it with 400 µL of pre-cooled 10% sterile glycerol, aliquot it into 50 µL / tube, and store it at -80 °C to obtain competent bacteria VNP20009 (the viable cell count is 5×10 8 ~ 6×10 8CFU / mL). The competent bacteria VNP20009 were placed on ice to melt. After melting, 1 μg of PD-1 plasmid was mixed with the competent bacteria VNP20009, and the whole process was carried out on ice. The mixture of PD-1 plasmid and competent bacteria VNP20009 was transferred to a pre-cooled electroporation cuvette (1 mm) at 4 °C and electroporated under the conditions of 2.5 KV and 4 ms. After the electroporation was completed, the engineered bacteria VNP-mPD-1 were obtained.

[0041] To verify whether the plasmid in the engineered bacteria VNP-mPD-1 was successfully transferred, the specific steps were as follows: 1 mL of pre-cooled LB liquid medium without antibiotics was added to the electroporation cuvette containing the engineered bacteria VNP-mPD-1. After gently pipetting and mixing evenly, the bacterial solution was transferred to a sterile EP tube. The bacterial solution was placed in a constant temperature shaker at 37 °C and shaken at 220 rpm for 1 h. After the culture was completed, the bacteria were collected by centrifugation at 4000 rpm for 5 min. After carefully discarding the supernatant, 100 μL of the medium was retained. The bacterial pellet was resuspended thoroughly with the remaining medium and evenly spread on the surface of LB solid medium containing ampicillin (Amp, 100 μg / mL), and then cultured in an inverted position in a constant temperature incubator at 37 °C for 16 h.

[0042] 2. Preparation of engineered bacteria VNP-mPD-1-N with azide groups on the surface 3 When the obtained engineered bacteria VNP-mPD-1 were cultured until their OD 600 was 0.3 - 0.45, the bacterial solution of the engineered bacteria (the viable cell count was 3×10 6 ~3.5×10 6 CFU / mL) was obtained. According to the volume ratio of 1:20, LB liquid medium (containing 5 mM D-alanine azide) was added to the bacterial solution of the engineered bacteria for culture. The culture temperature was 37 °C and the culture time was 12 h, and the bacterial solution VNP-mPD-1-N of the engineered bacteria with azide groups on the surface was obtained. 3 .

[0043] 3. Synthesis of photosensitizer IN The chemical structural formula of photosensitizer IN is: .

[0044] The synthesis steps of photosensitizer IN are as follows: S1. Add compound 1 (0.50 g, 0.81 mmol) to a flask equipped with a Dean-Stark trap, which contains 25 mL of toluene. Add compound 2 (1.24 g, 4.24 mmol), piperidine (2 mL), and 4-methylbenzenesulfonic acid (50 mg) to the solution, and heat it overnight under reflux conditions. Then evaporate the solvent under reduced pressure, add 100 mL of water to the residue, and extract with 300 mL of CHCl 3 Extract, combine the organic phases, and dry the organic phases over Na 2 SO 4 Then remove the solvent under vacuum. Then purify by silica gel column chromatography (CH 2 Cl 2 / PE 5:1) to obtain product 1 (0.57 g, 60%) as a green powder.

[0045] Among them, the structural formula of compound 1 is: ; the structural formula of compound 2 is: .

[0046] The structural formula of product 1 is: .

[0047] S2. Add product 1 (0.48 g, 0.41 mmol) and tetrahydrofuran (THF) (12 mL) to a flask to make a solution, cool the resulting solution to 0 °C in an ice bath, add a solution of LiOH (0.10 g, 4.18 mmol) in water (4 mL) at 0 °C, stir and react for 1 h, and then warm up to room temperature. Then acidify to pH 3.0 with a saturated KHSO 4 solution, extract with EtOAc (ethyl acetate, 300 mL), combine the organic phases, dehydrate using MgSO 4 and then concentrate by vacuum distillation to obtain product 2 (0.40 g, 92%).

[0048] Among them, the structural formula of product 2 is: .

[0049] S3. Dissolve product 2 (10 mg, 0.01 mmol) in dimethyl sulfoxide (5 mL) to make a solution, then add bis(pyrrolidin-1-yl)methanone N-succinimidyl carbonate (HSPyU) (25 mg, 0.06 mmol) and N,N-diisopropylethylamine (DIPEA) (10 μL, 0.06 mmol) to this solution, stir at room temperature for 30 min, then add taurine (6 mg, 0.05 mmol) and dibenzocyclooctyne-amine (DBCO-NH 2)(4 mg, 0.01 mmol), stirred for 12 h in the dark. Then the crude product was precipitated in diethyl ether to obtain a green solid (10 mg, 61%), and further purified by reverse-phase high performance liquid chromatography (RP-HPLC) to obtain the DBCO-modified photosensitizer (abbreviated as photosensitizer IN, yield 61%). 1 H NMR: (600 MHz, DMSO-d 6 , δ ppm): 8.15 (m, 4H), 7.63 (m, 2H), 7.52 (m, 2H), 7.45 (d, 1H, J = 7.9 Hz), 7.23 - 7.33 (m, 5H), 7.20 (d, 1H, J = 7.8 Hz), 7.07 (m, 1H), 6.97 (s, 2H), 6.72 (m, 4H), 4.64 (s, 2H), 4.32 (m, 6H), 4.30 (s, 2H), 3.91 - 3.99 (m, 6H), 3.78 (t, 2H, J = 7.1 Hz), 3.19 - 3.27 (m, 6H), 2.71 (t, 2H, J = 7.3 Hz), 2.36 (s, 3H), 2.08 (s, 6H), 1.46 (s, 3H), and 1.44 (s, 3H). The 1H NMR spectrum of photosensitizer IN is shown as Figure 6 shown.

[0050] 4. Synthesis of controllable self-destructing engineered bacteria VNP-mPD-1@IN Photosensitizer IN was added to the bacterial solution of engineered bacteria with azide groups on the surface. 0.3 mg of photosensitizer IN was added to each milliliter of the bacterial solution, and then the reaction was carried out at 37 °C for 12 h. After the reaction was complete, it was washed with PBS and centrifuged to remove the supernatant to obtain the controllable self-destructing engineered bacteria VNP-mPD-1@IN.

[0051] I. Verification of the controllable self-destructing engineered bacteria prepared in the present invention 1. Photoanalysis of attenuated Salmonella typhimurium VNP20009 and VNP-mPD-1 cultured on solid LB agar medium containing ampicillin (Amp): The construction of VNP-mPD-1 engineered bacteria was verified by the ampicillin resistance plate screening method. Based on the experimental design principle, after the successful transformation of the PD-1 plasmid with Amp resistance, the strain will acquire Amp resistance. VNP20009 (abbreviated as VNP) and VNP-mPD-1 engineered bacteria were simultaneously inoculated on LB solid medium containing Amp (100 µg / mL) and observed after inverted culture at 37 °C for 16 hours, as Figure 1As shown, no colony formation was observed in the VNP control group, while typical single colony growth was presented in the VNP-mPD-1 group, indicating the successful construction of VNP-mPD-1.

[0052] 2. Near-infrared fluorescence imaging analysis: Since the photosensitizer IN emits fluorescence in the second near-infrared region, but the engineered bacteria VNP-mPD-1 alone does not emit fluorescence, the controllable self-destructing engineered bacteria VNP-mPD-1@IN prepared by the present invention was subjected to near-infrared fluorescence imaging analysis. The results are as Figure 2 shown. Fluorescence appeared in VNP-mPD-1@IN, indicating that IN was loaded on the surface of VNP-mPD-1, proving the successful construction of the controllable self-destructing engineered bacteria.

[0053] 3. Fluorescence microscopy imaging analysis: After the successful synthesis of the controllable self-destructing engineered bacteria VNP-mPD-1@IN, since the photosensitizer IN emits fluorescence under the microscope, a fluorescence microscope was used to further observe IN loaded on the surface of the bacteria. As Figure 3 shown, fluorescence was found on the surface of the bacteria, indicating its successful synthesis.

[0054] The above research results all indicate the successful synthesis of the controllable self-destructing engineered bacteria.

[0055] II. Antitumor performance of the controllable self-destructing engineered bacteria prepared by the present invention 1. Cell viability and cytotoxicity staining analysis Cancer cells B16F10 were respectively inoculated into 96-well plates and cultured for 12 h, with the number of cells inoculated in each well being 5×10 3 cells; fresh sample medium (RPMI1640 complete medium containing 10% fetal bovine serum) containing the anticancer drug of the controllable self-destructing engineered bacteria VNP-mPD-1@IN was added to the wells, and cultured in an incubator at 37 °C for 4 h; after the culture was completed, irradiated with 808 nm laser at 1 W / cm 2 for 5 min; after the irradiation was completed, a live / dead cell staining reagent was used to observe the death of cancer cells. The results are as Figure 4 shown. All cancer cells died, indicating that the controllable self-destructing engineered bacteria prepared by the present invention have good tumor cell killing ability.

[0056] 2. CCK8 cell viability analysis Cancer cells B16F10 were respectively inoculated into 96-well plates and cultured for 12 h, with the number of cells inoculated in each well being 5×10 3Individuals were randomly divided into a control group and an experimental group. In the wells of the experimental group, fresh sample medium containing the anti-cancer drug of the controllable self-destructing engineered bacteria VNP-mPD-1@IN (RPMI1640 complete medium containing 10% fetal bovine serum) was added. In the wells of the control group, an equal amount of fresh sample medium (RPMI1640 complete medium containing 10% fetal bovine serum) was added. They were cultured in an incubator at 37 °C for 4 h. After the culture was completed, they were irradiated with 808 nm laser at 1 W / cm 2 for 5 min. After the irradiation was completed, the CCK8 reagent was used to observe the death of cancer cells. The results are as Figure 5 shown. The survival rate of cancer cells in the control group was 100%, and the survival rate of cancer cells in the experimental group was 16% (compared with the control group, ), indicating that the experimental group could kill 84% of tumor cells. It shows that the anti-cancer drug of the controllable self-destructing engineered bacteria prepared by the present invention has significant tumor cell killing ability.

[0057] Finally, it should be noted that the above are only the preferred embodiments of the present invention and are not used to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A controllable self-destructive engineering bacteria, characterized in that: The engineered bacteria are attenuated Salmonella typhimurium carrying a plasmid and containing an azide group on the surface. The plasmid is a plasmid containing a PD-1 gene, and the azide group is covalently linked to a photosensitizer.

2. The controllable self-destructive engineering bacteria according to claim 1, characterized in that: The plasmid containing the PD-1 gene is pCMV-Pdcd1(mouse)-3×FLAG-Neo plasmid.

3. The controllable self-destructive engineering bacteria according to claim 1, characterized in that: The source of the azide group is a substance having an azide group, and the substance having an azide group is D-alanine azide.

4. The controllable self-destructive engineering bacteria according to claim 1, characterized in that: The photosensitizer is a dibenzocyclooctyne modified photosensitizer, and its structural formula is: 。 5. The controllable self-destructive engineering bacteria according to claim 4, characterized in that: The preparation method of the dibenzocyclooctyne-modified photosensitizer comprises the following steps: S1, reflux compound 1 and compound 2 overnight to obtain product 1; S2, stirring the product 1 and LiOH for 40-80 min in an ice bath, and acidifying the obtained substance to pH 3.0 to obtain the product 2; S3, the product 2 is mixed evenly with dipyrrolidino (N-succinimidyloxy) carbonyl hexafluorophosphate and N, N-diisopropylethylamine, and stirred for reaction for 20 to 60 minutes, and then taurine and dibenzocyclooctyne-amino are added, and the reaction is stirred for 8 to 20 hours in the dark, and the obtained product is precipitated in diethyl ether, and then purified to obtain a photosensitizer modified with dibenzocyclooctyne; In step S1, the molar ratio of compound 1 to compound 2 is 1:(3-6); in step S2, the molar ratio of product 1 to LiOH is 1:(8-12); in step S3, the molar ratio of product 2, dipyrrolidino (N-succinimidyloxy) carbonyl hexafluorophosphate, N,N-diisopropylethylamine, taurine and dibenzocyclooctyne-amino is 1:(4-8):(4-8):(3-7):(0.8-1.5); Wherein, the structural formula of compound 1 is: ; The structural formula of compound 2 is: .

6. A method for preparing the controllable self-destructive engineering bacteria according to any one of claims 1 to 5, characterized in that: The following steps are involved: (1) Preparation of engineered bacteria: Using attenuated Salmonella typhimurium as a vector, a plasmid containing the PD-1 gene is introduced into the attenuated Salmonella typhimurium by electroporation to obtain an engineered bacterium; (2) Preparing engineered bacteria with azide groups on their surfaces: co-incubating the engineered bacteria with substances having azide groups through a sugar metabolism method to obtain engineered bacteria with azide groups on their surfaces; (3) Synthesis of controllable self-destructive engineered bacteria: The photosensitizer is covalently linked to the engineered bacteria containing azide groups on the surface through click chemistry to obtain controllable self-destructive engineered bacteria.

7. The method for preparing the controllable self-destructive engineering bacteria according to claim 6, characterized in that: Step (1) specifically comprises: inoculating attenuated Salmonella typhimurium into LB liquid medium for cultivation, centrifuging, washing, and resuspending in sterile glycerol to obtain a competent bacterial resuspension, wherein the number of viable bacteria in the competent bacterial resuspension is 3×10 8 ~8×10 8 CFU / mL, add 0.005~0.05 mg of plasmid containing PD-1 gene to each ml of competent bacterial resuspension, mix well, and perform electroporation at 2~3 KV and 3~6ms to obtain engineered bacteria.

8. The method for preparing the controllable self-destructive engineering bacteria according to claim 6, characterized in that: Step (2) specifically comprises: culturing the engineered bacteria obtained in step (1) to a viable bacterial count of 0.5×10 6 ~5.5×10 6 CFU / mL of an engineered bacterial liquid, add a culture medium containing a substance having an azide group to the engineered bacterial liquid at a volume ratio of 1:10-1:50, the concentration of the substance having an azide group in the culture medium is 2-10 mM, and then culture at 30-37°C for 4-20h to obtain engineered bacteria containing azide groups on the surface.

9. The method for preparing the controllable self-destructive engineering bacteria according to claim 6, characterized in that: Step (3) specifically comprises: adding 0.1-0.5 mg of DBCO-modified photosensitizer to each milliliter of engineered bacteria containing azide groups on the surface, and reacting at 30-37° C. for 2-16 hours to obtain controllable self-destructive engineered bacteria.

10. Use of the controllable self-destructive engineering bacteria as claimed in claim 1 in the preparation of anti-tumor drugs.

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