Bionic T cell secreting type engineering bacterium lung targeted delivery system based on respiratory tract atomization and construction method and application of bionic T cell secreting type engineering bacterium lung targeted delivery system

Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to a bionic T cell secreting type engineering bacterium lung targeted delivery system based on respiratory tract atomization and a construction method and application. The delivery system comprises engineering bacteria and a coated bionic biological membrane, the coated bionic biological membrane is a T cell membrane, and the engineering bacteria can secrete IL-12. The delivery system disclosed by the invention can realize intelligent, precise, visual and non-invasive efficient delivery of exogenous engineering bacteria drugs; by utilizing a gradient freeze-drying technology and an advanced aerosol inhalation technology, local delivery of an exogenous engineering bacterium drug in a host lung is realized, and focus targeted colonization of the engineering bacterium drug is realized; by utilizing an accurate cell membrane coating technology, monitoring and killing of immune cells are reduced, and the colonization efficiency of engineering bacteria is improved; and an engineering bacterium secretion system after biological modification and optimization is synthesized, so that the space-time accuracy of immune regulation of an exogenous engineering bacterium medicine can be further realized in a synergistic manner.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and specifically relates to a bionic T cell secretory engineered bacteria lung targeted delivery system based on respiratory tract atomization, a construction method and an application thereof. Background Art

[0002] With the in-depth development of biotechnology and medical research, the application of cytokines in tumor treatment is becoming more and more extensive. However, systemic administration of cytokines is often accompanied by problems such as large side effects and easy clearance by the immune system, which in turn causes adverse reactions related to systemic immunotherapy and affects the treatment effect, ultimately affecting the patient's prognosis. Therefore, for local delivery of drugs for lung diseases, the method of respiratory aerosol administration has emerged based on nanotechnology, biomaterial technology, and synthetic biology technology. According to current research reports, nanoparticles loaded with naftifine are delivered to the infected area of ​​the lungs by respiratory aerosol administration, inhibiting the biosynthesis of Staphylococcus aureus antioxidant staphyloxanthin (STX), thereby enhancing the chemotaxis of neutrophils to effectively eliminate Staphylococcus aureus. The results showed that it effectively increased the bactericidal effect on Staphylococcus aureus, improved the survival rate of mice, and reduced the toxic side effects of drugs on other organs and tissues. As an active carrier with a particle size of 0.5 μm-3 μm, facultative anaerobic engineered bacteria have a suitable particle size for preparing aerosol inhalation preparations. After inhalation, they can reach the middle airways and bronchi at all levels, achieving a long-term retention effect.

[0003] With the continuous breakthroughs in the research and clinical application of microbial tumor prevention and treatment, important progress has been made in the engineered microbial preparations that can predict the host immune response using synthetic biology technology. Studies have reported that the mitochondrial targeting domain (MTD) of Noxa is considered to be a cargo protein with anti-tumor activity. The research team synthesized the gene encoding MTD into Escherichia coli with a type III secretion system, and used its own secretion system to secrete the MTD protein with anti-tumor activity into the tumor site, further improving the anti-tumor therapeutic efficacy of the drug.

[0004] Genetic engineering technology can be used to build customized engineered bacteria from the molecular level to the overall functional level, which involves the precise decomposition of target genes, innovative design, fine assembly and final construction. These engineered bacteria not only retain the natural immunogenic molecules carried by the bacteria themselves, such as lipopolysaccharide (LPS) and peptidoglycan (PGN), which play an important role in the host's innate immune response in both health and disease. For example, by binding to innate immune receptors such as NOD1 / 2, it regulates immune and inflammatory responses. At the same time, genetic engineering technology can be used to enable these engineered bacteria to express a variety of bioactive molecules with anti-tumor potential. These include cytokines, such as interleukin-12 (IL-12), which can significantly enhance the activation and proliferation of T cells and promote anti-tumor immune responses; cytotoxic factors, such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which can specifically induce apoptosis of tumor cells without affecting normal cells; or the expression of specific siRNA, such as siPD-1 RNA targeting PD-1 (programmed death receptor 1), which downregulates PD-1 expression on the surface of tumor cells through RNA interference mechanism, thereby relieving immunosuppression and promoting T cells to recognize and kill tumor cells. By precisely regulating the expression of these bioactive molecules, engineered bacteria can target multiple key steps of the anti-tumor immune response, produce synergistic effects with the host's innate immune system, and jointly reshape the tumor immune microenvironment. Not only does it enhance the anti-tumor ability of the engineered bacteria itself, it also promotes the activation and amplification of the body's overall anti-tumor immune response, significantly improving the anti-tumor efficacy.

[0005] Although the co-delivery of immune checkpoint inhibitors and anti-inflammatory cytokines by engineered bacteria can increase the anti-tumor effect, after administration, the engineered bacteria are easily killed and eliminated by immune cells in the body, reducing the retention time of the engineered bacteria and the drug concentration of the engineered bacteria in the local tumor, thus affecting its therapeutic effect on the tumor. Coating with biomimetic biofilms, such as T cell membranes, neutrophil membranes, and red blood cell membranes, can make the engineered bacteria have better biocompatibility and enhance the ability to escape immunity, protect the engineered bacteria from the attack of the immune system, prolong the retention time of the engineered bacteria in the tumor tissue, and reduce the clearance rate of the engineered bacteria.

[0006] The applicant envisions developing a lung-targeted delivery of engineered bacteria aerosol inhalation preparations that secrete cytokines by coating T cell membranes, with the goal of increasing the partial targeting and concentration accumulation of anti-tumor cytokines in lung cancer tissues and reducing the adverse reactions associated with systemic immunotherapy of engineered bacteria drugs. The autologous T cell membranes are coated with engineered bacteria active biological drugs that secrete anti-tumor cytokine interleukin-12 (IL-12) transformed using synthetic biology technology, and colonize in tumor lesions through hypoxia tropism to construct an engineered bacteria aerosol preparation with immune regulation.

[0007] (1) Engineered bacteria can deliver engineered bacteria drugs locally to the lungs based on respiratory aerosol preparations, avoiding systemic side effects of drugs; respiratory aerosol administration and long-term retention in the trachea promote the colonization of engineered bacteria in the lungs and achieve local delivery; (2) After the engineered bacteria colonize in the trachea, they tend to lung tumors through their own hypoxic characteristics and colonize in the core hypoxic area of ​​the tumor. Through the modified engineered bacteria secretion system such as type I and type III secretion systems, they secrete anti-tumor cytokines such as interleukin-12 (IL-12), achieving tumor targeting of engineered bacteria drugs and precise controlled drug release; (3) Engineered bacteria use cell membranes, such as T cell membranes, to coat themselves, camouflage themselves, escape recognition and killing by the immune system, further improve the colonization rate of engineered bacteria and prolong retention time, and improve and maintain drug blood concentrations. Summary of the invention

[0008] The purpose of the present invention is to overcome the deficiencies in the prior art and to provide a local lung delivery system based on respiratory tract atomized engineered bacteria and a construction method and application thereof.

[0009] The present invention first provides a bionic T cell secretory engineered bacteria lung targeted delivery system based on respiratory tract atomization, and the delivery system comprises: engineered bacteria and a coated bionic biomembrane.

[0010] In certain embodiments, the coated biomimetic biological membrane is selected from one or more of a T cell membrane, a neutrophil membrane, and a red blood cell membrane.

[0011] In certain embodiments, the coated biomimetic biological membrane is a T cell membrane.

[0012] In certain embodiments, the engineered bacteria are capable of secreting the anti-tumor cytokine interleukin-12 (IL-12).

[0013] In certain embodiments, the engineered bacteria contain pRSF-J23119-T1SS HlyA-IL-12 or p15A-J23119-HlyB-HlyD plasmid.

[0014] In certain embodiments, the engineered bacteria contain pRSF-J23119-T1SS HlyA-IL-12 and p15A-J23119-HlyB-HlyD plasmids.

[0015] In certain embodiments, the pRSF-J23119-T1SS HlyA-IL-12 and p15A-J23119-HlyB-HlyD plasmid sequences are shown as SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0016] In certain embodiments, the system has a reconstitution function after freeze-drying and can achieve respiratory tract aerosol inhalation administration.

[0017] In certain embodiments, the engineered bacteria containing the secretion system@T cell membrane grow well after being coated.

[0018] In certain embodiments, the secretion system-engineered bacteria@T cell membrane has a particle size of 3-6 μm.

[0019] The present invention also provides an application of the above-mentioned bionic T cell secretory engineered bacteria lung targeted delivery system based on respiratory tract atomization, wherein the application is one of a) or b);

[0020] a) Screening for drugs to treat or prevent lung diseases;

[0021] b) Preparation of medicines for the treatment or prevention of lung diseases.

[0022] The present invention also provides a method for preparing a bionic T cell secretory engineered bacteria lung targeted delivery system based on respiratory tract atomization, the method comprising the following steps:

[0023] (1) Preparation of engineered bacteria;

[0024] (2) Preparation of coated biomimetic biofilm;

[0025] (3) The engineered bacteria and the coated biomimetic biofilm are mixed in proportion, and the engineered bacteria particles coated with the biofilm are produced using a cell membrane extruder to form micron-sized particles.

[0026] In some embodiments, the specific method is as follows:

[0027] (1) Construction of engineered strains of HlyA, HlyB, HlyD and IL-12 including type I secretion system signal peptide and secretion structure: Gene sequences of HlyA, HlyB, HlyD and IL-12 were synthesized by gene synthesis technology, and plasmids were constructed and amplified using homologous recombination technology with Escherichia coli DH5α strain as the host. The sequences of HlyA and IL-12 amplified by PCR were introduced into the pRSF vector, and the sequences of HlyB and HlyD amplified by PCR were introduced into the p15A vector. Finally, the synthesized plasmids were transferred into Escherichia coli Nissle 1917ΔΔ knocked out the cryptic plasmid, and the engineered strains EcNΔΔ-A12-BD (containing pRSF-J23119-T1SS HlyA-IL-12 and p15A-J23119-HlyB-HlyD plasmids) were constructed. The primers and gene sequences used were synthesized by GENEWIZ, Inc. (Suzhou, China).

[0028] (2) Preparation of secretory engineered bacteria@T cell membrane system: CTLL-2 mouse T cells were cultured in T75 culture flasks, and the cells were lysed by four degrees of hypotonicity overnight with 0.25% PBS solution, repeated freezing and thawing, centrifuged at 1000 rpm for 10 min, and the supernatant was removed. The T lymphocyte membrane was obtained by centrifugation at 45000 g / min in a high-speed centrifuge for 40 min, and the supernatant was discarded and resuspended in 100 µL PBS solution and stored at -80°C. The engineered bacteria biofilm-coated particles were prepared in batches by repeated extrusion using a membrane extruder with 10 μm, 8 μm, 5 μm, and 1 μm filter membranes. The number of cells of the engineered bacteria mixture containing the secretion system and IL-12 was 1.0·10 8 CFU; the number of T cells in the T lymphocyte membrane PBS mixture is 1.0·10 7 The ratio of the two is about 10:1. A cell membrane squeezer is used, which is controlled by two injection tubes. Under the shear force of the T cell membrane mixed liquid fluid shearing the engineering bacteria mixed liquid fluid, the engineering bacteria aggregate forms a uniform micro-droplet outer layer that uniformly covers the T cell membrane. The number of squeezes of each filter membrane with a large and small pore size is about 15-20 times, and micron-sized biofilm-coated engineering bacteria particles are prepared.

[0029] (3) Construction of a local lung delivery system for engineered bacteria based on respiratory inhalation: When constructing a respiratory inhalation engineered bacteria system for local lung delivery, the gradient freeze-drying program of the capping freeze dryer (model SCIENTZ-50F / B) was first set to ensure that the engineered bacteria reached an ideal dehydrated state while maintaining activity. This program gradually lowered the temperature, specifically set to maintain the temperature at 0°C for a period of time, then lowered to -20°C and extended the freeze-drying time, and finally cooled to -40°C and continued freeze-drying. The duration of each temperature stage was optimized to 30-60 min, 30-240 min, and 30-360 min, respectively, to accurately control the water removal rate, and finally obtain freeze-dried engineered bacteria particles with a water content precisely controlled in the range of 4%-10%. In order to effectively deliver these freeze-dried engineered bacteria particles to the lungs, respiratory atomization inhalation was used. The freeze-dried powder was evenly dispersed in physiological saline to restore its certain dispersibility and suspension. Subsequently, the commercial nebulizer (model Jiaruikang JLN-2311HDD) was adaptively modified by adjusting the nebulization parameters, such as air flow velocity, nebulization chamber pressure, etc., to ensure that the particle size distribution of the generated engineered bacterial particle aerosol was suitable for the respiratory system characteristics of the small animal model, thereby achieving efficient and safe local lung delivery.

[0030] Furthermore, in step (3), the gradient freeze-drying and atomization inhalation platform is used to inhale the prepared secretory engineered bacteria@T cell membrane microparticles with uniform particle size through the respiratory tract to achieve local delivery of the engineered bacteria to the lungs.

[0031] Finally, the present invention provides an application of the method for preparing the above-mentioned bionic T cell secretory engineered bacteria lung targeted delivery system based on respiratory tract atomization, wherein the application is to prepare drugs for treating or preventing lung diseases.

[0032] Compared with the prior art, the present invention has at least the following beneficial effects:

[0033] 1. The present invention innovatively proposes an engineered bacteria local lung delivery system based on respiratory inhalation. The core of this system is the integration of engineered bacteria @ T cell bionic particles with a type I secretion system. These bionic particles exhibit highly uniform morphology and excellent dispersion properties, ensuring their effective distribution and deposition in the respiratory tract and lungs. More importantly, the engineered bacteria in this system are cleverly embedded with specific secretion system genes. This design enables the engineered bacteria to accurately release exogenous anti-tumor cytokines locally in the lungs. These cytokines can directly act on lung lesions, such as lung cancer lesions or lung inflammation sites, and achieve significant relief and effective treatment of lung diseases through multiple mechanisms such as regulating immune responses, inhibiting tumor growth or promoting tissue repair.

[0034] 2. The core of the system of the present invention is to use bionic particles carrying type I secretion system engineered bacteria @ T cell membrane to achieve local delivery through respiratory inhalation. This innovative strategy ensures that the engineered bacteria bionic particles can be accurately implanted in the lung lesions, forming a close interaction with the lung mucosal immune system, triggering a synergistic response mechanism. The T cell membrane on the engineered bacteria bionic particles not only ensures its excellent targeting and penetration. It also promotes recognition and fusion with local immune cells in the lungs, thereby achieving targeted and effective colonization of exogenous engineered bacteria drugs in the host lungs. While improving the bioavailability of the drug, it also reduces the exposure of non-targeted tissues, reduces potential side effects, achieves spatiotemporal precision of therapeutic effects, helps to enhance the anti-tumor or anti-inflammatory ability of the immune system, inhibits the progression of the disease within a specific time and space, prevents the recurrence of lung diseases to the greatest extent, promotes the effect of in situ treatment, and effectively inhibits the development of the disease.

[0035] 3. The present invention is based on the local lung delivery system of engineered bacteria inhaled through the respiratory tract, and realizes the recurrence prevention, in situ treatment and course inhibition of lung diseases by exogenous engineered bacteria drugs through local positioning, precise secretion and coordinated response of the lung mucosal immune system. The present invention innovatively integrates "synthetic biology-cell membrane coating technology" and develops a new strategy that can achieve targeted and effective colonization of exogenous engineered bacteria drugs in the host lungs. The genetically modified engineered bacteria carry an optimized secretion system that can accurately control the release of therapeutic factors. It also uses advanced cell membrane coating technology to cover the surface of the engineered bacteria with cell membranes (such as T cell membranes) that have a high affinity for the target lung cells, thereby significantly improving its positioning accuracy and colonization efficiency in the lung lesion area. After the engineered bacteria are successfully colonized in the lungs, the precise secretion system they carry will release synergistic therapeutic factors at an appropriate time and space according to the preset program. These therapeutic factors interact closely with the lung mucosal immune system, activate and enhance local immune responses, and then achieve targeted treatment of lung diseases. This process not only improves the therapeutic effect, but also minimizes the potential impact of drugs on non-target tissues.

[0036] 4. The secretory engineered bacteria@T cell membrane delivery system of the present invention can realize intelligent, precise, visualized and non-invasive efficient delivery of exogenous engineered bacteria drugs; utilize gradient freeze-drying technology and advanced atomization inhalation technology to deliver exogenous engineered bacteria, realize local delivery of exogenous engineered bacteria drugs in the host lungs, and realize targeted colonization of engineered bacteria drugs in lesions; utilize precise cell membrane coating technology to reduce the surveillance and killing of immune cells and improve the colonization efficiency of engineered bacteria; the engineered bacteria secretion system optimized by synthetic biology can further coordinately realize the spatiotemporal precision of exogenous engineered bacteria drugs in exerting immune regulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a verification diagram of the secretion of IL-12 by the engineered bacteria;

[0038] Figure 2 This is a verification diagram of T cell membrane protein activity;

[0039] Figure 3 This is a process diagram for the preparation of secretory engineered bacteria@T cell membrane aerosol preparations;

[0040] Figure 4 This is the immunofluorescence verification image of secretory engineered bacteria@T cell membrane;

[0041] Figure 5 This is a verification diagram of the growth of secretory engineered bacteria @T cell membrane engineered bacteria;

[0042] Figure 6 This is a diagram showing the validation of lung colonization based on the local lung delivery system of engineered bacteria inhaled through the respiratory tract; DETAILED DESCRIPTION

[0043] The embodiments of the present invention are described in detail below. It should be noted that the embodiments are descriptive rather than restrictive and cannot be used to limit the protection scope of the present invention.

[0044] The raw materials used in the present invention, unless otherwise specified, are conventional commercially available products; the methods used in the present invention, unless otherwise specified, are conventional methods in the art.

[0045] Example 1

[0046] Construction of HlyA, HlyB, HlyD and IL-12 engineered strains including type I secretion system signal peptide and secretion structure: HlyA, HlyB, HlyD and IL-12 gene sequences were synthesized by GENEWIZ, Inc. (Suzhou, China). The synthesized sequence plasmids, vectors pRSF and p15A were linearized by PCR technology, and the original promoter region was avoided. The HlyB and HlyD fragments were connected to the p15A vector by homologous recombination to construct the plasmid p15A-J23119-HlyB-HlyD; the IL-12 fragment and HlyA were connected to the pRSF vector to construct the plasmid pRSF-J23119-T1SS HlyA-IL-12. The constructed plasmid was transformed into DH5α competent strain, and the possible positive colonies were screened out after preliminary detection by gel electrophoresis, and sent for Sanger sequencing. The positive colonies were screened out according to the sequencing results, and the plasmids in the positive colonies were extracted by a plasmid extraction kit. The extracted plasmids were transferred into Escherichia coli Nissle 1917 (containing GFP), and the engineered strain EcN ΔΔ-A12-BD (containing pRSF-J23119-T1SS HlyA-IL-12 and p15A-J23119-HlyB-HlyD plasmids) was obtained.

[0047] Example 2

[0048] Preparation of secretory engineered bacteria@T cell membrane: CTLL-2 mouse T cells were cultured in T75 culture flasks, and the cells were lysed by hypotonicity overnight at 4°C with 0.25% PBS solution and repeated freezing and thawing. The supernatant was taken after centrifugation at 1000 rmp / min for 10 min, and the supernatant was discarded after centrifugation at 45000 g / min for 40 min in a high-speed centrifuge to obtain T lymphocyte membranes, which were resuspended in 100 µL PBS solution and stored at -80°C. The engineered bacteria biofilm-coated particles were prepared in batches by repeated extrusion using a membrane extruder using 10 µm, 8 µm, 5 µm, and 1 µm filter membranes. The cell volume of the engineered bacteria mixture containing the secretion system and IL-12 was 1.0·10 8CFU; the number of T lymphocytes in the T lymphocyte membrane PBS mixture is 1.0·10 7 The ratio of the two is about 10:1. A cell membrane squeezer is used, which is controlled by two injection tubes. Under the shear force of the T lymphocyte membrane mixed fluid shearing the engineered bacteria mixed fluid, the engineered bacteria aggregate forms a uniform micro-droplet outer layer that uniformly covers the T cell membrane. The number of squeezes for each pore size is about 15-20 times, and micron-sized biofilm-coated engineered bacteria particles are prepared.

[0049] Example 3

[0050] Construction of a local lung delivery system based on respiratory inhalation engineered bacteria: When constructing a respiratory inhalation engineered bacteria system for local lung delivery, the gradient freeze-drying program of the capping freeze dryer (model SCIENTZ-50F / B) was first set to ensure that the engineered bacteria reached an ideal dehydrated state while maintaining activity. This program gradually lowered the temperature, specifically set to maintain the initial 0°C for a period of time, then drop to -20°C and extend the freeze-drying time, and finally cool to -40°C and continue freeze-drying. The duration of each temperature stage was optimized to 30-60 min, 30-240 min, and 30-360 min, respectively, to accurately control the water removal rate, and finally obtain freeze-dried engineered bacteria particles with a water content precisely controlled in the range of 4%-10%. In order to effectively deliver these freeze-dried engineered bacteria particles to the lungs, respiratory atomization inhalation was adopted. The freeze-dried powder was evenly dispersed in physiological saline to restore its certain dispersibility and suspension. Subsequently, the commercial nebulizer (model Jiaruikang JLN-2311HDD) was adaptively modified by adjusting the nebulization parameters, such as air flow velocity, nebulization chamber pressure, etc., to ensure that the particle size distribution of the generated engineered bacterial particle aerosol was suitable for the respiratory system characteristics of the small animal model, thereby achieving efficient and safe local lung delivery.

[0051] Example 4

[0052] The functional experimental techniques for evaluating the p15A-J23119-HlyB-HlyD and pRSF-J23119-T1SS HlyA-IL-12 plasmids prepared by the present invention are as follows:

[0053] The constructed plasmid was transformed into DH5α competent strain, and the possible positive colonies were screened out after preliminary detection by gel electrophoresis and sent for Sanger sequencing. The positive colonies were screened out according to the sequencing results, and the plasmids in the positive colonies were extracted by a plasmid extraction kit. The extracted plasmids were transferred into Escherichia coli Nissle 1917 (containing GFP) to obtain the engineered strain. The engineered strain containing p15A-J23119-HlyB-HlyD and pRSF-J23119-T1SS HlyA-IL-12 plasmids was inoculated into LB medium at a ratio of 1:100 and cultured to the logarithmic phase (OD 600=0.5). The biological activity of interleukin-12 (IL-12) was detected by Western blot. Compared with wild-type Escherichia coli, the engineered bacteria with IL-12 gene, secretion system and IL-12 gene can secrete IL-12 normally. The results are shown in Figure 1 .

[0054] Example 5

[0055] The techniques for evaluating the activity of the membrane protein of the secretion system engineered bacteria@T cells prepared by the present invention and the biofilm coating are as follows:

[0056] CTLL-2 mouse T cells were cultured in T75 culture flasks, and the cells were lysed by hypotonic overnight and repeated freezing and thawing. The supernatant was collected after centrifugation at 1000 rpm for 10 min, and the supernatant was discarded after centrifugation at 45000 g / min for 40 min to obtain T cell membranes. Western blot was used to detect the activity of T cell membrane surface proteins such as CD3. The results are shown in Figure 2 , it can be seen that compared with LLC cells and their cell membranes, the coated T cells and cell membrane surface CD3 protein expression is positive. Using DID dye, the T cell membrane is stained dark red. Using a liposome extruder, the engineered bacterial solution containing the secretion system and IL-12 and the PBS mixed solution containing the T cell membrane are squeezed back and forth to prepare biofilm-coated engineered bacterial particles, including the schematic diagram of the preparation process of secretory engineered bacteria @ T cell membrane, and the results are shown in Figure 3 Take 2 µL of the mixed solution after squeezing, put it on a glass slide, observe it under a fluorescence microscope and take pictures to verify the coating of the T cell membrane engineered bacteria. E. coli can be seen under bright field, and the red fluorescence of the cell membrane and the green fluorescence of E. coli can be synthesized. The overlap can be clearly seen, indicating that the engineering bacteria are successfully coated. The results are shown in Figure 4 .

[0057] Example 6

[0058] The experimental technique for evaluating the growth of the atomized inhaled secretory engineered bacteria@T cell membrane engineered bacteria prepared by the present invention is as follows:

[0059] The wild-type E. coli and the engineered bacteria were inoculated into LB medium and incubated at 37°C with gentle shaking. 100 µL of samples were taken at 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 h, and fixed with 100 µL of polymethanol. After all samples were fixed, the concentration of the bacterial solution (OD value) was measured at a wavelength of 600 nm. The measured OD values ​​were plotted against the corresponding culture time, and the growth curve of the bacteria was drawn to verify the growth of the engineered bacteria. It can be observed that the growth curve trend of the engineered bacteria loaded with plasmids and coated with T cell membranes is consistent with that of normal unmodified E. coli, indicating that the plasmid and T cell membrane do not affect its growth. The results are shown in Figure 5 .

[0060] Example 7

[0061] The experimental techniques for evaluating the lung colonization of the local lung delivery system based on respiratory inhalation engineered bacteria prepared by the present invention are as follows:

[0062] After the secretory system engineered bacteria@T cell membrane engineered bacteria prepared after extrusion are fully mixed, male C57BL / 6J mice aged 6 to 8 weeks are selected, and anesthesia is induced using 3% isoflurane equipped with a dedicated gas anesthesia device. After anesthesia, 50-100 µL of the engineered bacteria mixture is sucked with a 200 µL pipette, the mouse body is vertical, and the engineered bacteria mixture is dripped into the left and right nostrils in turn. After the liquid is inhaled, it is slowly dripped to prevent the mouse from choking and coughing to death. After all the mixed bacterial liquid is dripped, the mouse is put back into the cage. After 30 minutes, the mouse is killed by dislocating the neck. The lungs are dissected from the body and placed in a small animal in vivo imaging device. Appropriate imaging parameters are selected, and then imaging and recording of the distribution and intensity of the fluorescent signal in the mouse lungs are started. It can be clearly seen that the fluorescent signal is enriched in the mouse lungs, indicating the successful colonization of the engineered bacteria in the lungs. The results are shown in Figure 6 .

[0063] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A bionic T cell secretory engineered bacteria lung targeted delivery system based on respiratory tract atomization, characterized in that: The delivery system comprises: engineering bacteria and a coated biomimetic biofilm.

2. The delivery system according to claim 1, characterized in that The coated biomimetic biological membrane is selected from one or more of T cell membrane, neutrophil membrane and red blood cell membrane.

3. The delivery system according to claim 2, characterized in that The coated biomimetic biological membrane is a T cell membrane.

4. The delivery system according to claim 1, characterized in that The engineered bacteria can secrete the anti-tumor cytokine interleukin-12 (IL-12).

5. The delivery system according to claim 4, characterized in that The engineering bacteria contain pRSF-J23119-T1SSHlyA-IL-12 or p15A-J23119-HlyB-HlyD plasmid.

6. The delivery system according to claim 4, characterized in that The engineering bacteria contain pRSF-J23119-T1SSHlyA-IL-12 and p15A-J23119-HlyB-HlyD plasmids.

7. The delivery system according to claim 4, characterized in that The plasmid sequences of pRSF-J23119-T1SS HlyA-IL-12 and p15A-J23119-HlyB-HlyD are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

8. An application of the bionic T cell secretory engineered bacteria lung targeted delivery system based on respiratory tract atomization according to any one of claims 1 to 7, characterized in that: The application is one of a) or b); a) For screening drugs for treating or preventing lung diseases; b) Preparation of medicines for the treatment or prevention of lung diseases.

9. A method for preparing a bionic T cell secretory engineered bacteria lung targeted delivery system based on respiratory tract atomization, characterized in that: The method comprises the following steps: (1) Preparing engineered bacteria; (2) Preparation of coated biomimetic biofilm; (3) The engineered bacteria and the coated biomimetic biofilm are mixed in proportion, and the engineered bacteria particles coated with the biofilm are produced in micrometer size using a cell membrane extruder.

10. An application of the method for preparing the bionic T cell secretory engineered bacteria lung targeted delivery system based on respiratory tract atomization as claimed in claim 9, characterized in that: The application is to prepare medicine for treating or preventing lung diseases.

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