Genetically engineered bacterium for synthesizing CD47 antibody through hypoxia response, construction and application of genetically engineered bacterium in preparation of tumor immune checkpoint blocking preparation
Genetically engineered bacteria that synthesize CD47 antibodies through hypoxic responses efficiently express and secrete CD47 antibodies in the hypoxic tumor microenvironment, solving the inadequate application of monoclonal antibodies in tumor immune checkpoint blocking therapy. , achieving efficient tumor immune checkpoint blocking effect.
Patent Information
- Application Number
- CN202411461411.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-18
- Publication Date
- 2024-12-27
AI Technical Summary
In the existing tumor immune checkpoint blocking therapy, immune checkpoint inhibitors mainly based on monoclonal antibodies face common limitations of protein drugs, such as high clearance rate in vivo, low targeting accuracy, difficulty in maintaining local concentrations and high costs. Magphage-based tumor immune checkpoint blocking strategy has been blocked.
A genetically engineered bacteria that synthesizes CD47 antibodies in an hypoxic response was designed. By carrying the hypoxic promoter and CD47 antibody expression gene, it efficiently expresses and secretes CD47 antibodies in the hypoxic tumor microenvironment, blocks the CD47-SIRP pathway, and promotes the giant Phages kill tumor cells.
The efficient expression and local efficient supply of immune checkpoint inhibitors under hypoxia conditions have been achieved, which extends the drug action time, reduces the clearance rate in the body, improves the therapeutic effect, and monitors the distribution of bacteria in the body through tracer genes and colonization situation.
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Figure CN119193450A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of tumor immune checkpoint blocking preparations, and more specifically, to a genetically engineered bacterium that synthesizes CD47 antibodies in response to hypoxia, and the application of the construction and preparation of tumor immune checkpoint blocking preparations. Background Art
[0002] Tumor immune checkpoint blockade therapy has made significant progress in the past decade. It enhances anti-tumor immune response by intervening in key signal exchanges between tumor cells and immune cells. At present, tumor immune checkpoint blockade therapy focuses on activating adaptive immune mechanisms represented by T lymphocytes. However, the complex tumor immune microenvironment and T lymphocyte activity and targeting caused by specific antigen screening barriers have severely restricted its development. The innate immune system represented by macrophages has rapid immune response, a wide range of anti-tumor targets and relatively independent antigen presentation capabilities, and is gradually becoming the research focus of the next generation of immunotherapy and a medium for anti-cancer therapy. The key to regulating the phagocytic mechanism of macrophages on tumor cells lies in recognizing and responding to the delicate balance between "phagocytosis" and "anti-phagocytosis" signals on the surface of tumor cells. Among them, the CD47-SIRPa signaling axis, as a classic anti-phagocytic pathway, has a regulatory effect on this process. Unfortunately, tumor cells form immune checkpoints by upregulating CD47 expression (3-5 times higher than normal cells), thereby escaping the recognition and clearance of macrophages. Therefore, interfering with the interaction between CD47 on the surface of tumor cells and SIRPα, a receptor on macrophages, has become an effective strategy to relieve tumor immune escape and promote phagocytosis. However, the current immune checkpoint inhibitors, mainly monoclonal antibodies, face the common limitations of protein drugs, including high in vivo clearance, low targeting accuracy, difficulty in maintaining local concentration, and high costs, which hinder the macrophage-based tumor immune checkpoint blockade strategy.
[0003] With the rapid development of synthetic biology technology, protein-based immune checkpoint inhibitors with tumor treatment potential can be mass-produced with the help of engineered bacteria as an efficient and scalable biomanufacturing platform. At the same time, the tumor microenvironment unique to solid tumors is characterized by hypoxia, eutrophication, and immune privilege, which is very suitable for the enrichment and colonization of most anaerobic and facultative anaerobic bacteria in tumor sites (up to tens of thousands of times the level of normal tissue). The natural tropism of bacteria to the tumor microenvironment brings unique advantages to engineered bacteria as an innovative carrier for delivering immunotherapy drugs directly, overcoming the inherent limitations of traditional protein-based immune checkpoint inhibitors, such as rapid clearance in the body, low targeting efficiency, insufficient local drug concentration, and high cost. Engineered bacteria are designed with reprogrammed genetic modules, including gene control elements that sense and respond to specific signals and gene circuits that encode therapeutic proteins. Accurate and efficient spatiotemporal regulation can achieve efficient expression of immune checkpoint inhibitors in situ in the tumor, ensuring the precise delivery of therapeutic drugs and maximizing efficacy. Current genetic engineering strategies have enabled programmable engineered bacteria to respond to bacterial quorum sensing signals or external physical or chemical stimuli (IPTG, temperature, ultrasound, etc.), but precise regulation under tumor-specific microenvironmental factors (such as hypoxia, low pH or high GSH concentration) is still an underdeveloped area. Given the inherent adaptive mechanisms of bacteria, especially their high sensitivity to changes in oxygen concentration, incorporating hypoxia-responsive gene regulatory elements into the design of engineered bacteria will provide more precise temporal and spatial regulation for the treatment process.
[0004] Traditional bacterial activity regulation, safety and efficacy of tumor immune checkpoint blockade therapy are limited. Therefore, designing and reprogramming new engineered bacteria to address the in vivo application defects of protein immune checkpoint inhibitors remains a huge challenge. Summary of the invention
[0005] The present invention provides a genetically engineered bacterium that synthesizes CD47 antibodies in response to hypoxia. The engineered bacteria are designed to carry a reprogrammed genetic module, including a gene control element (hypoxia promoter) that senses and responds to specific signals and a gene circuit (CD47 antibody expression gene) that encodes a therapeutic protein. The genetically engineered bacteria of the present invention start the expression of plasmids in a tumor microenvironment with low oxygen concentration, synthesize and secrete CD47 antibodies, bind to CD47 receptors on the surface of tumor cells, and block the CD47-SIRP pathway, prompting macrophages to effectively kill tumor cells. Thereby solving the technical problems faced by immune checkpoint inhibitors based on monoclonal antibodies in the prior art, such as high in vivo clearance rate of protein drugs, low targeting accuracy, and difficulty in maintaining local concentration.
[0006] According to a first aspect of the present invention, a genetically engineered bacterium for synthesizing CD47 antibodies in response to hypoxia is provided, characterized in that the genetically engineered bacterium contains a hypoxia promoter, an expression sequence of a pelB secretion signal, an expression sequence of a CD47 antibody and a terminator which are sequentially connected in series from upstream to downstream.
[0007] Preferably, the hypoxic promoter is promoter fdhF, promoter nirb, promoter vgb or promoter foca.
[0008] Preferably, the genetically engineered bacteria further include a tracer gene expression system, and the tracer gene expression system is located between the expression sequence of the hypoxic promoter and the pelB secretion signal, or is located downstream of the terminator.
[0009] Preferably, the tracer gene expression system is an mCherry tracer gene expression system.
[0010] According to another aspect of the present invention, a method for constructing a genetically engineered bacterium that synthesizes CD47 antibodies in response to hypoxia is provided, comprising the following steps:
[0011] S1: Using attenuated Escherichia coli genomic DNA as a template, amplification was performed using primers with nucleotide sequences of SEQ ID NO.1 and SEQ ID NO.2 to obtain the hypoxic promoter PfdhF sequence, wherein SEQ ID NO.1 contains a BamHI restriction site, and SEQ ID NO.2 contains a NotI restriction site;
[0012] S2: using a chemical synthesis method to obtain the sequence shown in SEQ ID NO.3, wherein the sequence shown in SEQ ID NO.3 includes a NotI sequence, an expression sequence of a pelB secretion signal, a linker, an expression sequence of a CD47 antibody, a terminator, and a NotI sequence connected in sequence;
[0013] S3: the sequence obtained in step S1 was digested with BamHI and NotI, the sequence obtained in step S2 was digested with NotI, and the pBV220-Tac-mCherry plasmid was digested with BamHI and NotI;
[0014] S4: The three sequences obtained in step S3 are connected, and sequenced and identified to obtain the hypoxia promoter, the expression sequence of the pelB secretion signal, the expression sequence of the CD47 antibody, the terminator, and the target plasmid of the mCherry gene expression system which are connected in series from upstream to downstream;
[0015] S5: The target plasmid obtained in step S4 is transformed into Escherichia coli, and the target genetically engineered bacteria are obtained by identifying the resistance gene in the pBV220-Tac-mCherry plasmid.
[0016] According to another aspect of the present invention, there is provided the use of genetically engineered bacteria that synthesize CD47 antibodies in response to hypoxia for preparing tumor immune checkpoint blocking preparations.
[0017] Preferably, the genetically engineered bacteria are used to specifically respond to and initiate the expression of CD47 antibodies in the hypoxic microenvironment of tumors to produce exogenous CD47 antibodies, which bind to CD47 receptors on the surface of tumor cells and block the CD47-SIRP pathway, thereby prompting macrophages to kill tumor cells.
[0018] In general, the above technical solution conceived by the present invention has the following technical advantages compared with the prior art:
[0019] (1) The precise and efficient spatiotemporal regulation of the genetically engineered bacteria in the present invention can achieve efficient expression of immune checkpoint inhibitors under hypoxic conditions.
[0020] (2) In the present invention, the genetically engineered bacteria locally synthesize and secrete immune checkpoint inhibitors in the tumor microenvironment, and the drugs do not quickly enter the systemic circulation, thereby reducing the clearance rate in the body. This not only prolongs the duration of drug action, but also reduces systemic side effects.
[0021] (3) The genetically engineered bacteria of the present invention can continuously synthesize and secrete immune checkpoint inhibitors, ensuring that the local drug concentration at the tumor site remains at an effective level. This continuous local supply avoids the problem of insufficient local concentration caused by rapid clearance of traditional protein drugs, thereby improving the therapeutic effect.
[0022] (4) Preferably, the genetically engineered bacteria further include a tracer gene expression system, and the tracer gene expression system is located between the expression sequence of the hypoxic promoter and the pelB secretion signal, or downstream of the terminator. The tracer gene can be used to monitor the distribution and colonization of the genetically engineered bacteria in the body in real time, and visualize the enrichment of bacteria at the tumor site. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 Representative SDS-PAGE images of aCD47 and mCherry proteins expressed by the genetically engineered bacteria of the present invention under normoxic and hypoxic environments.
[0024] Figure 2 These are in vitro photos and fluorescent images of the genetically engineered bacteria of the present invention in an oxygen-deficient environment.
[0025] Figure 3 It is the fluorescence intensity of aCD47 and mCherry of the genetically engineered bacteria of the present invention detected by flow cytometry.
[0026] Figure 4 This is a quantitative analysis of the aCD47 protein production of the genetically engineered bacteria of the present invention at different time points.
[0027] Figure 5 These are confocal microscope images of aCD47 and mCherry proteins expressed by the genetically engineered bacteria of the present invention in normoxic and hypoxic environments.
[0028] Figure 6 The figure shows the distribution of the genetically engineered bacteria of the present invention in CT26 tumor spheres after being cultured for 2 hours and 6 hours.
[0029] Figure 7 The confocal image is a three-dimensional structure and intensity surface map of the distribution of the genetically engineered bacteria of the present invention after being cultured in CT26 tumor spheres for 6 hours.
[0030] Figure 8 Representative bioluminescent images of mice with orthotopic colon cancer treated with genetically engineered bacteria described in the present invention.
[0031] Fig. 9 The tumor sections are TUNEL-stained and Ki67-stained in mice with orthotopic colon cancer treated with the genetically engineered bacteria of the present invention.
[0032] Fig.10 This is an immunofluorescence staining image of tumor tissue in mice with orthotopic colon cancer treated with the genetically engineered bacteria of the present invention.
[0033] Fig.11 This is a flow chart of the genetically engineered bacteria of the present invention blocking the CD47-SIRP pathway to prompt macrophages to kill tumor cells. DETAILED DESCRIPTION
[0034] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.
[0035] The invention discloses a genetically engineered bacterium for synthesizing CD47 antibody in response to hypoxia. The genetically engineered bacterium contains a hypoxia promoter, an expression sequence of a pelB secretion signal, an expression sequence of a CD47 antibody and a terminator which are sequentially connected in series from upstream to downstream.
[0036] The method for constructing a genetically engineered bacterium for synthesizing CD47 antibodies in response to hypoxia of the present invention comprises the following steps:
[0037] S1: Using attenuated E. coli MG1655 genomic DNA as a template, primers with nucleotide sequences of SEQ ID NO.1 and SEQ ID NO.2 were used for amplification to obtain the hypoxic promoter PfdhF sequence, wherein SEQ ID NO.1 contains a BamHI restriction site and SEQ ID NO.2 contains a NotI restriction site;
[0038] S2: using a chemical synthesis method to obtain the sequence shown in SEQ ID NO.3, wherein the sequence shown in SEQ ID NO.3 includes a NotI sequence, an expression sequence of a pelB secretion signal, a linker, an expression sequence of a CD47 antibody, a terminator, and a NotI sequence connected in sequence;
[0039] S3: the sequence obtained in step S1 was digested with BamHI and NotI, the sequence obtained in step S2 was digested with NotI, and the pBV220-Tac-mCherry plasmid was digested with BamHI and NotI;
[0040] S4: Sterilized water, the product in step S3, buffer and ligase were added to the centrifuge tube in sequence. The target fragment was not added to the control group to test the self-ligation of the vector. After reacting at room temperature for 2 hours, the sample was sent for sequencing to obtain the target plasmid;
[0041] S5: transforming the target plasmid obtained in step S4 into Escherichia coli, and identifying the target genetically engineered bacteria using the resistance gene in the pBV220-Tac-mCherry plasmid;
[0042] Specifically, the competent E. coli MG1655 (the bacteria have natural tropism) was taken out from -80℃ and placed on ice. After 5 minutes, the target plasmid obtained in step S4 was added, and the bottom of the 1.5mL EP tube was gently moved to promote mixing. After the 1.5mL EP tube was placed in ice for 25 minutes, it was heat-shocked in a water bath (42℃) for 45 seconds and then placed on ice for 2 minutes. 700μLLB medium was added to the 5mL EP tube and placed in a shaking incubator for 1 hour (200rpm, 37℃);
[0043] The bacterial suspension was centrifuged at 4000 rpm for 1 minute, and the bacterial pellet was spread on LB agar plates (50 μg / mL ampicillin) and placed in a 37°C incubator overnight to obtain a monoclonal strain. A single colony was picked out and amplified in LB medium at 37°C, 220 rpm overnight, and the solution was diluted to OD600 = 0.5 for further experiments.
[0044] The sequence of the pBV220-Tac-mCherry plasmid is mainly composed of fragments such as the AmpR resistance gene and the ColEI replicon, and there are EcoRI / NotI restriction enzyme sites at both ends of its promoter.
[0045] The principle of the present invention is: the target nucleic acid fragment is connected to the pBV220-Tac-mCherry vector by using restriction endonucleases and DNA ligases, and then the recombinant vector is introduced into the competent Escherichia coli MG1655 by heat shock method, and finally the desired genetically engineered bacteria are screened by resistance.
[0046] The genetically engineered bacteria of the present invention specifically respond to and initiate the expression of CD47 antibodies in the hypoxic microenvironment of tumors, and the produced exocytic CD47 antibodies have affinity for CD47 proteins.
[0047] The genetically engineered bacteria of the present invention are used to specifically respond to and initiate the expression of CD47 antibodies in the hypoxic microenvironment of tumors, thereby producing exocytic CD47 antibodies that bind to CD47 receptors on the surface of tumor cells and block the CD47-SIRP pathway, thereby prompting macrophages to kill tumor cells.
[0048] The genetically engineered bacteria of the present invention form an interactive regulation that is dependent on the inducible response of the genetically engineered bacteria and the tumor microenvironment.
[0049] In some embodiments, the genetically engineered bacteria themselves have tropism for the tumor microenvironment.
[0050] As shown in Table 1, the present invention provides a list of sequences for constructing hypoxia-responsive genetically engineered bacteria. The target nucleic acid fragment is connected to the pBV220-Tac-mCherry vector by using restriction endonucleases and DNA ligases, and then the recombinant vector is introduced into the competent Escherichia coli MG1655 by heat shock method, and finally the target genetically engineered bacteria are screened by resistance.
[0051] Table 1
[0052]
[0053] like Figure 1The embodiment of the present invention provides representative SDS-PAGE images of CD47 antibody (aCD47) and mCherry protein expressed by hypoxia-responsive genetically engineered bacteria (HRB) under normoxic and hypoxic environments. In order to verify the hypoxic logic circuit of HRB, we placed HRB in normoxic and hypoxic incubation for 24 hours and observed the expression of mCherry and aCD47 to illustrate. SDS-PAGE gel electrophoresis analysis showed that compared with the bacterial lysate and supernatant of MG1655, aCD47 protein appeared in large quantities in the lysate and supernatant of HRB incubated under hypoxia, while negligible CD47 protein appeared in the lysate and supernatant of HRB incubated under normoxic conditions, confirming that hypoxia induces HRB to produce aCD47. In addition, regardless of whether HRB is incubated under normoxic or hypoxic conditions, compared with MG1655, the mCherry tracer protein is stably expressed, confirming the stable tracing ability of mCherry.
[0054] like Figure 2 The embodiment of the present invention provides in vitro photos and fluorescence images of hypoxia-responsive genetically engineered bacteria in an oxygen-free environment. Through real-time visual recording and fluorescence imaging, HRB was incubated at 37°C under hypoxia for 24 hours, and the red light level and corresponding fluorescence intensity increased significantly over time. The above phenomenon proves the feasibility and effectiveness of the designed sequence of the present invention.
[0055] like Figure 3 The embodiment of the present invention provides the fluorescence intensity of aCD47 and mCherry in the flow cytometer detection of hypoxia-responsive genetically engineered bacteria under normoxic and hypoxic environments. In order to explore the expression amount and affinity of aCD47 protein produced by HRB in a hypoxic environment, the present invention treats HRB under different oxygen content environments by flow analysis, and uses Escherichia coli MG1655 as a negative control. The flow analysis results show that Escherichia coli MG1655 detects a weak green fluorescence signal under both normoxic (B4) and hypoxic (B2) conditions, indicating that Escherichia coli MG1655 has a small amount of non-specific binding with hFc-CD47 protein. Compared with groups B4 and B2, HRB detected enhanced green fluorescence signals under normoxic (B3) and hypoxic (B1), and the green fluorescence signal of group B1 was significantly higher than that of group B3, indicating that the expression of aCD47 gene is inhibited under normoxic conditions, and the inhibition of aCD47 expression can be relieved under hypoxia induction. In addition, strong red fluorescence signals were detected in HRB (B1 and B3) compared with E. coli MG1655 (B2 and B4), indicating the normal expression of the mCherry tracer gene.
[0056] like Figure 4The embodiment of the present invention provides a quantitative analysis of the aCD47 protein production of hypoxia-responsive genetically engineered bacteria at different time points. The aCD47 protein expression level was further calculated by detecting the CD47 protein content after aCD47 binding by ELISA. The results showed that when the hypoxia incubation time was extended from 30 min to 24 h, the expression level of aCD47 protein increased to 43.33 pmol / mL, and was time-dependent.
[0057] like Figure 5 The embodiment of the present invention provides confocal microscopy images of aCD47 and mCherry proteins expressed by hypoxia-responsive genetically engineered bacteria in normoxic and hypoxic environments. The present invention uses a confocal laser scanning microscope to observe immunofluorescence staining of aCD47, which shows the same results as flow analysis, with almost no green fluorescence in groups B2 and B4, and a large amount of green fluorescence in group B1 compared to group B3, indicating that aCD47 is highly expressed in hypoxia-induced HRB, thereby confirming the controllability of the hypoxia logic circuit of HRB.
[0058] like Figure 6 The embodiment of the present invention provides the distribution of hypoxia-responsive genetically engineered bacteria in CT26 tumor spheres after 2 hours and 6 hours of culture. In order to evaluate the penetration ability of HRB, the present invention first used the CT26 tumor sphere model for further testing and evaluation, and found that over time, the HRB group gradually observed stronger mCherry fluorescence in the center of the tumor sphere.
[0059] like Figure 7 The embodiment of the present invention provides a confocal image of the three-dimensional structure and intensity surface map of the distribution of hypoxia-responsive genetically engineered bacteria in CT26 tumor spheres after 6 hours of culture. Figure 3 Fluorescence intensity analysis was performed after 3D imaging, and spatial visualization also confirmed the deep penetration of HRB into the tumor spheroids. This indicates that the hypoxic tropism of the bacteria itself makes it easier for HRB to target the center of the hypoxic tumor spheroids.
[0060] like Figure 8 The present invention embodiment provides representative bioluminescent images of hypoxia-responsive genetically engineered bacteria for the treatment of mice with orthotopic colon cancer. Similar fluorescence signals were observed in each group of data on day 0, indicating successful development of orthotopic colon cancer. After two treatments, the MG1655 group had a slight tumor inhibition effect compared with the PBS group, while the HRB group had a relatively better tumor inhibition effect.
[0061] like Fig. 9The embodiment of the present invention provides TUNEL staining and Ki67 staining tumor sections of mice with in situ colon cancer treated with hypoxia-responsive genetically engineered bacteria. By terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) detection, an enhancement of the green fluorescence signal in the HRB group was also found, indicating that the treatment had stronger tumor cell apoptosis relative to the MG1655 group. In addition, the expression of Ki67 was reduced to varying degrees in immunofluorescence staining relative to the PBS and MG1655 groups. The HRB group showed the greatest reduction in Ki67 expression, indicating that the tumor proliferation potential was significantly reduced.
[0062] like Fig.10 The embodiment of the present invention provides an immunofluorescence staining image of tumor tissue of mice with orthotopic colon cancer treated with hypoxia-responsive genetically engineered bacteria. F4 / 80+mmp9+ inflammatory macrophages were co-localized by immunofluorescence staining of tumor tissue sections. Compared with the other groups, HRB can be observed to have enhanced green fluorescence and red fluorescence overlap, inducing polarization of macrophages in tumors to M1.
[0063] like Fig.11 The embodiment of the present invention provides a flow chart of hypoxia-responsive genetically engineered bacteria blocking the CD47-SIRP pathway to encourage macrophages to kill tumor cells. The engineered bacteria are designed with reprogrammed genetic modules and start the expression of plasmids in a tumor microenvironment with low oxygen concentrations. CD47 antibodies are synthesized and secreted to bind to the CD47 receptors on the surface of tumor cells, blocking the CD47-SIRP pathway, thereby enabling macrophages to effectively identify and kill tumor cells.
[0064] It will be easily understood by those skilled in the art that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.
Claims
1. A genetically engineered bacterium that synthesizes CD47 antibodies in response to hypoxia, characterized in that: The genetically engineered bacteria contain a hypoxic promoter, an expression sequence of a pelB secretion signal, an expression sequence of a CD47 antibody and a terminator which are sequentially connected in series from upstream to downstream.
2. The genetically engineered bacteria for synthesizing CD47 antibodies in response to hypoxia according to claim 1, characterized in that: The hypoxic promoter is promoter fdhF, promoter nirb, promoter vgb or promoter foca.
3. The genetically engineered bacteria for synthesizing CD47 antibodies in response to hypoxia according to claim 1 or 2, characterized in that: The genetically engineered bacteria also include a tracer gene expression system, which is located between the expression sequence of the hypoxic promoter and the pelB secretion signal, or downstream of the terminator.
4. The genetically engineered bacteria for synthesizing CD47 antibodies in response to hypoxia according to claim 3, characterized in that: The tracer gene expression system is an mCherry tracer gene expression system.
5. The method for constructing a genetically engineered bacterium capable of synthesizing CD47 antibodies in response to hypoxia according to any one of claims 1 to 4, characterized in that: The following steps are involved: S1: Using attenuated Escherichia coli genomic DNA as a template, amplification was performed using primers with nucleotide sequences of SEQ ID NO.1 and SEQ ID NO.2 to obtain the hypoxic promoter PfdhF sequence, wherein SEQ ID NO.1 contains a BamHI restriction site, and SEQ ID NO.2 contains a NotI restriction site; S2: using a chemical synthesis method to obtain the sequence shown in SEQ ID NO.3, wherein the sequence shown in SEQ ID NO.3 includes a NotI sequence, an expression sequence of a pelB secretion signal, a linker, an expression sequence of a CD47 antibody, a terminator, and a NotI sequence connected in sequence; S3: the sequence obtained in step S1 was digested with BamHI and NotI, the sequence obtained in step S2 was digested with NotI, and the pBV220-Tac-mCherry plasmid was digested with BamHI and NotI; S4: The three sequences obtained in step S3 are connected, and sequenced and identified to obtain the hypoxia promoter, the expression sequence of the pelB secretion signal, the expression sequence of the CD47 antibody, the terminator, and the target plasmid of the mCherry gene expression system which are connected in series from upstream to downstream; S5: The target plasmid obtained in step S4 is transformed into Escherichia coli, and the target genetically engineered bacteria are obtained by identifying the resistance gene in the pBV220-Tac-mCherry plasmid.
6. The use of genetically engineered bacteria for synthesizing CD47 antibodies in response to hypoxia according to any one of claims 1 to 4, characterized in that: Used to prepare tumor immune checkpoint blockade preparations.
7. The use according to claim 6, characterized in that The genetically engineered bacteria are used to specifically respond to and initiate the expression of CD47 antibodies in the hypoxic microenvironment of tumors, thereby producing exogenous CD47 antibodies that bind to CD47 receptors on the surface of tumor cells and block the CD47-SIRP pathway, thereby prompting macrophages to kill tumor cells.