An intelligent engineered bacterial system, its preparation, and its application in anti-tumor drugs

Abstract

The present invention provides an intelligent engineered bacterial system, its preparation, and application in anti-tumor drugs. The intelligent engineered bacterial system is a bacterium loaded with a plasmid, which has a tropism for tumor tissue. The plasmid contains a gene fragment encoding a fusion protein, and the fusion protein is secreted and expressed by the bacteria. The fusion protein comprises protein A, an anti-tumor peptide, and a matrix metalloenzyme-2 sensitive peptide that connects the two and can be targeted and cleaved by matrix metalloenzyme-2. The protein A can inactivate the anti-tumor peptide. After inducing the expression of the fusion protein in vitro, the intelligent engineered bacterial system is injected into tumor-bearing mice. Driven by hypoxia and immunosuppression, the bacteria can tend to and colonize the tumor site. The matrix metalloenzyme-2 highly expressed in the tumor site is used to cut off the sensitive peptide, so that the activity of the anti-tumor peptide can be restored and accurately released locally in the tumor, greatly improving the anti-tumor effect of the engineered bacteria.

Description

Technical Field

[0001] The present invention belongs to the technical field of nano-biomedicine, and specifically relates to an intelligent engineering bacterial system, a preparation method thereof, and an application thereof in anti-tumor drugs. Background Art

[0002] Cancer has always been a major threat to human life and health. Currently, treatments for cancer, such as surgery, radiotherapy, chemotherapy, and more recently, immunotherapy, gene therapy, and cell therapy, can alleviate cancer symptoms to a certain extent, but each inevitably has its own drawbacks. Among them, the non-targeted accumulation and release of loaded drugs is a major obstacle to achieving safe and effective treatment. With the advent of the concept of precision medicine, the use of multifunctional carriers for targeted tumor therapy has been extensively researched and rapidly developed, showing potential for clinical application.

[0003] In recent years, research on bacterial-based cancer therapy has grown exponentially, achieving significant progress and demonstrating significant clinical potential. Due to the presence of cytokines and the hypoxic microenvironment in tumor tissue, facultative anaerobes and anaerobic bacteria tend to migrate to tumor sites. Furthermore, the immunosuppressive microenvironment of tumor tissue also facilitates bacterial colonization. Compared to traditional cancer treatments, novel bacterial therapy offers a novel, targeted, and highly effective approach, effectively avoiding the challenges of early insensitivity and late-stage drug resistance associated with traditional therapies. With the rapid development of synthetic biology, researchers are actively exploring ways to further optimize bacterial cancer therapy, providing a more diverse range of strategies for cancer treatment. Bacterial genomes are simple and easily engineered. Using genetic engineering techniques to reshape bacterial synthetic gene networks, or even transplant entire chemically synthesized genomes into bacteria, offers a promising approach to targeted cancer therapy. Tumor-targeted bacteria expressing anti-tumor proteins can reduce pathogenicity and toxic side effects in the host, thereby optimizing treatment options.

[0004] As a foreign substance, the in vivo biosafety of bacterial-based tumor therapy has always been a focus of concern. The pathogenicity of bacteria and the strong inflammatory response of the body caused by the lipopolysaccharide components of the bacterial wall are both problems in the process of bacterial therapy. At present, the commonly adopted response strategy to these problems is to use non-pathogenic bacteria or attenuate the strains. However, the residual bacteria and their whereabouts in the tumor tissue after the treatment are still a mystery, and the potential safety hazards they pose to the body cannot be ignored. In addition, the use of genetic recombination technology to make bacteria express anti-tumor proteins. The precise synthesis and release of anti-tumor proteins in this process and whether they will damage normal tissues are also details that need to be considered in our design process. Summary of the Invention

[0005] In response to the shortcomings of existing tumor bacterial therapies, the present invention aims to construct an intelligent engineered bacterial system that overcomes the problems of non-targeted drug enrichment, poor drug efficacy, and safety of in vivo drug delivery systems that occur in traditional tumor treatments, and has significant anti-tumor effects and high safety.

[0006] In order to achieve the above object, the technical solution of the present invention is as follows:

[0007] A smart engineered bacterial system, specifically bacteria loaded with a plasmid, which has a tropism for tumor tissue. The plasmid contains a gene fragment encoding a fusion protein, and the fusion protein is secreted and expressed by the above-mentioned bacteria. The above-mentioned fusion protein contains protein A, an anti-tumor peptide, and a matrix metalloenzyme-2 sensitive peptide that connects the two and can be targeted and cleaved by matrix metalloenzyme-2, wherein protein A has the function of inactivating the function of the anti-tumor peptide.

[0008] In the above scheme, bacteria serve as carriers, and the tumor targeting of the intelligent engineered bacterial system is achieved through the tropism of the carrier bacteria to the tumor tissue. Moreover, the carrier bacteria can colonize in the tumor tissue by utilizing the immunosuppressive microenvironment of the tumor tissue. Therefore, the carrier bacteria should be facultative anaerobes or anaerobic bacteria, such as Escherichia coli.

[0009] In this scheme, the fusion protein in the intelligently engineered bacterial system is completely secreted and expressed by the carrier bacteria. Protein A in the fusion protein inactivates the anti-tumor peptide, rendering the system non-toxic in non-tumor tissues. However, when the system reaches the tumor site, the matrix metalloenzyme-2 (MMP-2)-sensitive peptide is cleaved by the highly expressed matrix metalloenzyme-2 (MMP-2) in the tumor. This releases the inhibitory effect of protein A on the anti-tumor peptide, allowing the anti-tumor peptide to exert its anti-tumor effect and kill tumor cells. Therefore, this intelligently engineered bacterial system achieves targeted delivery and precise release of the anti-tumor peptide, enhancing its effectiveness in killing tumor cells.

[0010] In a specific embodiment of the present invention, the fusion protein specifically comprises superfolded green fluorescent protein GFP and melittin (Melittin, amino acid sequence is GIGAVLKVLTTGLPALISWIKRKRQQ), and GFP is coupled to the N-terminus of Melittin via a matrix metalloenzyme-2 sensitive peptide. GFP at the N-terminus of the fusion protein acts as a non-signal peptide to guide the secretion of the target protein, which can significantly reduce the cytotoxicity of Melittin and improve the solubility of the fusion protein. At the same time, GFP will attach to the cell membrane to shield the toxicity of the bacterial endotoxins themselves, reducing the inflammatory response caused by bacterial toxins to the body. When the matrix metalloenzyme-2 sensitive peptide is cut off, the inhibitory effect of GFP on the function of Melittin is released, and Melittin exerts an anti-tumor effect. Moreover, Melittin is also an antimicrobial peptide and has a significant anti-inflammatory effect. After being released, it can also kill bacteria and cause the death of immunogenic tumor cells, thereby enhancing the anti-tumor effect. Therefore, the intelligent engineering bacterial system expressing this fusion protein can not only achieve targeted delivery and precise release of melittin, but also eliminate the bacteria themselves, achieving efficient and safe treatment of tumors in vivo.

[0011] Furthermore, in the above embodiment, when the amino acid sequence of the matrix metalloenzyme-2 sensitive peptide is PLGVR and the N-terminus of the fusion protein is modified with a 6*His tag, the amino acid sequence of the fusion protein (denoted as GPM) is shown in SEQ ID NO.1, and the nucleotide sequence encoding the fusion protein is shown in SEQ ID NO.2.

[0012] The present invention further provides a method for constructing an intelligent engineering bacterial system for secreting and expressing a fusion protein represented by a sequence such as SEQ ID NO. 1, specifically:

[0013] Step 1: Amplify the target gene fragment and the linearized vector using PCR technology, wherein the nucleotide sequence of the target gene fragment is as shown in SEQ ID NO. 4, the linearized vector is a vector containing 6*His-GFP, and the target gene fragment and the linearized vector have homology regions;

[0014] Step 2: Use T5 exonuclease to cut the 5' homology arms of the linearized vector and the target gene fragment, connect them into a circular plasmid, heat-shock transform them into Escherichia coli, use bacterial ligase to fill the gap, and then screen and identify the recombinants;

[0015] Step 3: Transform the recombinant plasmid into the recipient bacteria and induce expression.

[0016] In the above construction method, the target gene fragment is synthesized by using primers with sequences such as SEQ ID NOs. 9 to 10 as templates, annealing and pairing them into a double-stranded DNA, and then synthesizing the double-stranded DNA as a template under the guidance of the primer pair with sequences such as SEQ ID NOs. 11 to 12.

[0017] Preferably, the recipient bacteria in step 3 is Escherichia coli BL21 (DE3) plysS and IPTG is used to induce expression.

[0018] The intelligent engineered bacterial system provided by the present invention can be used to prepare anti-tumor drugs. The obtained drugs can be used to treat solid tumors, and the administration method is injection.

[0019] The beneficial effects of the present invention are:

[0020] (1) Efficient expression and intelligent response: The intelligent engineering bacterial system E.coli@GPM provided by the present invention utilizes E. coli to efficiently secrete and express the fusion protein GPM. GPM carrying a matrix metalloenzyme-sensitive peptide can be activated in response to the environment at the tumor site, thereby restoring the antibacterial and antitumor activities of bee venom and accurately releasing it locally at the tumor site.

[0021] (2) Significant anti-tumor effect: The intelligent engineered bacterial system E.coli@GPM utilizes the characteristics of E. coli gravitating towards tumor tissues, allowing the engineered bacteria to accumulate at the tumor site before releasing and activating Melittin, which can significantly inhibit tumor growth. The tumor inhibition rate for Babl / c mice inoculated with colon cancer cells is as high as 80%.

[0022] (3) It has significant biosafety and is non-toxic: the hemolytic effect of the fusion protein GPM is significantly improved compared to the naked bee venom protein; and no physiological and pathological changes were found in the major organ tissues of tumor-bearing mice after injection of E. coli@GPM; blood biochemical analysis of tumor-bearing mice at different times after injection showed that the liver and kidney function indicators showed slight fluctuations within the normal range and then returned to the initial values. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of the structures of the two fusion expression vectors pET23a-GPM (top) and pET23a-GM (bottom) constructed in Example 1;

[0024] Figure 2 This is a gel image of the linearized vector pET23a-GFP in Example 1, wherein lane M is a marker and lane 1 is the linearized pET23a-GFP vector;

[0025] Figure 3This is a gel image of the target gene fragment PLGVR-Melitttin constructed in Example 1, wherein lane M is a marker, lanes 1 and 2 are primer fusion PCR products, and lanes 3 and 4 are target gene fragment PLGVR-Melitttin;

[0026] Figure 4 This is a PCR gel image of the recombinant pET23a-GPM colony in Example 1, where lane M is the marker and lane 1 is the colony PCR result;

[0027] Figure 5 This is a gel image of the linearized vector pET23a-GPM in Example 1, wherein lane M is a marker, lane 1 is a plasmid template, and lane 2 is a linearized vector pET23a-GPM;

[0028] Figure 6 This is a PCR gel image of the recombinant pET23a-GM colony in Example 1, where lane M is the marker and lane 1 is the colony PCR result;

[0029] Figure 7 This is a flow chart for constructing the intelligent engineering bacteria system E.coli@GPM in Example 1;

[0030] Figure 8 This is an SDS-PAGE gel image of the fusion proteins expressed by each engineered bacteria in Example 1, wherein lane M is a marker, lanes 1, 4, and 7 are the lysate, supernatant, and precipitate of E. coli@G, respectively; lanes 2, 5, and 8 are the lysate, supernatant, and precipitate of E. coli@GM, respectively; and lanes 3, 6, and 9 are the lysate, supernatant, and precipitate of E. coli@GPM, respectively;

[0031] Figure 9 This is a Western Blot analysis of the fusion proteins expressed by the engineered bacteria in Example 1;

[0032] Figure 10 The blue light analyzer is used to analyze the distribution of proteins expressed by smart engineering bacteria in bacterial solution, culture supernatant, precipitation, and membrane washing solution;

[0033] Figure 11 This is the result of the inhibition zone experiment in Example 1;

[0034] Figure 12 This is a diagram showing the results of the live-dead cell staining experiment in Example 1;

[0035] Figure 13 Graph showing the results of hemolysis experiments before and after cleavage of each fusion proteinase in Example 1;

[0036] Figure 14This is a diagram showing the distribution of bacteria in various tissues in mice in Example 2;

[0037] Figure 15 This is a statistical graph showing changes in body weight and tumor volume of mice after treatment with different materials in Example 2;

[0038] Figure 16 This is a statistical diagram of tumor mass in mice after treatment with different materials in Example 2;

[0039] Figure 17 This is a photo of mouse tumors after treatment with different materials in Example 2;

[0040] Figure 18 This is a graph showing the results of blood biochemical analysis of liver and kidney function in mice after tail vein injection of E. coli@GPM in Example 2. DETAILED DESCRIPTION

[0041] 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 examples and experimental data. 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.

[0042] In the following examples, unless otherwise specified, all methods are conventional methods; the reagents and materials described, unless otherwise specified, can be obtained from commercial sources.

[0043] Example 1

[0044] The intelligent engineering bacterial system constructed in this example is denoted as E.coli@GPM, that is, the carrier bacteria used are Escherichia coli, and the fusion protein (GPM) is composed of superfolded green fluorescent protein (GFP), anti-tumor peptide-melittin (Melittin) and matrix metalloenzyme-2 (MMP-2) sensitive peptide (PLGVR).

[0045] (1) Construction of expression vector

[0046] The linearized vector and target gene PLGVR-Melittin were obtained by in vitro PCR and primer construction, and two clones were constructed as follows: Figure 1 The expression vector shown in FIG. 1 , wherein the primer sequences used are shown in Table 1.

[0047] Table 1

[0048]

[0049] The expression vector pET23a-GFP was used as a template and F-23A-GFP and R-23A-GFP were used as primers to linearize the vector (the gel image after linearization is shown in FIG. Figure 2With primers F-PLGVR-M-1 and R-PLGVR-M-1 as templates, they were annealed to form a complementary DNA double strand. Then, with the double strand as a template, the target gene chain PLGVR-Melittin was synthesized under the guidance of primers F-PLGVR-M-2 and R-PLGVR-M-2 (the gel image of the gene fragment is shown in FIG). Figure 3 The nucleotide sequence is shown in SEQ ID NO.4, and the amino acid sequence of the encoded peptide is shown in SEQ ID NO.3). The linearized vector and the 5' end of the target gene contain a 15 bp homology region, and a homology arm connection interface is formed using T5 exonuclease. Competent cells E. coli DH5α (i.e., Escherichia coli DH5α, from the School of Life Sciences, Hubei University) are added and heat-shocked to complete the transformation. Recombinants are picked and colony PCR is performed using F-PLGVR-M-2 and R-PLGVR-M-2 to obtain positive recombinants (the recombinant PCR test results are shown in FIG. Figure 4 As shown), and sequenced to construct pET23a-GPM plasmid (i.e. Figure 1 pET-23a-sfGFP-PLGVG-Melittin in PBS).

[0050] Using pET23a-GPM as template and F-SSSSG-M and R-SSSSG-M as primers, the plasmid pET23a-GPM was linearized (the gel image after linearization is shown in FIG. Figure 5 As shown), the linearized vector 5' contains a 15bp homology region. The homology arm connection interface is formed using T5 exonuclease, and the competent cells E. coli DH5α are added and heat-shocked to complete the transformation. Recombinants are picked and colony PCR is performed using F-PLGVR-M-2 and R-PLGVR-M-2 to obtain positive recombinants (PCR test results are shown in Figure 6 As shown), and sequenced to construct pET23a-GM plasmid (i.e. Figure 1 pET-23a-sfGFP-SSSSG-Melittin in the pET-23a-sfGFP-SSSSG-Melittin, wherein the amino acid sequence of SSSSG-Melittin is shown as SEQ ID NO.5, and the nucleotide sequence encoding the peptide is shown as SEQ ID NO.6).

[0051] The plasmid pET23a-GFP was transformed into competent cells of Escherichia coli BL21(DE3)plysS (i.e., Escherichia coli BL21(DE3)plysS, purchased from Beijing Qingke Biotechnology Co., Ltd.) to obtain the engineered bacteria E.coli@G; the plasmid pET23a-GM was transformed into competent cells of Escherichia coli BL21(DE3)plysS to obtain the intelligent engineered bacteria system E.coli@GM; the plasmid pET23a-GPM was transformed into competent cells of Escherichia coli BL21(DE3)plysS to obtain the intelligent engineered bacteria system E.coli@GPM (the complete construction process is shown in Figure 7 ).

[0052] (2) Induced expression and characterization of fusion protein.

[0053] The engineered bacteria after transformation were spread on the plate and the positive strains screened by antibiotics were selected. E.coli@G, E.coli@GM and E.coli@GPM were inoculated into 100 mL of liquid culture medium containing ampicillin and placed in a shaking incubator at 37°C. When the OD 600 When the concentration of β-D-thiogalactopyranoside (IPTG) was ≈0.6, the induction agent was added and the expression was induced at 18°C ​​for 18 hours. After the expression was completed, the following characterization was performed:

[0054] ① Collect the cells and perform ultrasonic disruption. Perform SDS-PAGE on the supernatant and precipitate to determine whether the fusion protein is expressed. The results show that the molecular size of the expressed fusion protein is consistent with the expected value (e.g. Figure 8 shown).

[0055] ② Perform SDS-PAGE electrophoresis on the supernatant after lysis, transfer to the membrane, and perform Western Blot analysis. The target protein band is consistent with the SDS-PAGE result (such as Figure 9 shown).

[0056] From the results of SDS-PAGE detection and Western blotting analysis after the intelligent engineering bacteria E.coli@GPM was induced to express the recombinant protein, it can be seen that GPM was successfully expressed efficiently in BL21.

[0057] ③ To determine whether the fusion protein is secreted, the bacteria were induced to express the fusion protein by adding the inducer IPTG and then the cells were collected. After adding the membrane washing buffer, the cells were placed in a 4°C silent mixer for 48 hours and the supernatant was collected. The bacterial solution of the smart engineered bacteria, the pellet after centrifugation, the culture medium supernatant, and the supernatant after the cell membrane was washed were subjected to blue light analysis. The results are as follows Figure 10 As shown, it was shown that the fusion protein was secreted and could be collected without lysing the bacteria.

[0058] (3) In vitro antibacterial activity detection of GPM fusion protein.

[0059] E.coli@GM and E.coli@GPM were respectively coated on plates, filter paper was soaked in MMP-2 solution of different concentrations, and attached to the plates. After overnight incubation, obvious inhibition zones were observed (see Figure 11 This indicates that PLGVR is cut by MMP-2 enzyme, the inactivation effect of GFP on melittin is relieved, and the antibacterial activity is restored.

[0060] (4) In vitro anti-tumor activity detection and hemolysis test of GPM fusion protein.

[0061] Live and dead cell staining: CT26 cells were seeded in 6-well plates and cultured until the cells covered 90% of the bottom of the chamber. 100 μL of collected GFP, GM, and GPM protein solutions of different concentrations were added to each well. After incubation for 18 hours, the culture medium was discarded and the cells were washed. Each group of cells was stained with Calcein-AM and PI in PBS buffer for 30 minutes and observed under an inverted fluorescence microscope. The results are shown in Figure 2. Figure 12 As shown, it shows that GPM protein exhibits good tumor killing effect.

[0062] The hemolytic reaction of the smart engineered bacteria was then evaluated: 150 μL of H2O, PBS, GFP protein solution, GM protein solution, and GPM protein solution were placed in 1.5 mL EP tubes. An experimental group and a negative control group were set up for each sample. 1 μL of MMP-2 was added to the experimental group and mixed evenly (final enzyme concentration 3200 ng mL -1 The control group and the experimental group were incubated at 37°C for 4 hours. 150 μL of mouse blood was taken from the anticoagulant tube and added to the above EP tube. The tube was incubated at 37°C for 4 hours. After centrifugation at 2500 rpm for 10 minutes, photos were taken and the supernatant was taken to measure the absorbance at 570 nm. The results are shown in Figure 2. Figure 13 As shown: After treatment with exogenous MMP-2 (right figure), GPM showed obvious hemolytic effect; while without exogenous MMP-2 treatment (left figure), the hemolytic effect was not obvious.

[0063] The control group was transformed with the plasmid pET23a-GM, and the recombinant protein GFP-SSSSG-Melittin (GM) expressed was not cleaved by MMP-2. The above test results show that without exogenous MMP-2 treatment, both E.coli@GM and E.coli@GPM exhibited low cytotoxicity; however, after exogenous MMP-2 treatment, E.coli@GPM exhibited significant cytotoxicity.

[0064] Example 2

[0065] In this example, the E. coli@GPM constructed in Example 1 was administered to tumor-bearing mice to observe its anti-tumor effect. The animal experiments in this example strictly adhered to international standards for experimental animal welfare ethics, and the experimental protocol was carried out under the supervision of the Hubei University Animal Ethics Committee.

[0066] (1) Establishment of CT26 tumor-bearing nude mouse model.

[0067] Newly purchased Balb / c female mice were acclimated for one week. CT26 cells were cultured and digested with 0.25% trypsin until the cells covered 80-90% of the bottom of the culture dish. Digestion was terminated by adding 2 mL of serum-containing DMEM and the cells were pipetted to a single-cell suspension. The cells were collected by centrifugation at 1000 rpm for 2 minutes, the supernatant was discarded, and the cells were resuspended in PBS buffer and counted. The cell concentration was adjusted to 10 7 pcs / mL for use. 6 CT26 cells were injected into the right side of the back of mice to establish a tumor-bearing mouse model.

[0068] (2) Biodistribution of bacteria in vivo.

[0069] The dose of 10 9 CFU E. coli@GPM were fed for 0, 12, 24, 48, and 72 hours. After reaching the designated feeding time points, mice were euthanized. Major organs such as heart, liver, spleen, lung, kidney, and tumor tissue were collected. These samples were weighed, homogenized, and diluted to 1 mg mL in sterile PBS. -1 Suspension, tumor tissue serially diluted to 0.1 mg mL -1 The tissue suspension (100 μL) was evenly spread on a solid LB plate containing ampicillin and penicillin, and then cultured at 37°C for 24 hours and the colonies were counted. The results were as follows: Figure 14 shown.

[0070] The results showed that the bacteria in the main organs of mice were gradually metabolized and eliminated by the body over time; in contrast, the amount of bacterial colonization in the tumor site showed a clear growth trend.

[0071] (3) Evaluation of the anti-tumor efficacy of engineered bacteria.

[0072] When the tumor volume of the tumor-bearing mice was about 100 mm 3 At the same time, 24 mice were randomly divided into 4 groups: PBS, E.coli@G, E.coli@GM, and E.coli@GPM. As a control, the first group of mice was injected with PBS through the tail vein, the main material group was injected with E.coli@GPM through the tail vein, and the other two groups of mice were injected with E.coli@G and E.coli@GM through the tail vein, respectively. Each mouse was injected with 10 9CFU of engineered bacteria.

[0073] During the treatment period, the weight of each mouse was monitored daily using an electronic balance, and the length and width of each mouse's tumor were recorded using a vernier caliper to calculate the tumor volume. Figure 15 After treatment, the mice were euthanized, the tumor tissues were removed, and the tumor mass of each mouse was recorded (as shown in Figure 16 ), and take photos (as shown Figure 17 shown).

[0074] Figure 16 and 17 The results showed that E.coli@GPM exhibited a significant tumor-suppressing effect, while the protein expressed by E.coli@G had almost no anti-tumor activity; however, E.coli@GM also had certain anti-tumor activity. The reason was that the molecular weight of melittin was relatively small, and the large molecular weight GFP protein at the N-terminus of the fusion protein could prevent melittin from exerting its active effect. However, the C-terminus of the melittin fusion protein was not modified, so its function could not be completely inactivated.

[0075] (4) Biosafety analysis.

[0076] To evaluate the physiological effects of E. coli@GPM injection on tumor tissues and major organs in mice, blood samples were collected from mice at designated time points (0, 12, 24, 48, 96, and 120 h) for blood biochemical analysis. The results are shown in Figure 18 As shown: Liver and kidney functions returned to their initial values ​​after slight fluctuations within the normal range.

[0077] The above test results indicate that the intelligent engineered bacterial system E. coli@GPM constructed in Example 1 demonstrated significant tumor tropism and tumor inhibition in the in vivo efficacy evaluation experiment, with a tumor inhibition rate of up to 80% in Babl / c mice inoculated with colon tumor cells, and exhibited good in vivo biosafety.

[0078] In summary, the intelligent engineered bacterial system provided by the present invention can overcome the problems of non-targeted drug enrichment and poor drug efficacy that occur in traditional tumor treatment processes, achieve targeted delivery and precise release of drugs, and use bee venom with anti-tumor and antibacterial functions to kill tumor cells while eliminating the bacteria themselves, solving the safety issues of existing bacterial therapies.

[0079] The above description is a preferred embodiment of the present invention, which cannot be used to limit the scope of rights of the present invention. It should be pointed out that for ordinary technicians in this technical field, any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Application of an intelligent engineering bacterial system in the preparation of anti-tumor drugs, characterized in that: The intelligent engineering bacterial system is a recombinant Escherichia coli loaded with a plasmid, wherein the plasmid contains a gene segment encoding a fusion protein. The fusion protein is secreted and expressed by the recombinant Escherichia coli, and the fusion protein is composed of superfolded green fluorescent protein (GFP), melittin, and a matrix metalloenzyme-2 sensitive peptide that connects the superfolded green fluorescent protein (GFP) and melittin and can be targeted and cleaved by matrix metalloenzyme-2. The GFP is coupled to the N-terminus of melittin via a matrix metalloenzyme-2 sensitive peptide, and the amino acid sequence of the fusion protein is shown in SEQ ID NO.1; The drug is administered by intravenous injection after inducing expression in the recombinant Escherichia coli; The N-terminus of the fusion protein is modified with a 6*His tag, and the nucleotide sequence encoding the fusion protein is shown in SEQ ID NO.2; The construction method of the intelligent engineering bacterial system includes the following steps: Step 1: Amplify the target gene fragment and the linearized vector using PCR technology, wherein the nucleotide sequence of the target gene fragment is as shown in SEQ ID NO. 4, the linearized vector is a vector containing 6*His-GFP, and the target gene fragment and the linearized vector have homology regions; Step 2: Use T5 exonuclease to cut the 5' homology arms of the linearized vector and the target gene fragment, connect them into a circular plasmid, heat-shock transform them into Escherichia coli, and then screen and identify the recombinants; Step 3: Transform the recombinant plasmid into Escherichia coli BL21 (DE3) plysS and induce expression using IPTG.

2. The use according to claim 1, characterized in that The method for synthesizing the target gene fragment is as follows: using primers with sequences as shown in SEQ ID NOs. 9 to 10 as templates, annealing and pairing them into a DNA double strand, and then synthesizing the double strand as a template under the guidance of primer pairs with sequences as shown in SEQ ID NOs. 11 to 12.

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