Escherichia coli outer membrane vesicle with tumor targeting effect and construction method thereof
By modifying the surface of E. coli outer membrane vesicles with GX1 targeting peptides to form a fusion protein with the membrane protein ClyA, tumor-targeting E. coli outer membrane vesicles were constructed, solving the problem of insufficient GX1 targeting and achieving efficient targeted delivery to cancer cells.
Patent Information
- Application Number
- CN202511350942.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-22
- Publication Date
- 2026-02-10
AI Technical Summary
Existing targeted chemotherapy drugs are prone to secondary drug resistance, and the tumor angiogenesis-targeting peptide GX1 has a short residence time in the body and insufficient targeting, which limits its application in tumor treatment.
By linking the GX1 targeting peptide with the membrane protein cytolysin A (ClyA) to form a fusion protein, and then modifying it onto the surface of E. coli outer membrane vesicles, E. coli outer membrane vesicles with tumor-targeting activity were constructed, and E. coli outer membrane vesicles were used as targeted delivery carriers.
It significantly improved the half-life and targeting of GX1 targeting peptides, achieving specific targeting of cancer cells and improving the efficiency and effectiveness of drug delivery.
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Figure CN121494993A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of engineering technology of outer membrane vesicles of Escherichia coli, and particularly relates to an outer membrane vesicle of Escherichia coli with tumor targeting effect and a construction method thereof. BACKGROUND
[0002] Gastric cancer is the fifth most common cancer worldwide and the third most common cause of cancer death. Risk factors for the disease include Helicobacter pylori infection, age, high salt intake, and a diet low in fruits and vegetables. The main treatment for early gastric cancer is endoscopic resection, lymph node dissection, or adjuvant chemotherapy. Advanced gastric cancer is treated with chemotherapy, targeted chemotherapy drugs. Because the early manifestations of gastric cancer are not obvious, most patients show advanced disease at the time of diagnosis, missing the best time for surgery and radiotherapy. Traditional targeted chemotherapy drugs are prone to secondary drug resistance, therefore, developing new drug delivery systems with high efficiency, low toxicity, strong targeting and good biocompatibility is a key direction for tumor treatment research.
[0003] Bacterial outer membrane vesicles (OMVs) are mainly membrane vesicles secreted by gram-negative bacteria. Escherichia coli is a human parasitic bacterium that does not cause disease in normal environments. OMVs from Escherichia coli can regulate the host's innate immune system and also serve as a delivery system, with good biocompatibility. Compared with artificial carriers, OMVs from Escherichia coli are less likely to be rapidly cleared by the body, extending the circulation time. In addition, OMVs have the characteristics of easy engineering, and by introducing tumor-specific antibodies, peptide segments or fluorescent labels through genetic engineering or chemical coupling, the properties of OMVs can be changed directionally, allowing them to have functions such as targeting, tracing, and stimulating immunity. Studies have shown that OMVs can deliver bioactive molecules carried by them to cells through endocytosis or membrane fusion, so engineering OMVs to have the ability to target tumor cells is expected to become a new drug delivery system for delivering drugs.
[0004] Studies have shown that inhibiting tumor angiogenesis to suppress cancer cell proliferation is a highly effective method. The tumor angiogenesis-targeting peptide GX1 can bind to specific receptors on gastric cancer tumor vascular endothelial cells, acting as a recognition signal and binding to specific sites on tumor vascular cells. Therefore, GX1-mediated drugs exhibit stronger targeting compared to the original drug. Furthermore, attaching its terminal N-group to the drug itself or the surface of the drug carrier can effectively improve therapeutic efficiency and reduce drug toxicity. Currently, research on GX1-modified carrier drugs mainly focuses on prodrugs and nanomedicine delivery systems. In addition, numerous studies have shown that GX1-targeting peptides can significantly enhance the antitumor activity of drugs without revealing serious toxic side effects; therefore, GX1-targeting peptides are ideal modified ligands. However, because the binding receptor of GX1-targeting peptides is located on the cell membrane, its residence time in vivo is short, its half-life is short, and its targeting is insufficient, limiting its application development.
[0005] Based on the above background, this invention utilizes the ease of surface modification of bacterial outer membrane vesicles to modify the surface of GX1 targeting peptides, aiming to improve the problems of short half-life and insufficient targeting of GX1 targeting peptides, while providing conditions for bacterial outer membrane vesicles to serve as targeted drug delivery carriers. Summary of the Invention
[0006] (a) Technical problems to be solved
[0007] To address the shortcomings of existing technologies, this invention provides an Escherichia coli outer membrane vesicle with tumor-targeting activity and a method for constructing it.
[0008] (II) Technical Solution
[0009] To achieve the above objectives, the present invention provides the following technical solution:
[0010] In a first aspect, the present invention provides a fusion protein, wherein the fusion protein is composed of a targeting peptide GX1 linked to a membrane protein cytolysin A (ClyA), the amino acid sequence of the fusion protein is shown in SEQ ID NO.1, and the nucleotide sequence of the fusion protein is shown in SEQ ID NO.2.
[0011] Specifically, the fusion protein is formed by linking the C-terminus of the ClyA protein (as shown in SEQ ID NO.3) to the N-terminus of the 3×MYC tag protein (as shown in SEQ ID NO.5) via Linker1 (as shown in SEQ ID NO.4), and then linking the N-terminus of the targeting peptide GX1 (as shown in SEQ ID NO.7) to the C-terminus of the 3×MYC protein via Linker2 (as shown in SEQ ID NO.6).
[0012] The second method is that the present invention provides an Escherichia coli outer membrane vesicle with tumor-targeting activity, wherein the Escherichia coli outer membrane vesicle expresses a fusion protein with a sequence as shown in SEQ ID NO.1 or SEQ ID NO.2.
[0013] Thirdly, the present invention provides a method for constructing Escherichia coli outer membrane vesicles with tumor-targeting activity, wherein the Escherichia coli outer membrane vesicles are prepared according to the following steps:
[0014] Step 1: Design fusion protein sequence
[0015] Linker1 (sequence shown in SEQ ID NO. 4), MYC tag protein sequence (sequence shown in SEQ ID NO. 5), Linker2 (sequence shown in SEQ ID NO. 6), and GX1 protein sequence (sequence shown in SEQ ID NO. 7) were sequentially inserted into the 3' end of the ClyA protein (sequence shown in SEQ ID NO. 3) of the natural Escherichia coli W3110 strain. An Ncol site and an Xhol site were added to the 5' end of the fusion protein, resulting in the fusion protein with the nucleotide sequence shown in SEQ ID NO. 2.
[0016] Step 2: Constructing a recombinant plasmid expressing the fusion protein
[0017] The fusion protein with the sequence shown in SEQ ID NO.2 was cloned into the multiple cloning site on the pGEX-6P-1 vector to construct the recombinant plasmid ClyA-GX1-Pgex-6P-1;
[0018] Step 3: Constructing recombinant bacteria expressing the fusion protein
[0019] The ClyA-GX1-Pgex-6P-1 plasmid was transformed into W3110 Escherichia coli competent cells to obtain a monoclonal strain that stably expresses the ClyA-GX1 fusion protein, which is referred to as C-GX1.
[0020] Step 4: Preparation of E. coli outer membrane vesicles with tumor-targeting activity
[0021] C-GX1 bacterial culture was inoculated into ampicillin-resistant LB medium and shaken at 220 rpm for 4 h. The OD value of the bacterial culture was measured every 20 minutes. When the OD value reached 0.5-0.6, IPTG was added to bring the final IPTG concentration in the medium to 1 mM. After adding IPTG, the culture was incubated overnight at 21 ℃ and 200 rpm to induce the expression of the exogenous protein, obtaining a C-GX1 bacterial culture that stably expresses the ClyA-GX1 fusion protein. The bacterial culture was centrifuged at 5000×g for 15 min at 4 ℃, and the supernatant was collected. The culture was then filtered through a 0.45 μm filter membrane, and the filtrate was concentrated 20 times using an ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kD. The culture was then filtered again through a 0.22 μm filter membrane and centrifuged at 150,000×g for 3 hours. The supernatant was discarded, and the precipitate was the outer membrane vesicle of E. coli with tumor-targeting activity.
[0022] Fourthly, the present invention provides the application of the above-mentioned Escherichia coli outer membrane vesicles with tumor-targeting effects as targeted delivery carriers in the preparation of drugs for treating tumors.
[0023] (III) Beneficial Effects
[0024] This invention modifies the surface of bacterial outer membrane vesicles with the tumor angiogenesis-targeting peptide GX1, successfully obtaining *E. coli* outer membrane vesicles with tumor-targeting activity. These *E. coli* outer membrane vesicles not only achieve specific targeting of cancer cells but also significantly improve the insufficient targeting ability of the tumor angiogenesis-targeting peptide GX1. The *E. coli* outer membrane vesicles provided by this invention hold promise for further development and research as carriers for targeted delivery of anti-tumor drugs. Attached Figure Description
[0025] Figure 1 The gene sequencing map of the recombinant plasmid ClyA-GX1-Pgex-6P-1.
[0026] Figure 2 Agarose gel electrophoresis image of recombinant plasmid ClyA-GX1-Pgex-6P-1 (A) and Western blot analysis of the expression of ClyA-GX1 fusion protein in recombinant bacteria (B).
[0027] Figure 3 Transmission electron microscopy images of W3110-OMVs and C-GX1-OMVs.
[0028] Figure 4 The particle size (A) and polydispersity index (B) of W3110-OMVs and C-NGR-OMVs were determined by dynamic light scattering (DLS).
[0029] Figure 5To detect the expression of the fusion protein ClyA-GX1 on the membrane surface of C-GX1-OMVs using Western blot.
[0030] Figure 6 The targeting binding ability of W3110-OMVs and C-GX1-OMVs to HGC27 cells and GES-1 cells, respectively, after co-incubation for 2 h by cell flow cytometry was detected. Note: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
[0031] Figure 7 The targeting binding ability of W3110-OMVs and C-GX1-OMVs to HGC27 cells and GES-1 cells after co-incubation for 4 h was detected by cell flow cytometry. Note: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
[0032] Figure 8 Results of cell fluorescence microscopy detection of the targeting binding ability of W3110-OMVs and C-GX1-OMVs with HGC27 cells and GES-1 cells, respectively; Note: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
[0033] Figure 9 The effect of co-incubating C-GX1-OMVs and GX1 peptide with HGC27 cells for h on the HGC27 signaling pathway; Note: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Detailed Implementation
[0034] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0035] Example 1
[0036] Construction and identification of Escherichia coli outer membrane vesicles with tumor-targeting activity
[0037] 1. Escherichia coli outer membrane vesicles with tumor-targeting activity were prepared according to the following steps:
[0038] Step 1: Design fusion protein sequence
[0039] Linker1 (sequence shown in SEQ ID NO. 4), MYC tag protein sequence (sequence shown in SEQ ID NO. 5), Linker2 (sequence shown in SEQ ID NO. 6), and GX1 protein sequence (sequence shown in SEQ ID NO. 7) were sequentially inserted into the 3' end of the ClyA protein (sequence shown in SEQ ID NO. 3) of the natural Escherichia coli W3110 strain. An Ncol site and an Xhol site were added to the 5' end of the fusion protein. The 3×MYC protein was used for the expression identification of the ClyA-GX1 fusion protein, resulting in the fusion protein with the nucleotide sequence shown in SEQ ID NO. 2.
[0040] Step 2: Constructing a recombinant plasmid expressing the fusion protein
[0041] The fusion protein with the sequence shown in SEQ ID NO.2 was cloned into the multiple cloning site on the pGEX-6P-1 vector to construct the recombinant plasmid ClyA-GX1-Pgex-6P-1;
[0042] Step 3: Constructing recombinant bacteria expressing the fusion protein
[0043] The ClyA-GX1-Pgex-6P-1 plasmid was transformed into W3110 Escherichia coli competent cells to obtain a monoclonal strain that stably expresses the ClyA-GX1 fusion protein, which is referred to as C-GX1.
[0044] Step 4: Preparation of E. coli outer membrane vesicles with tumor-targeting activity
[0045] C-GX1 bacterial culture was inoculated into ampicillin-resistant LB medium and shaken at 220 rpm for 4 h. The OD value of the bacterial culture was measured every 20 minutes. When the OD value reached 0.5-0.6, IPTG was added to bring the final IPTG concentration in the medium to 1 mM. After adding IPTG, the culture was incubated overnight at 21 ℃ and 200 rpm to induce the expression of the exogenous protein, obtaining a C-GX1 bacterial culture that stably expresses the ClyA-GX1 fusion protein. The bacterial culture was centrifuged at 5000×g for 15 min at 4 ℃, and the supernatant was collected. The supernatant was then filtered through a 0.45 μm filter membrane, and the filtrate was concentrated 20 times using an ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kD. The supernatant was then filtered again through a 0.22 μm filter membrane and centrifuged at 150,000×g for 3 hours. The supernatant was discarded, and the precipitate was the outer membrane vesicle of E. coli with tumor-targeting activity, abbreviated as C-GX1-OMVs.
[0046] Wild-type bacterial outer membrane vesicles (W3110-OMVs) were prepared using the same method as in step four above, except that the bacterial solution used was wild-type W3110 bacterial solution.
[0047] 2. Identification of Escherichia coli outer membrane vesicles with tumor-targeting activity
[0048] We first performed gene sequencing on the constructed recombinant plasmid ClyA-GX1-Pgex-6P-1, and the results showed that the gene sequence was consistent with the expected sequence. Figure 1 As shown in the image. Subsequent agarose gel electrophoresis confirmed that the band of the recombinant plasmid ClyA-GX1-Pgex-6P-1 was located approximately 1000 bp on the DNA DL 5000 Marker, consistent with the theoretical value of 1095 bp. The results are as follows. Figure 2 As shown in A. Western blot analysis then confirmed that the ClyA-GX1 fusion protein was expressed in the C-GX1 strain, but not in the wild-type W3110 strain, consistent with expectations. Figure 2 As shown in B. The above results demonstrate that the recombinant plasmid ClyA-GX1-Pgex-6P-1 was successfully constructed in this invention, and a single clone of Escherichia coli stably expressing the ClyA-GX1 fusion protein was successfully screened.
[0049] We then performed transmission electron microscopy on W3110-OMVs and C-GX1-OMVs, and the results are as follows: Figure 3 As shown, both W3110-OMVs and C-GX1-OMVs exhibit a distinct saucer-like bilayer membrane structure, appearing as single vesicles. Furthermore, dynamic light scattering (DLS) was used to determine the vesicle size, and the results are as follows... Figure 4 As shown in Figure A, W3110-OMVs and C-GX1-OMVs particles are concentrated at approximately 64.02 nm and 58.64 nm, respectively, and their particle size distribution exhibits a unimodal normal distribution. The polydispersity index (PDI) is... Figure 4 B) The W3110-OMVs and C-GX1-OMVs are 0.406 and 0.458 respectively, indicating that the particle dispersibility is good.
[0050] Finally, we further elucidated the expression of the ClyA-GX1 fusion protein in bacterial exomodal vesicles. Western blotting was used to detect the marker membrane protein ompA and the fusion protein tag MYC in bacterial OMVs, identifying the expression location of the target protein in C-GX1-OMVs. The results are as follows: Figure 5As shown, W3110-OMVs, C-GX1-OMVs, and OMVs and PDL1-OMVs treated with proteinase K all expressed the membrane protein ompA. When W3110-OMVs and C-GX1-OMVs had their lipid membranes disrupted by SDS and were simultaneously digested with proteinase K, the membrane protein ompA was destroyed and no longer expressed. However, the MYC-tagged protein of C-GX1-OMV with an intact lipid membrane could be digested by proteinase K, and the expression of the MYC-tagged protein could be detected in C-GX1-OMVs that had not undergone any treatment.
[0051] In summary, the outer membrane vesicle structure of E. coli prepared by the method of the present invention conforms to the typical structural characteristics of OMVs. The introduction of exogenous proteins did not destroy its unique structure. At the same time, the present invention successfully anchored the GX1 targeting peptide to the surface of E. coli outer membrane vesicles, providing a structural and functional basis for its use as a carrier for targeted delivery of tumor drugs.
[0052] Example 2
[0053] Targeting validation of C-GX1-OMV E. coli outer membrane vesicles
[0054] First, W3110-OMVs and C-GX1-OMVs were stained and labeled. The specific method was as follows: W3110-OMVs and C-GX1-OMVs obtained from 1,000 mL of bacterial culture were resuspended in 900 μL PBS buffer, and DiR staining working solution was added at a 1:1 volume ratio and mixed by pipetting. The mixture was then incubated at 37℃ in the dark for 30 min. After incubation, the mixture was centrifuged at 150,000×g for 3 hours, the supernatant was discarded, and the supernatant was allowed to dry completely. The stained W3110-OMVs and C-GX1-OMVs were then resuspended in 500 μL PBS.
[0055] We then co-incubated W3110-OMVs and C-GX1-OMVs with HGC27 gastric cancer cells that highly express the receptor gene VEGF and GES-1 human gastric mucosal epithelial cells that lowly express the receptor gene VEGF. We used flow cytometry and fluorescence microscopy to observe the uptake of W3110-OMVs and C-GX1-OMVs by different cells and evaluated the targeting effect of W3110-OMVs and C-GX1-OMVs on cancer cells.
[0056] The specific method is as follows:
[0057] 1. Flow cytometry experiment
[0058] HGC27 and GES-1 were used at 2×10 5Cells were evenly seeded into 12-well plates at a uniform density and cultured in complete culture medium for 2 days. Two wells were then incubated with 20 μL of DIR-labeled W3110-OMVs and 20 μL of C-GX1-OMVs for 4 h each. Similarly, two more wells were incubated with 20 μL of DIR-labeled W3110-OMVs and 20 μL of C-GX1-OMVs for 2 h each. Cells were digested with 100 μL of trypsin in each well and transferred to a 1.5 mL sterile centrifuge tube. The tubes were centrifuged at 800 rpm for 3 min, and the supernatant was discarded. 400 μL of sterile PBS was added to each tube, and the mixture was centrifuged at 1000 rpm for 5 min. The supernatant was discarded, and 400 μL of sterile PBS buffer was added again and the mixture was remixed. Flow cytometry was used to detect the number and proportion of cells binding to C-GX1-OMVs. Each group was repeated in triplicate.
[0059] 2. Fluorescence microscope
[0060] HGC27 and GES-1 were used at 2×10 5 The cells were evenly seeded into each well of a 12-well plate and cultured in the corresponding complete culture medium for 2 days. 20 μL of DIR-labeled W3110-OMVs and 20 μL of C-GX1-OMVs were added to two wells respectively and co-incubated with the cells for 2 h. The uptake of vesicles by the cells was observed using a fluorescence microscope, and the fluorescence intensity was quantitatively analyzed.
[0061] Flow cytometry results showed that ( Figure 6 AC, Figure 7 As shown in AC, after incubation with different cells for 2 h or 4 h, the binding rates of C-GX1-OMVs to HGC27 gastric cancer cells with high expression of the receptor gene VEGF and GES-1 human gastric mucosal epithelial cells with low expression of the receptor gene VEGF were significantly higher than those to W3110-OMVs. This result indicates that C-GX1-OMVs provided by the present invention has a significant targeting effect on cells expressing the receptor gene VEGF (P<0.05, P<0.0001).
[0062] Furthermore, we calculated the differences in binding rates between W3110-OMVs, C-GX1-OMVs and GES-1 cells, and the differences in binding rates between W3110-OMVs, C-GX1-OMVs and HGC27 cells, and performed statistical analysis on the two differences. The results are as follows: Figure 6 D、 Figure 7As shown in D, the results indicate that the C-GX1-OMVs provided by this invention have a significantly higher targeting binding effect on HGC27 cells that highly express the receptor gene VEGF than on GES-1 cells that low express the receptor gene VEGF (P<0.0001).
[0063] Fluorescence microscopy observation results show that ( Figure 8 In GES-1 cells, the fluorescence intensity of the C-GX1-OMVs group was significantly higher than that of the W3110-OMVs group (P<0.05); in HGC27 cells, the fluorescence intensity of the C-NGR-OMVs group was significantly higher than that of the W3110-OMVs group, with an extremely significant difference (P<0.0001); furthermore, the fluorescence intensity of HGC27 cells after co-incubation with C-NGR-OMVs was significantly higher than that of GES-1 cells with C-NGR-OMVs (P<0.001).
[0064] The above results all demonstrate that the C-GX1-OMVs prepared in this invention can achieve specific targeting of cancer cells, thus providing conditions for C-GX1-OMVs to serve as a carrier for targeted delivery of tumor drugs.
[0065] Example 3
[0066] Comparison of the targeting properties of C-GX1-OMV E. coli outer membrane vesicles and GX1 monopeptides
[0067] The receptor gene VEGF is a downstream gene targeted by the GX1 peptide. The expression level of the VEGF receptor gene reflects the activation effect of the targeted substance, and further reflects the integrity and effectiveness of the targeted substance's function. Therefore, we used VEGF to target gastric cancer cells HGC27 at a concentration of 4 × 10⁻⁶ cells / year. 5 The C-GX1-OMVs and GX1 single peptide were evenly added to each well of a 6-well plate and cultured for 2 days. Then, 20 μL each of C-GX1-OMVs and GX1 single peptide were co-incubated with HGC27 for 2 h. RNA was extracted and reverse transcribed into cDNA, and PCR was performed to detect the expression level of the downstream gene VEGF.
[0068] The results are as follows Figure 9 As shown, after co-incubation with GX1 peptide for 2 hours, the mRNA content of the VEGF gene in gastric cancer cells HGC27 was significantly lower than that in C-GX1-OMVs (P<0.001). These results indicate that the C-GX1-OMVs provided by this invention have a stronger targeting effect on the target gene compared to GX1 peptide.
[0069] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims
1. A fusion protein, characterized in that, The fusion protein is composed of a targeting peptide GX1 linked to a membrane protein cytolysin A (ClyA). The amino acid sequence of the fusion protein is shown in SEQ ID NO.1, and the nucleotide sequence of the fusion protein is shown in SEQ ID NO.
2.
2. The fusion protein according to claim 1, characterized in that, The fusion protein is formed by linking the C-terminus of the ClyA protein (as shown in SEQ ID NO.3) to the N-terminus of the 3×MYC tag protein (as shown in SEQ ID NO.5) via Linker1 (as shown in SEQ ID NO.4), and then linking the N-terminus of the targeting peptide GX1 (as shown in SEQ ID NO.7) to the C-terminus of the 3×MYC protein via Linker2 (as shown in SEQ ID NO.6).
3. An Escherichia coli outer membrane vesicle with tumor-targeting activity, characterized in that, The outer membrane vesicles of the *E. coli* express a fusion protein with the sequence shown in SEQ ID NO.1 or SEQ ID NO.
2.
4. The method for constructing Escherichia coli outer membrane vesicles with tumor-targeting activity as described in claim 3, characterized in that, The E. coli outer membrane vesicles were prepared according to the following steps: Step 1: Design fusion protein sequence Linker1 (sequence shown in SEQ ID NO. 4), MYC tag protein sequence (sequence shown in SEQ ID NO. 5), Linker2 (sequence shown in SEQ ID NO. 6), and GX1 protein sequence (sequence shown in SEQ ID NO. 7) were sequentially inserted into the 3' end of the ClyA protein (sequence shown in SEQ ID NO. 3) of the natural Escherichia coli W3110 strain. An Ncol site and an Xhol site were added to the 5' end of the fusion protein, resulting in the fusion protein with the nucleotide sequence shown in SEQ ID NO.
2. Step 2: Constructing a recombinant plasmid expressing the fusion protein The fusion protein with the sequence shown in SEQ ID NO.2 was cloned into the multiple cloning site on the pGEX-6P-1 vector to construct the recombinant plasmid ClyA-GX1-Pgex-6P-1; Step 3: Constructing recombinant bacteria expressing the fusion protein The ClyA-GX1-Pgex-6P-1 plasmid was transformed into W3110 Escherichia coli competent cells to obtain a monoclonal strain that stably expresses the ClyA-GX1 fusion protein, which is referred to as C-GX1. Step 4: Preparation of E. coli outer membrane vesicles with tumor-targeting activity C-GX1 bacterial culture was inoculated into ampicillin-resistant LB medium and shaken at 220 rpm for 4 h. The OD value of the bacterial culture was measured every 20 minutes. When the OD value reached 0.5-0.6, IPTG was added to bring the final IPTG concentration in the medium to 1 mM. After adding IPTG, the culture was incubated overnight at 21 ℃ and 200 rpm to induce the expression of the exogenous protein, obtaining a C-GX1 bacterial culture that stably expresses the ClyA-GX1 fusion protein. The bacterial culture was centrifuged at 5000×g for 15 min at 4 ℃, and the supernatant was collected. The culture was then filtered through a 0.45 μm filter membrane, and the filtrate was concentrated 20 times using an ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kD. The culture was then filtered again through a 0.22 μm filter membrane and centrifuged at 150,000×g for 3 hours. The supernatant was discarded, and the precipitate was the outer membrane vesicle of E. coli with tumor-targeting activity.
5. The application of the Escherichia coli outer membrane vesicles with tumor-targeting activity as described in claim 3 as a targeted delivery carrier in the preparation of drugs for treating tumors.