An acid-sensitive peptide-labeled bacterial outer membrane vesicle and a preparation method and application thereof

Abstract

The application provides a kind of bacterial outer membrane vesicle based on acid-sensitive peptide label and its preparation method and application, it is related to the technical field of engineered bacterial outer membrane vesicle.The ClyA-pHLIP fusion protein is designed and synthesized in the application, and the pHA-W3110OMV stably expressing the ClyA-pHLIP fusion protein is successfully obtained by using genetic engineering technology.The pHA-W3110OMV effectively solves the problem of easy aggregation of pHLIP in acidic environment, has good stability and solubility, and provides conditions for the stable targeting performance of pHLIP.Meanwhile, the pHA-W3110OMV not only retains the characteristics of targeting tumor cells in acidic environment, but also realizes the characteristics of targeting tumor cells in non-acidic environment.The pHA-W3110OMV has high targeting tracing efficiency on tumor cells under different pH conditions, and can realize qualitative and quantitative detection of tumor cells in peripheral blood, and has application potential in circulating tumor cell detection and tumor primary and metastasis localization.

Description

Technical Field

[0001] This invention relates to the field of engineered bacterial outer membrane vesicle technology, specifically to a bacterial outer membrane vesicle based on acid-sensitive peptide labeling, its preparation method, and its application. Background Technology

[0002] Cancer is the leading cause of death worldwide, and cancer-related issues are a global public health concern. Cancer survival rates vary depending on the stage of the cancer, and early diagnosis and screening, treatment monitoring, and recurrence monitoring have always been important components in reducing cancer incidence and mortality and improving cancer treatment. These methods help to detect and remove precancerous lesions early, before patients develop symptoms, monitor treatment effectiveness in real time, and predict the rate of tumor progression and prognosis.

[0003] Currently, commonly used clinical technologies for early cancer screening and monitoring are mainly divided into invasive and non-invasive methods. Invasive methods primarily involve tissue biopsy, including percutaneous biopsy and bronchoscopic imaging. Non-invasive methods mainly utilize imaging techniques, biomarker detection, liquid biopsy, exhaled breath testing, and circulating tumor cell (CTC) detection. CTC detection is a technique that captures and analyzes tumor cells present in peripheral blood to assess tumor progression, treatment effectiveness, and prognosis. By capturing "flowing tumor signals" in the blood, CTC detection enables dynamic monitoring of early cancer screening, tumor metastasis, treatment response, and prognosis, making it an important tool for precision oncology diagnosis and treatment. Its significance lies not only in optimizing clinical decision-making but also in providing patients with more individualized and timely interventions, ultimately improving quality of life and prolonging survival.

[0004] Bacterial outer membrane vesicles (OMVs) are vesicles ranging from 10 to 300 nm secreted by bacteria. Combining the advantages of natural substances and nanotechnology, they possess biocompatibility, tumor targeting, and immunomodulatory functions, playing a vital role in drug delivery, cancer therapy, bacterial infection treatment, and vaccines. As bacterial-secreted nanovesicles, OMVs are easily modified, making them highly promising for applications in cancer treatment research. Modifying OMVs can not only enhance their tumor targeting and reduce their systemic toxicity but also endow them with specific functions as needed.

[0005] pHLIP peptides (pH-low insertion peptides) are pH-sensitive peptides that utilize the pH difference between healthy and diseased cells as targeting markers to deliver therapeutic and imaging agents to cells in acidic diseased tissues. Extensive evidence suggests that pHLIPs can be used as imaging carriers and therapeutic agents for early tumor diagnosis and treatment, showing broad application prospects in areas such as targeted tumor therapy. Furthermore, pHLIPs can be used for extracellular and intracellular delivery of various imaging and therapeutic payloads, and can also be localized to the surface of various nanoparticles for targeted intracellular delivery of payloads. While pHLIPs possess advantages such as high targeting specificity, good tumor type compatibility, and strong tissue penetration, the varying acidity levels of different tumor tissues and different regions within the same tumor can lead to inconsistent insertion efficiency and targeting effects, affecting the uniformity and effectiveness of treatment or imaging. In addition, pHLIPs suffer from poor in vivo stability, are easily hydrolyzed by proteases, tend to aggregate in acidic environments, and have poor solubility, limiting their application development.

[0006] Based on the above research background, we designed and prepared engineered OMVs carrying pHLIP using genetic engineering technology. By utilizing the characteristics of OMVs, we improved the stability and water solubility of pHLIP, while obtaining pH-sensitive bacterial outer membrane vesicles with high efficacy in targeting tumor cells, providing a new method and means for the detection of circulating tumor cells. Summary of the Invention

[0007] (a) Technical problems to be solved

[0008] To address the shortcomings of existing technologies, this invention provides a bacterial outer membrane vesicle based on acid-sensitive peptide labeling, its preparation method, and its application.

[0009] (II) Technical Solution

[0010] To achieve the above objectives, the present invention provides the following technical solution:

[0011] In a first aspect, the present invention provides a fusion protein comprising a ClyA protein amino acid sequence and a pHLIP protein amino acid sequence, the amino acid sequence of the fusion protein being shown in SEQ ID NO.1, and the fusion protein being abbreviated as ClyA-pHLIP.

[0012] Secondly, the present invention provides a recombinant plasmid expressing the fusion protein ClyA-pHLIP.

[0013] Thirdly, the present invention provides a recombinant bacterium expressing the fusion protein ClyA-pHLIP.

[0014] Fourthly, the present invention provides a bacterial outer membrane vesicle expressing the fusion protein ClyA-pHLIP, the bacterial outer membrane vesicle being abbreviated as pHA-W3110 OMV.

[0015] Fifthly, the present invention provides a method for preparing pHA-W3110 OMV, comprising the following steps:

[0016] (1) Design of ClyA-pHLIP fusion protein sequence

[0017] The amino acid sequences of a Linker protein, a MYC tag protein, and a pHLIP protein were sequentially added to the C-terminus of the ClyA protein amino acid sequence to obtain the ClyA-pHLIP fusion protein with the amino acid sequence shown in SEQ ID NO.1; wherein the amino acid sequence of the ClyA protein is shown in SEQ ID NO.2; the amino acid sequence of the Linker protein is shown in SEQ ID NO.3; the amino acid sequence of the MYC tag protein is shown in SEQ ID NO.4; and the amino acid sequence of the pHLIP protein is shown in SEQ ID NO.5.

[0018] (2) Construct a recombinant plasmid expressing the fusion protein ClyA-pHLIP.

[0019] The ClyA-pHLIP fusion protein gene was cloned into the multiple cloning site on the pGEX-6P-1 vector to obtain the recombinant plasmid pGEX-6P-1-ClyA-pHLIP, which is abbreviated as pHA.

[0020] (3) Construct recombinant bacteria expressing the fusion protein ClyA-pHLIP

[0021] The recombinant plasmid pHA was transformed into W3110 competent cells, and monoclonal strains were screened by PCR technology to obtain a monoclonal strain that stably expresses the fusion protein ClyA-pHLIP. The monoclonal strain is referred to as pHA-W3110.

[0022] (4) Preparation of pHA-W3110 OMV: After centrifuging the pHA-W3110 bacterial culture at 5,000×g for 15 min at 4℃, the supernatant was collected and filtered through a 0.45 μm filter membrane. The filtrate was concentrated 20 times using an ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kDa. It was then filtered again through a 0.22 μm filter membrane and centrifuged at 150,000×g for 3 h. The supernatant was carefully discarded. The precipitate was then resuspended in 200 μL of PBS buffer, centrifuged at 150,000×g for 3 h, and the supernatant was discarded. The precipitate was pHA-W3110 OMV.

[0023] Sixthly, this invention provides the application of pHA-W3110 OMV in the preparation of reagents for detecting circulating tumor cells. The reagents are those used in flow cytometry, fluorescence microscopy, ELISA, laser confocal microscopy, and in vivo imaging techniques.

[0024] Furthermore, the reagent also contains fluorescent staining working solution, red blood cell lysis buffer, and PBS buffer.

[0025] (III) Beneficial Effects

[0026] 1. This invention designs and synthesizes a ClyA-pHLIP fusion protein, and successfully obtains pHA-W3110 OMV that stably expresses the ClyA-pHLIP fusion protein using genetic engineering technology. The pHA-W3110 OMV still retains the structure and particle size distribution of bacterial outer membrane vesicles.

[0027] 2. The pHA-W3110 OMV provided by this invention effectively solves the problem of easy aggregation of pHLIP in acidic environment, and has good stability and solubility, providing conditions for pHLIP to stably exert its targeting efficacy.

[0028] 3. The pHA-W3110 OMV prepared by this invention not only retains the characteristic of targeting tumor cells in an acidic environment, but also achieves the characteristic of targeting tumor cells in a non-acidic environment. It has the potential to detect solid tumors or circulating tumor cells in plasma and has broad-spectrum tumor recognition characteristics.

[0029] 4. The pHA-W3110 OMV provided by this invention can effectively capture gastric cancer cells, breast cancer cells, and leukemia cells under different pH conditions, demonstrating high targeting and tracing efficacy against tumor cells. The pHA-W3110 OMV can achieve qualitative and quantitative detection of tumor cells in peripheral blood, and has application potential in the detection of circulating tumor cells and the localization of primary and metastatic tumor lesions. Attached Figure Description

[0030] Figure 1 This is a schematic diagram of the structure of the fusion protein ClyA-pHLIP.

[0031] Figure 2 This is a schematic diagram of the structure of the recombinant plasmid pGEX-6P-1-ClyA-pHLIP(pHA).

[0032] Figure 3 To detect the expression of the tag protein MYC in pHA-W3110 and wild-type W3110 using a Western blot assay with an anti-MYC antibody.

[0033] Figure 4The results are from transmission electron microscopy of W3110 OMV(A) and pHA-W3110 OMV(B).

[0034] Figure 5 Particle size distribution maps of W3110 OMV and pHA-W3110 OMV obtained by DLS detection.

[0035] Figure 6 To detect the expression of the tag protein MYC and the membrane protein OmpA in W3110 OMV and pHA-W3110 OMV using Western blotting assays with anti-MYC antibody and anti-OmpA antibody.

[0036] Figure 7 To detect the expression of the tag protein MYC and the membrane protein OmpA outside the W3110 OMV and pHA-W3110 OMV membranes using a Western blot assay with anti-MYC and anti-OmpA antibodies.

[0037] Figure 8 The particle size distribution of pHLIP peptide and pHA-W3110 OMV under different pH conditions is shown.

[0038] Figure 9 The relative binding rates of fluorescently labeled W3110 OMV and pHA-W3110 OMV at concentrations of 5 μL / mL, 10 μL / mL, 20 μL / mL, and 40 μL / mL to HGC-27 cells after co-incubation at pH 5.6, 6.4, and 7.4 for 2 h were determined using cell loss assay; ****P < 0.0001.

[0039] Figure 10 The results show the targeted binding of W3110 OMV and pHA-W3110 OMV to gastric cancer cells (A), breast cancer cells (B), and leukemia cells (C) under different pH conditions. Detailed Implementation

[0040] 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.

[0041] Example 1

[0042] Preparation of bacterial outer membrane vesicle pHA-W3110 OMV based on acid-sensitive peptide labeling

[0043] 1. Design of ClyA-pHLIP fusion protein sequence

[0044] The amino acid sequences of the Linker protein, MYC tag protein, and pHLIP protein were sequentially added to the C-terminus of the ClyA protein amino acid sequence to obtain the ClyA-pHLIP fusion protein with the amino acid sequence shown in SEQ ID NO.1.

[0045] The amino acid sequence of the ClyA protein is shown in SEQ ID NO.2; the amino acid sequence of the Linker protein is shown in SEQ ID NO.3; the amino acid sequence of the MYC tag protein is shown in SEQ ID NO.4, where MYC is used to identify the expression of the ClyA-pHLIP fusion protein; the amino acid sequence of the pHLIP protein is shown in SEQ ID NO.5; and a schematic diagram of the structure of the ClyA-pHLIP fusion protein is shown in [image missing]. Figure 1 As shown.

[0046] 2. Construct a recombinant plasmid expressing the fusion protein ClyA-pHLIP.

[0047] The ClyA-pHLIP fusion protein gene was cloned into the multiple cloning site on the pGEX-6P-1 vector to obtain the recombinant plasmid pGEX-6P-1-ClyA-pHLIP, which is abbreviated as pHA.

[0048] A schematic diagram of the structure of pHA is shown below. Figure 2 As shown, the ClyA-pHLIP fusion protein sequence is 1170 bp. The insertion site of the ClyA-pHLIP fusion protein into the pGEX-6P-1 vector and the vector selection marker are information such as ampicillin resistance.

[0049] 3. Construct recombinant bacteria expressing the fusion protein ClyA-pHLIP

[0050] The recombinant plasmid pHA was transformed into W3110 competent cells, and monoclonal strains were screened by PCR technology to obtain a monoclonal strain that stably expresses the fusion protein ClyA-pHLIP. The monoclonal strain is referred to as pHA-W3110.

[0051] The selected pHA-W3110 and wild-type W3110 E. coli were cultured to an OD value of 0.4-0.6, and 1 mM IPTG was added overnight to induce protein expression. Bacterial cells were extracted to prepare a protein loading system. The expression of the tag protein MYC in pHA-W3110 and wild-type W3110 was detected by Western blotting using an anti-MYC antibody. The results are as follows: Figure 3As shown, the ClyA-pHLIP fusion protein was expressed in pHA-W3110 bacteria, while wild-type W3110 bacteria did not express the MYC protein; indicating that a monoclonal strain pHA-W3110 stably expressing the ClyA-pHLIP fusion protein was successfully prepared.

[0052] 4. Preparation of pHA-W3110 OMV

[0053] After centrifuging the pHA-W3110 bacterial culture at 5,000×g for 15 min at 4°C, the supernatant was collected and filtered through a 0.45 μm filter membrane. The filtrate was concentrated 20-fold using an ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kDa. The solution was then filtered again through a 0.22 μm filter membrane and centrifuged at 150,000×g for 3 h. The supernatant was carefully discarded. The solution was then resuspended in 200 μL of PBS buffer, centrifuged at 150,000×g for 3 h, and the supernatant was discarded. The precipitate was pHA-W3110 OMV.

[0054] 6. Preparation of W3110 OMV

[0055] The W3110 bacterial culture was centrifuged at 5,000×g for 15 min at 4°C, and the supernatant was collected. The supernatant was then filtered through a 0.45 μm filter membrane. The filtrate was concentrated 20-fold using an ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kDa. The supernatant was then filtered again through a 0.22 μm filter membrane and centrifuged at 150,000×g for 3 h. The supernatant was carefully discarded. The culture was then resuspended in 200 μL of PBS buffer, centrifuged at 150,000×g for 3 h, and the supernatant was discarded. The precipitate was W3110OMV.

[0056] 7. Identification of pHA-W3110 OMV

[0057] (1) Morphological and particle size identification of pHA-W3110 OMV

[0058] Transmission electron microscopy was used to observe and acquire images of pHA-W3110 OMV and W3110 OMV. The results are as follows: Figure 4 As shown, the morphology of W3110 OMV and pHA-W3110 OMV is mostly a saucer-like double membrane structure, with single distribution and a diameter range of 10-300 nm, which is consistent with the size of bacterial OMV.

[0059] The particle size of W3110 OMV and pHA-W3110 OMV was determined by dynamic light scattering (DLS), and the results are as follows: Figure 5As shown, the W3110 OMV and pHA-W3110 OMV particles are concentrated at around 133.1 nm and 150.6 nm, respectively, and the particle size distribution shows a unimodal normal distribution. The polydispersity index (PDI) is around 0.269 and 0.297, respectively, indicating that the W3110 OMV and pHA-W3110 OMV particles have good dispersibility.

[0060] The defined diameter of bacterial OMV is 10-300 nm. The above results show that the particle size of pHA-W3110 OMV prepared in this embodiment conforms to the range of bacterial OMV.

[0061] (2) Identification of ClyA-pHLIP fusion protein expression in pHA-W3110 OMV

[0062] The expression of the tag protein MYC and the membrane protein OmpA of wild-type W3110 bacterial OMV and pHA-W3110 bacterial OMV were detected by Western blotting using a combination of differential centrifugation, ultrafiltration, and ultra-high speed centrifugation. This verified the successful expression of the ClyA-pHLIP fusion protein in pHA-W3110 bacterial OMV. The results are as follows: Figure 6 As shown, both wild-type W3110 OMV and pHA-W3110 OMV express the membrane protein OmpA, while only pHA-W3110 OMV expresses the tag protein MYC, verifying the successful display of the ClyA-pHLIP fusion protein at the OMV level in E. coli.

[0063] (3) The ClyA-pHLIP fusion protein was successfully expressed on the pHA-W3110 OMV membrane.

[0064] Three parallel groups were set up for the extracted W3110 OMV and pHA-W3110 OMV: a control group with no treatment, a proteinase K digestion group, and a group that underwent both SDS lipid membrane disruption and proteinase K digestion. The expression of the tag protein MYC and the membrane protein OmpA was detected by Western blotting using anti-MYC antibody and anti-OmpA antibody.

[0065] The results are as follows Figure 7As shown, both the wild-type W3110 OMV and pHA-W3110 OMV in the control group and the proteinase K-treated group expressed the membrane protein OmpA. When OMV was treated simultaneously with SDS and proteinase K, the intracellular OmpA protein was destroyed, and the MYC-tagged protein in pHA-W3110 OMV was also destroyed. When treated with proteinase K only, without SDS treatment, the intracellular OmpA protein was still present in both wild-type W3110 OMV and pHA-W3110 OMV, but the MYC protein in pHA-W3110 OMV was still degraded, indicating that the MYC protein exists outside the OMV membrane. These results indicate that the ClyA-pHLIP protein is displayed on the OMV surface. This example obtained pHA-W3110 OMV stably expressing the ClyA-pHLIP fusion protein, laying the foundation for further exploration of the cell binding characteristics and tumor targeting of pHA-W3110 OMV.

[0066] Example 2

[0067] Stability Study of pHA-W3110 OMV

[0068] pHLIP peptide molecules exhibit reduced net charge and increased hydrophobicity in acidic environments, leading to decreased electrostatic repulsion between molecules. This enhances their aggregation, a tendency that increases as the solution pH decreases. Aggregation alters their spatial structure, hindering their ability to accurately recognize and bind to target sites, thus reducing the precision of targeted delivery. Furthermore, aggregation forms larger aggregates, increasing molecular volume and making it difficult for them to penetrate interstitial spaces or cell membranes, ultimately affecting their targeting efficacy.

[0069] To determine the stability of pHLIP and pHA-OMV, DLS measurements were performed on a 5 μM pHLIP peptide solution and a pHA-W3110 OMV suspension containing 5 μM pHLIP at different pH conditions (7.4, 6.5, and 5.2). The results are as follows: Figure 8 As shown, the particle size distribution of the pHLIP solution and the pHA-W3110 OMV suspension exhibits a unimodal normal distribution. In a neutral environment, the particle size of both pHLIP and pHA-W3110 OMV is around 100 nm. When the acidity of the environment increases, the particle size of pHA-W3110 OMV remains stable at around 100 nm. However, in an acidic environment, the particle size of pHLIP increases significantly to over 1000 nm, indicating obvious aggregation. These results demonstrate that the pHA-W3110 OMV provided by this invention effectively solves the problem of easy aggregation of pHLIP in an acidic environment, exhibiting good stability and solubility, thus providing conditions for pHLIP to stably exert its targeting efficacy.

[0070] Example 3

[0071] Study on the Targeting Characteristics of pHA-W3110 OMV

[0072] 1. Experimental cells

[0073] Gastric cancer cells HGC-27; Culture conditions: Culture medium: RPMI-1640 complete medium (containing 20% ​​FBS); Culture environment: 37℃, 5% CO2 cell incubator.

[0074] 2. Experimental Methods

[0075] (1) OMV staining labeling: DiR probe stock solution was diluted 500-2,500 times with sterile PBS buffer to prepare staining working solution (to be used under light-protected conditions); pHA-W3110 OMV and W3110 OMV extracted from 500 mL of bacterial culture were resuspended in 900 μL of PBS buffer, and DiR staining working solution was added at a 1:1 volume ratio and mixed by pipetting; incubated at 37℃ for 30 min, and centrifuged at 150,000×g for 3 h after incubation; the supernatant was discarded, and the supernatant was allowed to dry completely. The stained pHA-W3110 OMV and W3110 OMV were resuspended in 500 μL of PBS to obtain the stained pHA-W3110 OMV and W3110 OMV for later use;

[0076] (2) HGC-27 at 2×10 5The cells were evenly seeded into 12-well plates (4 plates per well, labeled 5 μL, 10 μL, 20 μL, and 40 μL). The first column was labeled pH=5.6, the second column pH=6.4, and the third column pH=7.4. The cells were cultured in 1 mL of the corresponding complete RPMI-1640 medium for 2 days. After 2 days, the cells were replaced with complete medium at the corresponding pH values. In the 12-well plates labeled 5, 5 μL of W3110 OMV was added to each well in the two left columns, and 5 μL of pHA-W3110 OMV was added to each well in the two right columns. Similarly, in the 12-well plates labeled 10, 20, and 40, the corresponding volume of W3110 OMV was added to each well in the two left columns, and the corresponding volume of pHA-W3110 was added to each well in the two right columns. For OMV, ensure complete protection from light and aseptic operation throughout the process; incubate in a cell culture incubator for 2 hours; add 100 μL of trypsin to each well for digestion and transfer the cells to a 1.5 mL sterile centrifuge tube, centrifuge at 800 rpm for 3 min, and discard the supernatant; add 400 μL of sterile PBS buffer to each tube, mix well by pipetting, and centrifuge at 1,000 rpm for 5 min; discard the supernatant, add 400 μL of sterile PBS buffer again, mix well by pipetting, and use flow cytometry to detect the proportion and number of cells that target and bind pHA-W3110 OMV and W3110 OMV, and calculate the cell binding rate.

[0077] Statistical analysis was performed using GraphPad Prism9 software, and graphs were generated. Independent samples t-tests were used to compare the two groups, and a p-value < 0.05 was considered statistically significant. Statistical differences in the graphs are represented as follows: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0078] 3. Experimental Results

[0079] Figure 9To determine the relative binding rates of fluorescently labeled W3110 OMV and pHA-W3110 OMV at concentrations of 5 μL / mL, 10 μL / mL, 20 μL / mL, and 40 μL / mL to HGC-27 cells after co-incubation at pH 5.6, 6.4, and 7.4 for 2 h using leukocyte lysis, we used leukocyte lysis to analyze the relative binding rates of W3110 OMV and pHA-W3110 OMV to gastric cancer cells. The results showed that, under different pH conditions, the binding rates of W3110 OMV and pHA-W3110 OMV to gastric cancer cells increased with increasing OMV concentration in a dose-dependent manner. Importantly, under different pH conditions, the binding rate of pHA-W3110 OMV to gastric cancer cells was significantly higher than that of W3110 OMV (P < 0.0001). Furthermore, we surprisingly found that pHA-W3110 OMV not only maintained a high binding rate to gastric cancer cells under acidic conditions but also maintained a high binding rate under near-neutral conditions.

[0080] The formation of an acidic tumor microenvironment primarily relies on the rapid energy supply from tumor cell glycolysis, leading to lactic acid accumulation. Simultaneously, abnormal vascularization in tumor tissue hinders the clearance of metabolic waste, further lowering the pH value. Free tumor cells in the blood and early metastatic tumor cells struggle to form a low-acidity surface microenvironment, resulting in low accuracy of pH-based tumor marker detection methods. The pHA-W3110 OMV prepared in this invention not only retains its ability to target tumor cells in acidic environments but also achieves the ability to target tumor cells in non-acidic environments. It possesses the potential to detect solid tumors or circulating tumor cells in plasma, exhibiting broad-spectrum tumor-targeting recognition properties.

[0081] Example 4

[0082] Targeted tracking of tumor cells by pHA-W3110 OMV

[0083] 1. Experimental cells

[0084] Normal human gastric mucosal cells GES-1, gastric cancer cells HGC-27, human breast cancer cells MDA-MB-231, and leukemia cells THP-1.

[0085] Culture conditions: GES-1 cells: RPMI-1640 complete medium (containing 10% FBS); HGC-27 cells: RPMI-1640 complete medium (containing 20% ​​FBS); Culture environment: 37℃, 5% CO2 cell incubator. MDA-MB-231 cells: DMEM complete medium (containing 10% FBS); THP-1 cells: RPMI-1640 complete medium (containing 10% FBS, containing 0.05mM β-mercaptoethanol); Culture environment: 37℃, 5% CO2 cell incubator.

[0086] 2 Experimental Methods

[0087] The staining and labeling method for OMV is the same as in Example 3.

[0088] 2.1 Targeting effect of pHA-W3110 OMV on normal gastric mucosal cells GES-1 cells and gastric cancer cells HGC-27

[0089] GES-1 and HGC-27 cells were fed at a rate of 2 × 10⁻⁶ 5 The cells were evenly seeded into each well of a 12-well plate (one plate per well). The first column was labeled pH = 5.6, the second column pH = 6.4, and the third column pH = 7.4. The cells were cultured in 1 mL of the corresponding complete RPMI-1640 medium for 2 days. After 2 days, the cells were replaced with complete medium at the corresponding pH values. In the 12-well plate, 20 μL of W3110 OMV was added to each well in the two left columns, and 20 μL of pHA-W3110 was added to each well in the two right columns. For OMV, ensure complete protection from light and aseptic operation throughout the process; incubate in a cell culture incubator for 2 hours; add 100 μL of trypsin to each well for digestion and transfer the cells to a 1.5 mL sterile centrifuge tube, centrifuge at 800 rpm for 3 min, and discard the supernatant; add 400 μL of sterile PBS buffer to each tube, mix well by pipetting, and centrifuge at 1,000 rpm for 5 min; discard the supernatant, add 400 μL of sterile PBS buffer again, mix well by pipetting, and use flow cytometry to detect the proportion and number of cells that target and bind pHA-W3110 OMV and W3110 OMV, and calculate the cell binding rate.

[0090] 2.2 Targeting effect of pHA-W3110 OMV on breast cancer cells

[0091] MDA-MB-231 cells were used at a rate of 2 × 10⁻⁶ 5The cells were evenly seeded into each well of a 12-well plate (one plate per well). The first column was labeled pH = 5.6, the second column pH = 6.4, and the third column pH = 7.4. The cells were cultured in 1 mL of 10% DMEM medium for 2 days. After 2 days, complete medium with the corresponding pH values ​​was added for cell replacement. In the 12-well plates, 20 μL of W3110 OMV was added to each well in the two left columns, and 20 μL of pHA-W3110 was added to each well in the two right columns. For OMV, ensure complete protection from light and aseptic operation throughout the process; incubate in a cell culture incubator for 2 hours; add 100 μL of trypsin to each well for digestion and transfer the cells to a 1.5 mL sterile centrifuge tube, centrifuge at 800 rpm for 3 min, and discard the supernatant; add 400 μL of sterile PBS buffer to each tube, mix well by pipetting, and centrifuge at 1,000 rpm for 5 min; discard the supernatant, add 400 μL of sterile PBS buffer again, mix well by pipetting, and use flow cytometry to detect the proportion and number of cells that target and bind pHA-W3110 OMV and W3110 OMV, and calculate the cell binding rate.

[0092] 2.3 Targeting effect of pHA-W3110 OMV on leukemia cells

[0093] THP-1 cells were loaded at 2×10 5 The cells were evenly seeded into 12-well plates (one plate per well) at a density equal to pH 5.6, pH 6.4, and pH 7.4 in the first column. The cells were cultured in THP-1 complete medium for 2 days. After 2 days, complete medium at the corresponding pH was added to change the medium. In the 12-well plates, 20 μL of W3110 OMV was added to each well in the two left columns, and 20 μL of pHA-W3110 OMV was added to each well in the two right columns, ensuring complete protection from light and aseptic technique throughout the process. The cells were incubated in a cell culture incubator for 2 hours. 100 μL of trypsin was added to each well to digest the cells, and the cells were transferred to 1.5 mL sterile centrifuge tubes. The tubes were centrifuged at 800 rpm for 3 min, and the supernatant was discarded. 400 μL of sterile PBS buffer was added to each tube, and the mixture was mixed by pipetting and 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 mixed by pipetting. Flow cytometry was used to detect the targeted binding of pHA-W3110. The cell ratio and number of OMV and W3110 OMV were calculated, and the cell binding rate was determined.

[0094] Statistical analysis was performed using GraphPad Prism9 software, and graphs were generated. Independent samples t-tests were used to compare the two groups, and a p-value < 0.05 was considered statistically significant. Statistical differences in the graphs are represented as follows: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0095] 3 Experimental Results

[0096] 3.1 pHA-W3110 OMV has the characteristic of targeting gastric cancer cells.

[0097] The relative binding rates of W3110 OMV and pHA-W3110 OMV to GES-1 gastric mucosal epithelial cells and HGC-27 gastric cancer cells, respectively, after co-incubation for 2 hours under different pH conditions were detected by flow cytometry. The flow cytometry results were processed using Flowjo software. The results are as follows: Figure 10 As shown in Figure A, statistical analysis indicates that the binding rate of pHA-W3110 OMV to gastric cancer cells is significantly higher than that of W3110 OMV to gastric cancer cells, and the results are statistically significant (P < 0.0001). This suggests that pHA-W3110 OMV provided by this invention has a targeting effect on gastric cancer cells compared to wild-type bacteria OMV.

[0098] 3.2pHA-W3110 OMV has the property of targeting breast cancer cells.

[0099] The relative binding rates of W3110 OMV and pHA-W3110OMV to MDA-MB-231 breast cancer cells at different pH conditions for 2 h were detected by flow cytometry. The flow cytometry results were processed using Flowjo software. Statistical analysis showed that the binding rate of pHA-W3110 OMV to breast cancer cells was significantly higher than that of W3110 OMV (P < 0.0001), indicating that pHA-W3110 OMV has targeting properties for breast cancer cells compared to wild-type OMV. The results are as follows: Figure 10 As shown in B.

[0100] 3.3pHA-W3110 OMV has the property of targeting leukemia cells.

[0101] The relative binding rates of W3110 OMV and pHA-W3110OMV to THP-1 leukemia cells were detected by flow cytometry under different pH conditions after co-incubation for 2 h at 20 μL / mL. The flow cytometry results were processed using Flowjo software. Statistical analysis showed that the binding rate of pHA-W3110 OMV to leukemia cells was significantly higher than that of W3110 OMV (P < 0.0001), indicating that pHA-W3110 OMV has leukemia cell targeting characteristics compared to wild-type OMV. Results are as follows. Figure 10 As shown in C.

[0102] 4. Conclusion

[0103] The pHA-W3110 OMV provided by this invention can effectively capture gastric cancer cells, breast cancer cells, and leukemia cells under different pH conditions, demonstrating high targeting and tracing efficacy against tumor cells. The pHA-W3110 OMV can achieve qualitative and quantitative detection of tumor cells in peripheral blood, and has application potential in the detection of circulating tumor cells and the localization of primary and metastatic tumor lesions.

[0104] 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 comprises a ClyA protein amino acid sequence and a pHLIP protein amino acid sequence, the amino acid sequence of the fusion protein is shown as SEQ ID NO. 1, and the fusion protein is abbreviated as ClyA-pHLIP.

2. A recombinant plasmid for expressing the fusion protein ClyA-pHLIP according to claim 1.

3. A recombinant bacterium for expressing the fusion protein ClyA-pHLIP according to claim 1.

4. A bacterial outer membrane vesicle for expressing the fusion protein ClyA-pHLIP according to claim 1, which is abbreviated as pHA-W3110 OMV.

5. The method of producing bacterial outer membrane vesicle pHA-W3110 OMV according to claim 4, wherein, Comprising the following steps: (1) Designing a ClyA-pHLIP fusion protein sequence A Linker protein amino acid sequence, a MYC tag protein amino acid sequence and a pHLIP protein amino acid sequence are sequentially added to the C-terminal end of the ClyA protein amino acid sequence to obtain a ClyA-pHLIP fusion protein with an amino acid sequence shown as SEQ ID NO. 1; wherein the ClyA protein amino acid sequence is shown as SEQ ID NO. 2; the Linker protein amino acid sequence is shown as SEQ ID NO. 3; the MYC tag protein amino acid sequence is shown as SEQ ID NO. 4; and the pHLIP protein amino acid sequence is shown as SEQ ID NO. 5; (2) Constructing a recombinant plasmid for expressing the fusion protein ClyA-pHLIP The ClyA-pHLIP fusion protein gene is cloned into a multiple cloning site on a pGEX-6P-1 vector to obtain a recombinant plasmid pGEX-6P-1-ClyA-pHLIP, which is abbreviated as pHA; (3) Constructing a recombinant bacterium for expressing the fusion protein ClyA-pHLIP The recombinant plasmid pHA is transformed into a W3110 competent cell, and a single clone strain is screened by PCR technology to obtain a single clone strain stably expressing the fusion protein ClyA-pHLIP, which is abbreviated as pHA-W3110; (4) Preparation of pHA-W3110 OMV The pHA-W3110 bacterial solution is centrifuged at 5,000xg for 15 min at 4℃, and the supernatant is collected, then filtered with a 0.45μm filter membrane, and the filtrate is concentrated 20 times with an ultrafiltration centrifuge tube with a relative molecular mass of 100kDa; filtered again with a 0.22μm filter membrane, then centrifuged at 150,000xg for 3h, and the supernatant is carefully discarded; resuspended with 200μL PBS buffer, centrifuged at 150,000xg for 3h, and the supernatant is discarded, and the precipitate is pHA-W3110 OMV.

6. Use of the bacterial outer membrane vesicle pHA-W3110 OMV according to claim 4 in the preparation of a reagent for detecting circulating tumor cells.

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