TLR2 receptor-targeted escherichia coli outer membrane vesicle and construction method thereof

Abstract

The invention provides an escherichia coli outer membrane vesicle targeting a TLR2 receptor and a construction method of the escherichia coli outer membrane vesicle, and relates to the technical field of biological medicines. According to the present invention, the ClyA-HLYV fusion protein is firstly constructed, the recombinant plasmid ClyA-HLYV-pGEX-6P-1 capable of stably expressing the fusion protein is successfully obtained through the gene engineering technology, the endogenous expression of the recombinant plasmid ClyA-HLYV-pGEX-6P-1 in the Escherichia coli W3110 strain is induced, and the targeting bacterial outer membrane vesicles (HLYV-OMVs) with the ClyA-HLYV fusion protein expressed on the surface are prepared; the HLYV-OMVs prepared by the invention not only retains the specific recognition capability of HLYV on TLR2 receptors on the surfaces of breast cancer cells, but also has the potential of entrapment of drugs, and realizes the dual efficacy of targeted recognition and drug-loading treatment; besides, the HLYV-OMVs effectively solves the key problem of long onset time of the HLYV single peptide in targeting application, has better stability, obviously accelerates the effect starting speed after the HLYV is combined with a receptor, and lays an important foundation for efficient targeting treatment of tumors.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to an E. coli outer membrane vesicle targeting TLR2 receptor and a construction method thereof. BACKGROUND

[0002] The existing treatment methods (surgery, chemotherapy, targeted therapy and immunotherapy) for breast cancer face fundamental limitations: traditional chemotherapy drugs cause serious side effects due to the lack of tumor targeting, and the prognosis of metastatic and recurrent breast cancer is still not ideal, and there is an urgent need to develop a new targeted drug delivery system with high tumor killing efficiency, low systemic toxicity and excellent biocompatibility.

[0003] Under this background, bacterial outer membrane vesicles (OMVs) have become a promising carrier platform due to their unique biological characteristics. OMVs produced by outer membrane bubbling and cell lysis of Gram-negative bacteria (such as E. coli) are natural nanovesicles composed of phospholipid bilayers, which can encapsulate proteins, nucleic acids and small molecule drugs and evade extracellular degradation; their secretion is regulated by pH, oxygen concentration and antibiotic pressure, and genetic engineering modification can endow them with tumor targeting and reduce endotoxin toxicity; more importantly, based on the mature E. coli fermentation process, OMVs can be produced on a large scale to meet the needs of clinical transformation. Engineered OMVs can enhance their tumor targeting, reduce their systemic toxicity and endow them with specific functions, therefore, OMVs are widely used in antitumor drug delivery research.

[0004] Toll-like receptor 2 (TLR2) is involved in the development of various diseases including cancer. It can specifically recognize the pathogen-associated molecular patterns of invading microorganisms and the danger signal-associated molecular patterns in the body, thereby activating the innate immune response and playing a key role in tumor immunity. After specific ligands bind to the cell membrane TLR2 receptor by forming a heterodimer with the auxiliary receptor, the NF-κB kinase is activated through the signal pathway to induce the synthesis and release of inflammatory factors. Studies have found that TLR2 expression can be detected on the surface of various tumor cells, and the level of expression is related to the malignant degree of the tumor. Therefore, the development of anti-tumor drugs targeting TLR2 has good clinical application prospects. HLYVSPW is an artificial polypeptide sequence that can specifically bind to the TLR2 receptor, activate the TLR2 signal pathway and release cytokines, providing a lead compound for targeted anti-tumor therapy. Compared with other cell-penetrating peptides, HLYVSPW has the characteristics of short sequence and strong targeting, and has the potential to be used as a carrier to transport therapeutic substances. However, the binding time of HLYVSPW to the TLR2 receptor is relatively long, and the effect is slow, which is a problem that needs to be solved urgently for the clinical application of HLYVSPW. The strategy of engineering OMVs to display HLYVSPV can not only enhance the enrichment of drugs in the tumor site by targeting TLR2, but also improve the stability and delivery efficiency of peptides by taking advantage of the carrier protection effect, providing an innovative idea for breaking through the clinical bottleneck of breast cancer targeted therapy. SUMMARY

[0005] (I) Technical problems solved

[0006] In view of the deficiencies in the prior art, the present application provides an E. coli outer membrane vesicle targeting TLR2 receptor and a construction method thereof.

[0007] (II) Technical solutions

[0008] To achieve the above object, the present application is implemented by the following technical solutions:

[0009] In a first aspect, the present application provides an E. coli outer membrane vesicle (referred to as HLYV-OMVs) targeting TLR2 receptor, which comprises a fusion protein ClyA-HLYV formed by a targeting peptide HLYVSPW and a membrane protein ClyA on the surface of the E. coli outer membrane vesicle, wherein the amino acid sequence of the HLYVSPW is shown in SEQ ID NO. 1, and the amino acid sequence of the fusion protein ClyA-HLYV is shown in SEQ ID NO. 2.

[0010] In a second aspect, the present application provides a construction method of the HLYV-OMVs, which comprises the following steps:

[0011] (1) Engineering construction of ClyA-HLYV fusion gene

[0012] A linker 1 sequence, a 3xMYC tag protein coding sequence, a linker 2 sequence, and a HLYVSPW coding sequence were introduced in sequence at the 3' end of the ClyA coding region of the E. coli W3110 wild strain, and an NcoI enzyme cutting site was added at the 5' end of the fusion gene and an XhoI enzyme cutting site was added at the 3' end, to construct a recombinant gene with the structure NcoI-ClyA-Linker 1-3xMYC-Linker 2-HLYVSPW-XhoI; the amino acid sequence of the recombinant gene is shown in SEQ ID NO. 2, the amino acid sequence of the ClyA protein is shown in SEQ ID NO. 3, the amino acid sequence of the linker 1 protein is shown in SEQ ID NO. 4, the amino acid sequence of the 3xMYC tag protein is shown in SEQ ID NO. 5, the amino acid sequence of the linker 2 protein is shown in SEQ ID NO. 6, and the amino acid sequence of the HLYVSPW protein is shown in SEQ ID NO. 1;

[0013] (2) Construction of a recombinant plasmid

[0014] The fusion gene was directionally cloned into the multiple cloning site of the prokaryotic expression vector pGEX-6P-1, and after ligation, transformation, plasmid extraction, and sequencing verification, the recombinant plasmid ClyA-HLYV-pGEX-6P-1 was obtained, and the gene sequence of the recombinant plasmid is shown in SEQ ID NO. 7;

[0015] (3) Selection of a monoclonal strain

[0016] The recombinant plasmid ClyA-HLYV-pGEX-6P-1 was introduced into the W3110 E. coli strain, and a monoclonal colony was selected, and a monoclonal strain stably expressing the ClyA-HLYV fusion protein was obtained by PCR reaction of the bacterial liquid, positive banding of the product by agarose gel electrophoresis, and Western Blot screening, which is hereinafter referred to as HLYV-W3110.

[0017] (4) Extraction of E. coli outer membrane vesicles

[0018] The HLYV-W3110 strain was inoculated into LB medium containing ampicillin, and after shaking at 37°C and 220 rpm for 4 h, the OD value of the bacterial liquid was measured every 20 min, and when the OD value reached 0.5-0.6, IPTG was added to a final concentration of 1 mM, and the expression of the exogenous protein was induced at 21°C and 200 rpm overnight to obtain the HLYV-W3110 bacterial liquid; the bacterial liquid was concentrated by ultrafiltration and extracted by ultracentrifugation to obtain the HLYV-OMVs.

[0019] Thirdly, the present invention provides the application of HLYV-OMVs, which are used to target MDA-MB-231 tumor cells in vitro.

[0020] Fourthly, the present invention provides the HLYV-OMVs as delivery carriers for preparing targeted cancer drugs.

[0021] (III) Beneficial Effects

[0022] This invention provides a method for constructing *E. coli* outer membrane vesicles targeting the TLR2 receptor. First, a ClyA-HLYV fusion protein was designed and constructed. Then, a recombinant plasmid, ClyA-HLYV-pGEX-6P-1, stably expressing this fusion protein was successfully obtained through genetic engineering. This plasmid was then endogenously expressed in *E. coli* strain W3110 to prepare targeted bacterial outer membrane vesicles (HLYV-OMVs) expressing the ClyA-HLYV fusion protein. The HLYV-OMVs completely retain the morphological characteristics and particle size distribution of natural bacterial outer membrane vesicles. The HLYV-OMVs prepared by this invention not only retain the specific recognition ability of HLYV on the TLR2 receptor on the surface of breast cancer cells but also possess the potential to encapsulate drugs, achieving dual efficacy of targeted recognition and drug delivery therapy. Furthermore, HLYV-OMVs effectively solve the key problem of long onset time in targeted applications of HLYV monopeptides and exhibit good stability, significantly accelerating the effect initiation speed after HLYV binds to the receptor, laying an important foundation for efficient targeted therapy. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the structure of the fusion protein ClyA-HLYV.

[0024] Figure 2 The sequencing results are for the recombinant plasmid ClyA-HLYV-pGEX-6P-1.

[0025] Figure 3 The image shows the recombinant plasmid ClyA-HLYV-pGEX-6P-1.

[0026] Figure 4 Agarose gel electrophoresis image of recombinant plasmid ClyA-HLYV-pGEX-6P-1.

[0027] Figure 5 The expression status of the ClyA-HLYV fusion protein as a marker protein in Escherichia coli.

[0028] Figure 6 For W3110-OMVs ( Figure 6 A), HLYV-OMVs ( Figure 6 B) Transmission electron microscope.

[0029] Figure 7 Particle size (A) and PDI (B) distribution of W3110-OMVs, HLYV-OMVs. Figure 7 Figure 7

[0030] Figure 8 Western Blot analysis of HLYV-OMVs surface fusion protein expression.

[0031] Figure 9 Flow cytometry analysis of W3110-OMVs, HLYV-OMVs binding to MDA-MB-231 and 293T cells after 2h incubation.

[0032] Figure 10 Flow cytometry analysis of W3110-OMVs, HLYV-OMVs binding to MDA-MB-231 and 293T cells after 4h incubation.

[0033] Figure 11 Immunofluorescence microscopy analysis of W3110-OMVs, HLYV-OMVs binding to MDA-MB-231 and 293T cells.

[0034] Figure 12 TLR2 expression analysis of MDA-MB-231 cells after 1h and 24h incubation with HLYV-OMVs and HLYV single peptide. DETAILED DESCRIPTION

[0035] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described below in conjunction with the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0036] Embodiment 1

[0037] Construction and identification of HLYV-OMVs

[0038] 1. Construction of ClyA-HLYV fusion protein

[0039] ​​Linker1, MYC tag protein sequence, Linker2, HLYVSPW protein sequence were inserted into the 3' end of ClyA protein of natural E. coli W3110 strain in turn, and Ncol site was added at 5' end of the fusion protein and Xhol site was added at 3' end, to construct a fusion protein with structure of Ncol-ClyA-Linker1-3xMYC-Linker2-HLYV-Xhol, and the fusion protein gene sequence is shown as SEQ ID NO. 2.

[0040] 2 Construction of ClyA-HLYV fusion protein recombinant plasmid

[0041] The Ncol-ClyA-Linker1-3xMYC-Linker2-HLYV-Xhol fusion protein gene was cloned into the multiple cloning site on the pGEX-6P-1 vector suitable for fusion protein expression, and a recombinant plasmid ClyA-HLYV-pGEX-6P-1 was successfully constructed, and the nucleotide sequence of the recombinant plasmid is shown as SEQ ID NO. 7.

[0042] 3 Establishment of E. coli monoclonal strain stably expressing ClyA-HLYV fusion protein

[0043] The recombinant plasmid was transformed into W3110 competent cells, and agarose gel electrophoresis was used to screen monoclonal strains, to obtain a monoclonal strain stably expressing ClyA-HLYV fusion protein, which is referred to as HLYV-W3110.

[0044] The monoclonal strain was picked for PCR primary screening, and the product was analyzed by 1% agarose gel electrophoresis. Positive colonies were sent for sequencing to verify the correctness of the recombinant gene sequence, and the results are shown in Figure 2 The fusion gene sequencing results are consistent with the theoretical sequence, indicating that the plasmid ClyA-HLYV-pGEX-6P-1 is successfully constructed. Subsequently, the positive colonies with correct sequencing were selected for expansion culture, and the recombinant plasmid was extracted and identified by agarose gel electrophoresis, and the results are shown in Figure 4 The recombinant plasmid ClyA-iRGD-pGEX-6P-1 band is located near 1000 bp, which is consistent with the theoretical value of 1095 bp. To verify the expression of the fusion protein, HLYV-W3110 and wild type W3110 E. coli were shaken to an OD value of 0.4-0.6, and IPTG (final concentration of 1 mM) was added for overnight induction of foreign protein expression. Then the bacterial body was extracted to prepare the protein loading system, and the expression of the tag protein MYC in HLYV-W3110 and wild type W3110 was detected by WB test using anti-MYC antibody, and the results are shown in Figure 5As shown, HLYV-W3110 strain can express MYC-tagged protein, while wild-type W3110 strain does not express MYC protein; the above results show that the monoclonal strain HLYV-W3110 stably expressing ClyA-HLYV fusion protein is successfully constructed.

[0045] 4Extraction of E. coli outer membrane vesicles (OMVs)

[0046] According to the above method, IPTG was used to induce the expression of exogenous protein ClyA-HLYV in bacteria, and 500 mL of HLYV-W3110 and W3110 bacterial solution was prepared; after centrifugation of the bacterial solution at 5,000 x g at 4°C for 15 min, the supernatant was collected, then filtered with a 0.45 μm filter membrane, and the filtrate was concentrated 20 times with an ultrafiltration centrifuge tube with a relative molecular mass cut-off of 100 kD; the concentrated supernatant was filtered again with a 0.22 μm filter membrane, then centrifuged at 150,000 x g for 3 h, the supernatant was discarded, and resuspended with 200 μL PBS buffer, to obtain HLYV-OMVs and W3110-OMVs, respectively.

[0047] 5Morphological identification of OMVs

[0048] Transmission electron microscopy was used to observe the precipitates of W3110-OMVs and HLYV-OMVs obtained. As shown in Figure 6 , the observed vesicles had a diameter ranging from 20 to 250 nm, had a typical double-layer membrane morphology of a tea tray, and mostly existed as single particles. The field of view was clean and had few contaminants, indicating good sample purity.

[0049] 6Dynamic light scattering (DLS) determination of particle size

[0050] DLS was used to detect the particle size of W3110-OMVs and HLYV-OMVs. The results are shown in Figure 7 , the particle size of W3110-OMVs and HLYV-OMVs was concentrated at about 67 nm and 68 nm, and the polydispersity coefficient PDI was about 0.402 and 0.445, indicating good particle dispersion. The defined diameter of bacterial OMVs is 20-250 nm, and the particle size of microvesicles obtained in this experiment meets the range of bacterial OMVs.

[0051] 7Western Blot detection of OMVs marker proteins

[0052] WB was used to detect the expression of tag protein MYC and membrane protein OmpA in W3110-OMVs and HLYV-OMVs, to verify whether the ClyA-HLYV fusion protein was successfully expressed in HLYV-OMVs. The results are shown in Figure 8As shown, the membrane protein OmpA was expressed in both wild-type W3110-OMVs and HLYV-OMVs, regardless of whether they were treated with proteinase K; however, the OmpA signal disappeared when the OMVs were treated with proteinase K combined with SDS. In addition, the expression of the tag protein MYC was detected only in HLYV-OMVs, which confirmed that the ClyA-HLYV fusion protein was successfully displayed on the surface of E. coli OMVs, indicating that HLYV-OMVs have the potential to be used as a tumor-targeting drug delivery carrier.

[0053] Example 2

[0054] Flow cytometry verification of the targeting of HLYV-OMVs to tumor cells

[0055] 1. Experimental materials

[0056] Experimental cells: breast cancer cells MDA-MB-231, human embryonic kidney epithelial cells 293T;

[0057] Culture conditions: the cell culture medium was DMEM complete medium (containing 10% FBS), and the cells were routinely cultured at 37°C in a 5% CO2 cell incubator.

[0058] 2. Experimental methods

[0059] 2.1 Fluorescent labeling of bacterial outer membrane vesicles

[0060] Under light-protected conditions, the probe DiR stock solution was diluted with sterile PBS buffer to prepare the staining working solution (freshly prepared); then, 900 μL of PBS buffer was used to resuspend W3110-OMVs and HLYV-OMVs, and DiR staining working solution was added according to a 1:1 volume ratio, followed by mixing and blowing, incubation at 37°C for 30 min, centrifugation at 150,000 x g for 3 h after incubation, discarding the supernatant, resuspending the precipitate with 500 μL of PBS, and obtaining DiR-labeled W3110-OMVs and HLYV-OMVs, which were stored in the dark for later use.

[0061] 2.2 Cell treatment and flow cytometry detection

[0062] The density of MDA-MB-231 cells and 293T cells was adjusted to 2 x 10 5Cells / mL were seeded into 12-well plates (two plates for each cell type, labeled as 2h incubation group and 4h incubation group, respectively). The next day, in the 12-well plates labeled as 4h incubation group, 20μL of W3110-OMVs were added to each well in the two left columns and 20μL of HLYV-OMVs were added to each well in the two right columns. Care was taken to protect the cells from light and maintain aseptic technique throughout the process, and the cells were incubated for a total of 4h in a cell culture incubator. In the 12-well plates labeled as 2h incubation group, 20μL of W3110-OMVs were added to each well in the two left columns and 20μL of HLYV-OMVs were added to each well in the two right columns. Care was taken to protect the cells from light and maintain aseptic technique throughout the process, and the cells were incubated for a total of 2h in a cell culture incubator. After incubation, 100 μL of trypsin was added to each well to digest the cells, and the cell suspension was transferred to a 1.5 mL sterile centrifuge tube. The cells were centrifuged at 800 rpm for 3 min, and the supernatant was discarded. The pellet was resuspended in 400 μL of sterile PBS and centrifuged at 1,000 rpm for 5 min, and the supernatant was discarded. The cells were then resuspended in 400 μL of sterile PBS, and the proportion and number of cells that targeted and bound W3110-OMVs and HLYV-OMVs were detected by flow cytometry. The binding rate to the cells was calculated.

[0063] 2.3 Statistical Methods

[0064] Statistical analysis was performed using GraphPad Prism 10 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.

[0065] 3 Results

[0066] Flow cytometry results as follows Figure 9 , 10 As shown, under 2h and 4h incubation conditions, the binding rate of HLYV-OMVs to MDA-MB-231 breast cancer cells was significantly higher than that of wild-type W3110-OMVs (P<0.001), and the binding efficiency of HLYV-OMVs to MDA-MB-231 cells was significantly higher than that to 293T cells (P<0.0001). Furthermore, with prolonged incubation, the binding rate in the 4h incubation group was further increased compared to the 2h group. These results indicate that, compared to non-targeting W3110-OMVs, HLYV-OMVs have a significantly enhanced targeting binding ability to MDA-MB-231 cells and exhibit time-dependent enrichment characteristics.

[0067] 4. Conclusion

[0068] The HLYV-OMVs provided by the application can effectively capture breast cancer cells MDA-MB-231, have higher target tracing function, and the binding efficiency of the HLYV-OMVs and the target cells can be enhanced by prolonging the incubation time.

[0069] Example 3

[0070] Immunofluorescence microscope verification of the targeting of HLYV-OMVs to tumor cells

[0071] 1 Experimental materials

[0072] Experimental cells: breast cancer cells MDA-MB-231 and human embryonic kidney epithelial cells 293T;

[0073] Culture conditions: the cell culture medium is DMEM complete medium (containing 10% FBS), and the cells are cultured at 37℃ in a 5% CO2 cell incubator.

[0074] 2 Experimental method

[0075] The staining and labeling method of the bacterial outer membrane vesicles is the same as that in Example 3.

[0076] The density of MDA-MB-231 and 293T cells is adjusted to 2x10 5 cells / mL with DMEM complete medium, and the cells are inoculated into twelve-well plates and placed in a 37℃, 5% CO2 cell incubator; the next day, 20μL of W3110-OMVs is added to each well in the left two columns of the 12-well plate, and 20μL of HLYV-OMVs is added to each well in the right two columns, and attention is paid to light-free and sterile operation, and the plates are placed in a 37℃, 5% CO2 cell incubator for 4h; then the culture medium is discarded, pre-cooled PBS is used for washing, 4% paraformaldehyde solution is added to fix the cells in the incubator for 10min, the fixing solution is discarded, and after washing with PBS, 300μL of DAPIO staining solution is used for light-free staining for 10min; after washing with PBS for 2-3 times, the cell uptake is observed immediately under a fluorescence microscope.

[0077] 3 Results

[0078] As shown in Figure 11 , in the MDA-MB-231 cells with high expression of HLYV receptors, the fluorescence signal intensity of the HLYV-OMVs group is significantly higher than that of the W3110-OMVs group (P<0.0001); while in the 293T cells with low expression of HLYV receptors, the fluorescence signals of the two groups of OMVs are weak, and there is no significant difference (P>0.05). The above results show that the targeting effect of the HLYV-OMVs provided by the application on MDA-MB-231 breast cancer cells is obviously higher than that of the wild-type W3110-OMVs.

[0079] Example 4

[0080] PCR verification of the rapid activation effect of HLYV-OMVs on downstream signaling pathways

[0081] 1. Experimental materials

[0082] Experimental cells: breast cancer cells MDA-MB-231;

[0083] Culture conditions: the cell culture medium was DMEM complete medium (containing 10% FBS), and the cells were routinely cultured at 37°C in a 5% CO2 cell incubator.

[0084] 2. Experimental steps

[0085] The density of MDA-MB-231 cells was adjusted to 2x10 5 cells / mL with DMEM complete medium, and the cells were inoculated in a twelve-well plate, 20μL HLYV-OMVs or HLYV single peptide was added to each well, and the plate was placed in a 37°C, 5% CO2 cell incubator for culture. After 1h and 24h of culture, the supernatant in the well was aspirated and the cells were collected, total RNA was extracted using the TRIzol method and reverse transcribed into cDNA. The cDNA was added to a 96-well enzyme-free PCR plate, and after 3min of centrifugation, qRT-PCR was performed using a CFXConnect PCR instrument to measure the expression level of the target gene TLR2; β-Actin was used as an internal reference, and at least 3 replicates were set up, and the Cq value range between replicates should not be >0.5, and the Cq value between replicates was averaged.

[0086] 3. Experimental results

[0087] As Figure 12 shown, the results showed that after 1h of co-incubation, the expression level of TLR2 receptor gene in the HLYV-OMVs group was significantly higher than that in the HLYV single peptide group (P<0.001), indicating that it can rapidly activate the downstream signaling pathway; after 24h of co-incubation, there was no significant difference in the expression level of the target gene between the two groups (P>0.05), suggesting that the rapid activation effect of HLYV-OMVs may reach a peak in the early stage.

[0088] 4. Conclusion

[0089] In summary, compared with HLYV single peptide, HLYV-OMVs has a faster activation effect on downstream signaling pathways, and this rapid activation characteristic not only provides a time window optimization basis for tumor targeted therapy, but also may further realize the potential application value of reducing the frequency or dose of drug administration by shortening the onset time, and provides support for subsequent treatment plan design.

[0090] The above examples are only used to illustrate the technical solutions of the present application, and are not intended to limit the present application; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that the technical solutions recorded in the foregoing examples can be modified, or some technical features can be replaced by equivalent features; and these modifications or replacements 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 application.

Claims

1. An Escherichia coli outer membrane vesicle (HLYV-OMVs) targeting the TLR2 receptor, characterized in that, The E. coli outer membrane vesicles contain a fusion protein ClyA-HLYV formed by the targeting peptide HLYVSPW and the membrane protein cytolysin A (ClyA) on the surface of the E. coli outer membrane vesicles, wherein the amino acid sequence of HLYVSPW is shown in SEQ ID NO.1 and the amino acid sequence of the fusion protein ClyA-HLYV is shown in SEQ ID NO.

2.

2. The Escherichia coli outer membrane vesicle targeting the TLR2 receptor according to claim 1, characterized in that, The method for constructing HLYV-OMVs includes the following steps: (1) Engineering construction of ClyA-HLYV fusion gene A recombinant gene with the following structure was sequentially introduced into the 3' end of the ClyA coding region of wild-type Escherichia coli W3110: the Linker1 sequence, the 3×MYC tag protein coding sequence, the Linker2 sequence, and the HLYVSPW coding sequence. Simultaneously, an NcoI restriction site was added to the 5' end of the fusion gene and an XhoI restriction site was added to the 3' end, constructing a recombinant gene with the structure NcoI-ClyA-Linker1-3×MYC-Linker2-HLYVSPW-XhoI. The amino acid sequences of the recombinant gene are shown in SEQ ID NO.2, the ClyA protein amino acid sequence in SEQ ID NO.3, the Linker1 protein amino acid sequence in SEQ ID NO.4, the 3×MYC tag protein sequence in SEQ ID NO.5, the Linker2 protein amino acid sequence in SEQ ID NO.6, and the HLYVSPW protein amino acid sequence in SEQ ID NO.

1. (2) Construction of recombinant plasmids The above-mentioned fusion gene fragment was directionally cloned into the multiple cloning site of the prokaryotic expression vector pGEX-6P-1. After ligation transformation, plasmid extraction and sequencing verification, the recombinant plasmid ClyA-HLYV-pGEX-6P-1 was obtained. The gene sequence of the recombinant plasmid is shown in SEQ ID NO.

7. (3) Screening for single-clone strains The recombinant plasmid ClyA-HLYV-pGEX-6P-1 was introduced into the W3110 Escherichia coli strain, and single colonies were selected. Based on the primers, the strain was screened by bacterial PCR, agarose gel electrophoresis of the product showing positive bands, and Western Blot to obtain a single colony strain that stably expresses the ClyA-HLYV fusion protein, hereinafter referred to as HLYV-W3110. (4) Extraction of Escherichia coli outer membrane vesicles HLYV-W3110 strain was inoculated into LB medium containing ampicillin and incubated at 37℃ and 220 rpm for 4 h. The OD value of the bacterial solution was measured every 20 min. When the OD value reached 0.5-0.6, IPTG was added to a final concentration of 1 mM. The culture was incubated overnight at 21℃ and 200 rpm to induce the expression of exogenous protein and obtain HLYV-W3110 bacterial solution. The bacterial solution was concentrated by ultrafiltration and extracted by ultracentrifugation to obtain HLYV-OMVs.

3. The HLYV-OMVs application as described in any one of claims 1 to 2, characterized in that, The HLYV-OMVs are used to target MDA-MB-231 tumor cells in vitro.

4. The HLYV-OMVs application as described in any one of claims 1 to 2, characterized in that, The HLYV-OMVs are used to prepare delivery carriers for targeted cancer therapy drugs.

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