Bacterial outer membrane vesicle presenting Stoppin peptide as well as construction method and application of bacterial outer membrane vesicle

Abstract

The invention provides a bacterial outer membrane vesicle presenting Stoppin peptide as well as a construction method and application of the bacterial outer membrane vesicle, and relates to the technical field of biological medicines. According to the invention, a fusion protein ClyA-Stoppin is firstly designed and synthesized, and the fusion protein ClyA-Stoppin is displayed on the surface of the bacterial outer membrane vesicle by using a genetic engineering technology, so that the engineered bacterial outer membrane vesicle W-Stoppin OMV for targeting presentation of Stoppin peptide is successfully constructed. The W-Stoppin OMV provided by the invention has a remarkable inhibition effect on invasion and migration of tumor cells and can be used for remarkably promoting apoptosis of the tumor cells. According to the present invention, with the Stoppin monopeptide, the problems of insufficient Stoppin cell penetrability and difficult intracellular delivery are effectively solved, compared with the Stoppin monopeptide, the Stoppin monopeptide shows the excellent anti-tumor effect, and the migration inhibition, the invasion inhibition and the apoptosis promotion effects on the tumor cells are significantly increased. The W-Stoppin OMV provided by the invention has huge application potential in the aspect of preparation of anti-tumor drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to a bacterial outer membrane vesicle presenting Stoppin peptide, a construction method and application thereof. BACKGROUND

[0002] The development of safe and efficient anti-tumor drugs has become a major issue to be solved in current medicine.

[0003] Bioactive peptides are a class of biological resources derived from natural proteins, which have shown important potential in the field of cancer treatment in recent years due to their high efficiency and low toxicity. Stoppin peptide is an anti-tumor peptide from Buthus martensii Karsch, which can effectively kill tumor cells in a p53-dependent manner. Cancer proteins MDM2 and MDMX bind to the transcriptional activation domain of the p53 tumor suppressor protein through their N-terminal domains, negatively regulating the activity and stability of the protein. In various tumors, gene amplification and overexpression of MDM2 and MDMX can lead to p53 inactivation and promote tumorigenesis, which makes the p53-MDM2 / MDMX interaction site an important molecular target for anti-cancer therapy. Stoppin protein can kill tumor cells by competitively blocking the p53-MDM2 / MDMX interaction, and is a potential p53-targeted drug.

[0004] Due to the target located inside the cell, Stoppin peptide needs to be delivered into the cell to exert its anti-tumor effect. However, the lack of cell penetration ability and low delivery efficiency seriously affect the exertion of its anti-tumor efficacy, which limits the application and promotion of Stoppin peptide. With the development of material science and nanoscience, the emergence of nano-carrier system provides a basis for the delivery of Stoppin. At present, there are many types of nano-drug carriers, and the most widely studied are polymer micelles, liposomes, biological proteins and exosomes. However, these carrier systems generally have defects such as high cytotoxicity, short half-life, poor targeting, and complex preparation process, and currently there is no effective carrier for delivering Stoppin peptide reported.

[0005] Outer membrane vesicles (OMVs) are a kind of lipid nanocapsule membrane vesicles naturally shed during the growth of gram-negative bacteria, usually between 20-250 nanometers in diameter, mainly composed of phospholipids, lipopolysaccharides, proteins and nucleic acids and other components. It carries part of the active substances of the parent, has the ability to activate immune response, and therefore has broad application prospects in the field of tumor treatment and anti-tumor drug delivery. Compared with artificially synthesized nanocarriers, OMVs are simple to prepare, and the characteristics of OMVs can be changed by functionalizing the parent bacteria based on genetic engineering technology. In addition, OMVs have good drug loading capacity and cell barrier crossing ability, and are easy to mass-produce at low cost, making them ideal drug carriers. SUMMARY

[0006] (I) Technical problems solved

[0007] In view of the deficiencies of the prior art, the present application uses genetic engineering technology to present Stoppin peptide on the surface of OMV, and constructs an engineered bacterial outer membrane vesicle W-Stoppin OMV which targets the presentation of Stoppin peptide.

[0008] (II) Technical solutions

[0009] To achieve the above object, the present application is realized by the following technical solutions:

[0010] Firstly, the present application provides a bacterial outer membrane vesicle presenting Stoppin peptide, wherein the bacterial outer membrane vesicle expresses a fusion protein ClyA-Stoppin with an amino acid sequence as shown in SEQ ID NO. 1 or a nucleotide sequence as shown in SEQ ID NO. 2, and the bacterial outer membrane vesicle is referred to as W-Stoppin OMV.

[0011] Further, the fusion protein ClyA-Stoppin is constructed by a ClyA protein with an amino acid sequence as shown in SEQ ID NO. 3, a Linker protein with an amino acid sequence as shown in SEQ ID NO. 4, a Myc tag protein with an amino acid sequence as shown in SEQ ID NO. 5, a Linker2 protein with an amino acid sequence as shown in SEQ ID NO. 6, and a Stoppin protein with an amino acid sequence as shown in SEQ ID NO. 7.

[0012] Further, the W-Stoppin OMV is obtained by centrifugation, purification, concentration and ultrahigh-speed centrifugation of a recombinant bacteria expressing the fusion protein ClyA-Stoppin; and the recombinant bacteria is obtained by transforming a recombinant plasmid ClyA-Stoppin-pGEX-6P-1 into W3110 Escherichia coli.

[0013] Further, the nucleotide sequence of the recombinant plasmid ClyA-Stoppin-pGEX-6P-1 is shown as SEQ ID NO. 8.

[0014] In addition, the application also provides a method for constructing W-Stoppin OMV, which is prepared according to the following steps:

[0015] (1) Design and synthesis of ClyA-Stoppin fusion protein

[0016] A fusion protein of Linker protein, 3×Myc tag protein, Linker2 protein and Stoppin protein is designed to be inserted in sequence at the C-terminal end of E. coli ClyA protein; wherein the amino acid sequence of ClyA protein is shown as SEQ ID NO. 3; the amino acid sequence of Linker protein is shown as SEQ ID NO. 4; the amino acid sequence of 3×Myc tag protein is shown as SEQ ID NO. 5; the amino acid sequence of Linker2 protein is shown as SEQ ID NO. 6; the amino acid sequence of Stoppin protein is shown as SEQ ID NO. 7; the structure of the fusion protein ClyA-Stoppin is ClyA-Linker-3×Myc-Linker2-Stoppin, and the amino acid sequence of the fusion protein ClyA-Stoppin is shown as SEQ ID NO. 1, and the nucleotide sequence is shown as SEQ ID NO. 2;

[0017] (2) Construction of recombinant plasmid ClyA-Stoppin-pGEX-6P-1

[0018] The gene encoded by the nucleotide shown as SEQ ID NO. 2 is cloned into the multiple cloning site on the pGEX-6P-1 vector, and the recombinant plasmid ClyA-Stoppin-pGEX-6P-1 is successfully constructed; the sequence of the recombinant plasmid is shown as SEQ ID NO. 8;

[0019] (3) Construction of recombinant bacteria expressing fusion protein ClyA-Stoppin

[0020] The recombinant plasmid ClyA-Stoppin-pGEX-6P-1 is transformed into W3110 E. coli competent cells to obtain a monoclonal strain stably expressing ClyA-Stoppin fusion protein, which is referred to as W-Stoppin strain;

[0021] (4) Induced expression of ClyA-Stoppin fusion protein

[0022] A 1L conical flask was added with 500mL ampicillin-resistant LB medium, 10μL W-Stoppin bacterial liquid was inoculated into the medium, and was marked as W-Stoppin; after the W-Stoppin was shaken at 37℃ and 220rpm for 4h, the OD value of the bacterial liquid was measured every 20min, when the OD value reached 0.5-0.6, 500μL IPTG with a concentration of 1M was added to make the final concentration of IPTG in the medium 1mM; then the expression of the exogenous protein was induced overnight at 21℃ and 200rpm, and 500mL W-Stoppin bacterial liquid was obtained;

[0023] (5) Extraction of W-Stoppin OMV

[0024] After the W-Stoppin bacterial liquid was centrifuged at 4℃ and 5,000×g for 15min, the supernatant was collected and filtered by a 0.45μm filter membrane, and the filtrate was concentrated 20 times by using an ultrafiltration centrifuge tube with a relative molecular mass cut-off of 100kD; the filtrate was filtered again by using a 0.22μm filter membrane, and then centrifuged at 150,000×g for 3h, and the supernatant was discarded, and the precipitate was W-Stoppin OMV.

[0025] In addition, the present application also provides a medicine for treating tumors, and the effective component in the medicine is W-Stoppin OMV.

[0026] The tumors include, but are not limited to, gastric cancer, breast cancer, lung cancer, liver cancer, colorectal cancer, esophageal cancer, thyroid cancer and pancreatic cancer.

[0027] (Three) Beneficial Effects

[0028] The present application firstly designs and synthesizes a fusion protein ClyA-Stoppin, and displays the fusion protein on the surface of bacterial outer membrane vesicles by using genetic engineering technology, and successfully constructs an engineered bacterial outer membrane vesicle W-Stoppin OMV which targets and presents Stoppin peptide.

[0029] 1. The W-Stoppin OMV provided by the present application has a significant inhibitory effect on the invasion and migration of tumor cells, and can significantly promote the apoptosis of tumor cells.

[0030] 2. The W-Stoppin OMV provided by the present application effectively solves the problems of insufficient cell penetration and intracellular delivery of Stoppin, and has a more excellent anti-tumor effect than Stoppin monopeptide, and the migration inhibition, invasion inhibition and pro-apoptosis effects on tumor cells are significantly increased. The W-Stoppin OMV provided by the present application has great application potential in the preparation of anti-tumor drugs. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 Schematic diagram of the fusion protein ClyA-Stoppin.

[0032] Figure 2 Agarose gel electrophoresis identification of ClyA-Stoppin-pGEX-6P-1 prokaryotic expression plasmid.

[0033] Figure 3 Sequencing identification of pGEX-6P-1-ClyA-Stoppin prokaryotic expression plasmid.

[0034] Figure 4 Western-blot identification of Stoppin protein expression in W-Stoppin E. coli, using Myc antibody.

[0035] Figure 5 Transmission electron microscopy identification of W-Stoppin OMV and W3110 OMV structure and morphology.

[0036] Figure 6 W-Stoppin OMV and W3110 OMV particle size detection.

[0037] Figure 7 W-Stoppin OMV and W3110 OMV PDI distribution.

[0038] Figure 8 Western-blot identification of W-Stoppin OMV and W3110 OMV marker protein expression and Stoppin protein outer vesicle surface presentation, using OmpA and Myc antibodies.

[0039] Figure 9 W-Stoppin OMV inhibits gastric cancer cell (panel A) and breast cancer cell (panel B) migration. Independent sample t-test, *P<0.05, ***P<0.001.

[0040] Figure 10 W-Stoppin OMV inhibits gastric cancer cell (panel A) and breast cancer cell (panel B) invasion. Independent sample t-test, ***P<0.001.

[0041] Figure 11 W-Stoppin OMV promotes breast cancer cell (panel A, C) and gastric cancer cell (panel B, D) apoptosis. Independent sample t-test, ***P<0.001.

[0042] Figure 12W-Stoppin OMV, Stoppin peptide inhibits tumor cell migration effect comparison. Independent sample t test, *P<0.05.

[0043] Figure 13 W-Stoppin OMV, Stoppin peptide inhibits tumor cell invasion effect comparison. Independent sample t test, *P<0.05, **P<0.01.

[0044] Figure 14 W-Stoppin OMV, Stoppin peptide promotes tumor cell apoptosis effect comparison. Independent sample t test, ***P<0.001. DETAILED DESCRIPTION

[0045] In order to make the purpose, technical scheme and advantages of the embodiments of the present application more clear, the technical scheme in the embodiments of the present application will be described clearly and completely below in combination with the embodiments of the present application. Obviously, the described embodiments are part 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 skilled in the art without creative labor fall within the scope of protection of the present application.

[0046] Embodiment 1

[0047] Construction and preparation of a bacterial outer membrane vesicle W-Stoppin OMV presenting Stoppin peptide

[0048] (1) Design of ClyA-Stoppin fusion protein

[0049] ClyA is a highly expressed protein on the bacterial outer membrane vesicle, and we designed a fusion protein in which Linker protein, 3×Myc tag protein, Linker2 protein and Stoppin protein were inserted in turn at the C terminus of E. coli ClyA protein. The amino acid sequence of ClyA protein is shown in SEQ ID NO. 3; the amino acid sequence of Linker protein is shown in SEQ ID NO. 4; the amino acid sequence of 3×Myc tag protein is shown in SEQ ID NO. 5; the amino acid sequence of Linker2 protein is shown in SEQ ID NO. 6; and the amino acid sequence of Stoppin protein is shown in SEQ ID NO. 7. The structure of the fusion protein ClyA-Stoppin is ClyA-Linker-3×Myc-Linker2-Stoppin (structure diagram as shown in Figure 1 The amino acid sequence of the obtained fusion protein ClyA-Stoppin is shown in SEQ ID NO. 1, and the nucleotide sequence is shown in SEQ ID NO. 2.

[0050] (2) Construction of ClyA-Stoppin-pGEX-6P-1 recombinant plasmid

[0051] The nucleotide sequence of the ClyA-Stoppin fusion protein is shown as SEQ ID NO. 2. The sequence was cloned into the multiple cloning site of the pGEX-6P-1 vector to successfully construct the recombinant plasmid ClyA-Stoppin-pGEX-6P-1. The sequence of the recombinant plasmid is shown as SEQ ID NO. 8.

[0052] (3) Construction and screening of E. coli expressing ClyA-Stoppin fusion protein

[0053] The recombinant plasmid ClyA-Stoppin-pGEX-6P-1 was transformed into W3110 E. coli competent cells to obtain a monoclonal strain stably expressing the ClyA-Stoppin fusion protein, which is referred to as the W-Stoppin strain.

[0054] The strain was cultured overnight using LB medium supplemented with ampicillin. The next day, the plasmid was extracted using a plasmid extraction kit, and the plasmid molecular weight was identified by 1% agarose gel electrophoresis. The results are shown in Figure 2 The theoretical size of the recombinant plasmid ClyA-Stoppin-pGEX-6P-1 is 5416 bp, which is consistent with the size of the electrophoretic band. Subsequently, the recombinant plasmid ClyA-Stoppin-pGEX-6P-1 was sequenced, and the sequencing results are shown in Figure 3 The sequencing results are consistent with the theoretical sequence of the plasmid, indicating that the ClyA-Stoppin-pGEX-6P-1 recombinant plasmid was successfully constructed.

[0055] (4) Induced expression of ClyA-Stoppin fusion protein

[0056] A 1L conical flask was filled with 500mL of ampicillin-resistant LB medium, and 10μL of W-Stoppin bacterial solution was inoculated into the medium, labeled as W-Stoppin. A 1L conical flask was filled with 500mL of LB medium without antibiotics, and 10μL of W3110 bacterial solution was inoculated into the medium, labeled as W3110. W-Stoppin and W3110 were shaken at 37℃ and 220rpm for 4h, and then the OD value of the bacterial solution was measured every 20 minutes. When the OD value reached 0.5-0.6, 500μL of IPTG with a concentration of 1M was added to make the final concentration of IPTG in the medium 1mM. Subsequently, the expression of the foreign protein was induced overnight at 21℃ and 200rpm, and 500mL of W-Stoppin and W3110 bacterial solution was obtained.

[0057] The proteins of the bacteria after induction were extracted, and the expression of the foreign proteins was identified by Western-blot, and the results are shown in Figure 4 The Myc-tagged protein was identified in the W-Stoppin group, and the size was consistent with the expected molecular weight 42.7 kDa of the ClyA-Stoppin fusion protein, and no foreign protein band was identified in the W3110 group of bacterial proteins. It is shown that the fusion protein ClyA-Stoppin designed in the application is successfully induced and expressed in the bacteria.

[0058] (5) Extraction of bacterial outer vesicles

[0059] After centrifugation of the above bacterial solution at 4°C, 5,000xg for 15 min, the supernatant was collected, and then filtered with a filter membrane of 0.45 μm, and the filtrate was concentrated 20 times with an ultrafiltration centrifuge tube with a relative molecular mass of 100 kD; then filtered again with a filter membrane of 0.22 μm, and then centrifuged at 150,000xg for 3 hours, and the supernatant was discarded, and the precipitate was the outer membrane vesicle of E. coli.

[0060] (6) Identification of bacterial outer vesicles

[0061] The bacterial outer membrane vesicles were resuspended with 200 μL PBS, and then the morphology of the outer vesicles was identified by transmission electron microscopy Figure 5 ), and the results show that the W-Stoppin OMV and the wild-type E. coli strain W3110 OMV have clear outlines and complete structures, and both present a tea-tray-like double-membrane structure, indicating that the two bacterial outer membrane vesicles are successfully extracted. In addition, there is no difference in morphology and structure between the W-Stoppin OMV and the W3110 OMV, indicating that the functional modification of the anticancer peptide Stoppin on the bacterial outer membrane vesicle has no obvious effect on the structure.

[0062] Then, the particle size distribution of the W-Stoppin OMV and the W3110 OMV was measured by a particle size analyzer, and the results are shown in Figure 6 The particle sizes of the W-Stoppin OMV and the W3110 OMV are 71.7 nm and 83.9 nm, respectively, which are consistent with the theoretical particle size of OMV, and there is no significant difference between the two groups. The PDI of the W-Stoppin OMV group and the W3110 OMV group is 0.44 and 0.53, respectively, indicating that the particle dispersion is good Figure 7 .

[0063] Three parallel groups of the extracted W3110 OMV and W-Stoppin OMV were set, which were the control group without treatment, the protease K digestion treatment group, and the SDS lipid membrane destruction and protease K digestion treatment group. The expression of the tag protein Myc and the membrane protein OmpA was detected by Western-blot using anti-Myc antibody and anti-OmpA antibody. The results are shown inFigure 8 As shown, the OMV marker protein OmpA was identified in both W-Stoppin OMV and W3110 OMV, indicating that the purity of the extracted OMV samples met the requirements. Myc-tagged protein expression was identified in W-Stoppin OMV, but not in W3110 OMV, indicating that the fusion protein ClyA-Stoppin was successfully expressed in OMV. Furthermore, when W-Stoppin OMV was treated with proteinase K alone, the Myc band disappeared, while the OmpA band remained inside the bacterial outer membrane vesicles. However, when W-Stoppin OMV was treated simultaneously with SDS permeabilizing agent and proteinase K, both the Myc and OmpA bands disappeared, indicating that the fusion ClyA-Stoppin was presented on the surface of the bacterial outer membrane vesicles. This provides a basis for the development of W-Stoppin OMV as an anti-tumor drug.

[0064] Example 2

[0065] Investigation into the antitumor effects of W-Stoppin OMV

[0066] 1. Inhibitory effect of W-Stoppin OMV on tumor cell migration

[0067] We used a cell scratch assay to detect the inhibitory effect of W-Stoppin OMV on tumor cell migration. 1×10⁶ cells were seeded separately. 5 HGC-27 gastric cancer cells and MDA-MB-231 breast cancer cells were cultured in 6-well plates until adherence. The wells were then scratched using a pipette tip, and W-Stoppin OMV and an equal concentration of W3110 OMV were added to achieve a final Stoppin peptide concentration of 10 μg / mL. A negative control group was also included. Images of the scratched cell areas were recorded under a microscope at 0h, 12h, and 24h, and cell migration rate was calculated based on the scratch width.

[0068] The results are as follows Figure 9 As shown in the figure, after 12 hours, the migration rate of HGC-27 cells in the W-Stoppin OMV group was significantly lower than that in the control group and the W3110 OMV group (P < 0.05). In MDA-MB-231 breast cancer cells, the W-Stoppin OMV group showed extremely significant cell migration inhibition after 24 hours (P < 0.001). W3110 OMV did not show any cell migration inhibition effect in either cell type. These results indicate that W-Stoppin OMV has a significant inhibitory effect on tumor cell migration.

[0069] 2. Inhibitory effect of W-Stoppin OMV on tumor cell invasion

[0070] We used Transwell chamber experiment to detect the tumor invasion inhibition effect of W-Stoppin OMV. Transwell chamber was placed in 24-well plate to construct the upper and lower layers of the culture chamber, 1 x 10 5 HGC-27 cells and MDA-MB-231 cells were inoculated into the upper layer of the hole, and W-Stoppin OMV with a final concentration of 10 μg / mL of Stoppin peptide was added, and complete medium was added to the lower layer. The negative control group and wild type vesicle W3110 OMV control group were set up. After 24 h and 48 h of culture, crystal violet staining was performed, and the number of cells was counted under a microscope.

[0071] The results are shown in Figure 10 The wild type bacterial outer membrane vesicle W3110 OMV had no significant effect on the invasion ability of HGC-27 cells and MDA-MB-231 cells at 24 h and 48 h (P>0.05). However, the number of invasive cells of HGC-27 cells and MDA-MB-231 cells treated with W-Stoppin OMV decreased significantly at 24 h and 48 h (P<0.001), indicating that W-Stoppin OMV had a significant inhibitory effect on the invasion ability of HGC-27 and MDA-MB-231 cells.

[0072] (3) The promoting effect of W-Stoppin OMV on tumor cell apoptosis

[0073] We used flow cytometry to detect the promoting effect of W-Stoppin OMV on tumor cell apoptosis. 1 x 10 6 HGC-27 cells and MDA-MB-231 cells were inoculated into 6-well plates, and when the cell confluence reached 80%, W-Stoppin OMV was added to make the final concentration of Stoppin 10 μg / mL. The wild type vesicle W3110 OMV control group was set up, and the cells were digested after 6 h and 24 h of culture. Annexin V FITC / PI double staining was used to stain the cells, and then flow cytometry was used for detection.

[0074] The results are shown in Figure 11 After 6 h of co-incubation, the apoptosis rate of HGC-27 and MDA-MB-231 cells in the W-Stoppin OMV group did not increase significantly compared with the W3110 OMV group. However, when co-incubated for 24 h, the apoptosis rate of HGC-27 and MDA-MB-231 cells in the W-Stoppin OMV group was significantly up-regulated compared with the W3110 OMV group (P<0.001). The above results showed that W-Stoppin OMV could effectively promote the apoptosis of HGC-27 and MDA-MB-231 cells.

[0075] This invention constructs engineered bacterial outer membrane vesicles (W-Stoppin OMVs) that target and present Stoppin peptides by displaying the ClyA-Stoppin protein on the surface of bacterial outer membrane vesicles. The W-Stoppin OMVs provided by this invention significantly inhibit the invasion and migration of tumor cells and effectively promote tumor cell apoptosis. W-Stoppin OMVs hold promise for further development and research as an anti-tumor drug.

[0076] Example 3

[0077] Comparison of the antitumor effects of W-Stoppin OMV and Stoppin monopeptide

[0078] (1) Inhibitory effect of W-Stoppin and Stoppin monopeptide on tumor cell migration

[0079] We used a cell scratch assay to detect the inhibitory effects of W-Stoppin OMV and Stoppin monopeptide on tumor cell migration. 1×10⁶ cells were seeded respectively. 5 MDA-MB-231 breast cancer cells were cultured in 6-well plates until adherence. The wells were then scratched using a pipette tip, and W-Stoppin OMV containing 20 μg of Stoppin and Stoppin monopeptide containing 20 μg of Stoppin were added. A negative control group was also included. Images of the scratched areas were recorded under a microscope at 0 h and 24 h, and cell migration rate was calculated based on the scratch width.

[0080] The results are as follows Figure 12 As shown, the cell migration rates of both the W-Stoppin OMV and Stoppin monopeptide groups were lower than those of the negative control group (P < 0.05), indicating that both groups have an inhibitory effect on tumor cell migration. Meanwhile, the cell migration rate of the W-Stoppin group was significantly lower than that of the Stoppin monopeptide group (P < 0.05), indicating that W-Stoppin OMV can significantly enhance the inhibitory effect on tumor cell migration.

[0081] (2) Inhibitory effects of W-Stoppin and Stoppin monopeptide on tumor cell invasion

[0082] We used a Transwell chamber assay to detect the inhibitory effects of W-Stoppin OMV and Stoppin monopeptide on tumor invasion. Transwell chambers were placed in 24-well plates to construct two-layer culture chambers, and 1×10⁶ cells were cultured in each well. 5HGC-27 cells were inoculated into the upper wells, and 5 μg of W-Stoppin OMV containing Stoppin and 5 μg of Stoppin monopeptide were added, respectively. Complete medium was added to the lower layer. A negative control group was set up. After 24 h and 48 h of culture, crystal violet staining was performed, and the number of cells was counted under a microscope.

[0083] The results are shown in Figure 13 W-Stoppin OMV and Stoppin monopeptide groups significantly decreased the cell invasion ability after 24 h and 48 h of co-incubation (P < 0.05, P < 0.01), indicating that W-Stoppin OMV and Stoppin monopeptide can effectively inhibit cell invasion. In addition, the invasion efficiency of W-Stoppin OMV on tumor cells was significantly lower than that of the Stoppin monopeptide group after 24 h and 48 h (P < 0.05, P < 0.01), indicating that the W-Stoppin OMV significantly outperforms Stoppin monopeptide in inhibiting cell invasion.

[0084] (3) W-Stoppin and Stoppin monopeptide promote cell apoptosis

[0085] Flow cytometry was used to detect the promotion of tumor cell apoptosis by W-Stoppin OMV and Stoppin peptide. 1 × 10 6 MDA-MB-231 breast cancer cells were inoculated into a 6-well plate, and when the cell confluence reached 80%, W-Stoppin OMV and Stoppin monopeptide were added to make the final concentration of Stoppin 10 μg / ml. After 24 h of culture, the cells were digested, stained with Annexin V FITC / PI double staining, and then detected by flow cytometry.

[0086] The results are shown in Figure 14 The apoptosis rate of the W-Stoppin OMV group was 18.9%, and the apoptosis rate of the Stoppin monopeptide group was 8.4%. The apoptosis rate of the W-Stoppin OMV group was significantly higher than that of the Stoppin monopeptide group (P < 0.001), indicating that the W-Stoppin OMV significantly enhances the pro-apoptotic effect compared to the Stoppin peptide monomer.

[0087] The Stoppin peptide derived from Buthus martensii Karsch can kill tumor cells by competitively blocking the interaction between p53 and MDM2 / MDMX. However, due to the inability of the Stoppin peptide to penetrate the cell barrier, the intracellular delivery efficiency is low, and the anti-tumor efficacy cannot be effectively exerted. The engineered outer membrane vesicle W-Stoppin OMV constructed in the present application effectively solves the problems of insufficient cell penetration and intracellular delivery difficulty of Stoppin, and exhibits more excellent anti-tumor effect than the Stoppin single peptide, and the migration inhibition, invasion inhibition and pro-apoptosis effect on tumor cells are significantly increased. The W-Stoppin OMV provided in the present application has great application potential in the preparation of anti-tumor drugs.

[0088] The above examples are only used to illustrate the technical solutions of the present application, but not to limit it; 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 equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A bacterial outer membrane vesicle presenting a Stoppin peptide, characterized in that, The bacterial outer membrane vesicle expresses a fusion protein ClyA-Stoppin with an amino acid sequence as shown in SEQ ID NO. 1 or a nucleotide sequence as shown in SEQ ID NO. 2, and the bacterial outer membrane vesicle is referred to as W-Stoppin OMV.

2. The bacterial outer membrane vesicle presenting stoppin peptide as described in claim 1, characterized in that, The W-Stoppin is obtained by centrifugation, purification, concentration and ultracentrifugation of recombinant bacteria expressing the fusion protein ClyA-Stoppin; the recombinant bacteria are obtained by transforming W3110 Escherichia coli with a recombinant plasmid ClyA-Stoppin-pGEX-6P-1.

3. A bacterial outer membrane vesicle presenting stoppin peptide as described in claim 1, characterized in that, The ClyA-Stoppin fusion protein is constructed from a ClyA protein with an amino acid sequence as shown in SEQ ID NO. 3, a Linker protein with an amino acid sequence as shown in SEQ ID NO. 4, a Myc tag protein with an amino acid sequence as shown in SEQ ID NO. 5, a Linker 2 protein with an amino acid sequence as shown in SEQ ID NO. 6, and a Stoppin protein with an amino acid sequence as shown in SEQ ID NO.

7.

4. A bacterial outer membrane vesicle presenting stoppin peptide as described in claim 2, characterized in that, The nucleotide sequence of the recombinant plasmid ClyA-Stoppin-pGEX-6P-1 is shown in SEQ ID NO.

8.

5. The method of constructing a bacterial outer membrane vesicle presenting a Stoppin peptide according to claim 1, wherein, The W-Stoppin OMV is prepared according to the following steps: (1) Design and synthesis of the ClyA-Stoppin fusion protein A fusion protein of Linker protein, 3×Myc tag protein, Linker 2 protein and Stoppin protein is designed to be inserted in sequence at the C-terminal end of the ClyA protein of Escherichia coli; the amino acid sequence of the ClyA protein is shown in SEQ ID NO. 3; the amino acid sequence of the Linker protein is shown in SEQ ID NO. 4; the amino acid sequence of the 3×Myc 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; the amino acid sequence of the Stoppin protein is shown in SEQ ID NO. 7; the structure of the fusion protein ClyA-Stoppin is ClyA-Linker-3×Myc-Linker2-Stoppin, and the amino acid sequence of the fusion protein ClyA-Stoppin is shown in SEQ ID NO. 1, and the nucleotide sequence is shown in SEQ ID NO. 2; (2) Construction of the recombinant plasmid ClyA-Stoppin-pGEX-6P-1 The gene with the nucleotide sequence as shown in SEQ ID NO. 2 is cloned into the multiple cloning site on the pGEX-6P-1 vector to successfully construct the recombinant plasmid ClyA-Stoppin-pGEX-6P-1; the sequence of the recombinant plasmid is shown in SEQ ID NO. 8; (3) Construction of the recombinant bacteria expressing the fusion protein ClyA-Stoppin The recombinant plasmid ClyA-Stoppin-pGEX-6P-1 is transformed into W3110 E. coli competent cells to obtain a monoclonal strain stably expressing the ClyA-Stoppin fusion protein, which is referred to as W-Stoppin strain; (4) ClyA-Stoppin fusion protein induced expression A 1L conical flask is added with 500mL ampicillin-resistant LB medium, and 10μL W-Stoppin bacterial liquid is inoculated into the medium and marked as W-Stoppin; the W-Stoppin is shaken at 37℃ and 220rpm for 4h, and the OD value of the bacterial liquid is measured every 20min; when the OD value reaches 0.5-0.6, 500μL IPTG with a concentration of 1M is added to make the final concentration of IPTG in the medium 1mM; then the expression of the exogenous protein is induced overnight at 21℃ and 200rpm, and 500mL W-Stoppin bacterial liquid is obtained; (5) Extraction of W-Stoppin OMV The W-Stoppin bacterial liquid is centrifuged at 4℃ and 5,000×g for 15min, and the supernatant is collected and filtered with a 0.45μm filter membrane; the filtrate is concentrated 20 times with an ultrafiltration centrifuge tube with a relative molecular mass cut-off of 100kD; the filtrate is filtered again with a 0.22μm filter membrane, and then centrifuged at 150,000×g for 3h; the supernatant is discarded, and the precipitate is W-Stoppin OMV.

6. A medicament for treating a tumor, characterized by, The effective component in the medicine is W-Stoppin OMV.

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