Bacterial outer membrane vesicle based on genetic engineering modification as well as preparation and application of bacterial outer membrane vesicle

Abstract

The invention provides a bacterial outer membrane vesicle based on genetic engineering modification as well as preparation and application thereof, and relates to the technical field of biological medicines. According to the invention, through a genetic engineering technology, a bacterial outer membrane protein ClyA from escherichia coli W3110 is combined with a tumor killing peptide KLAK, and a recombinant plasmid ClyA-KLAK-pGEX-6P-1 for expressing a ClyA-KLAK fusion protein is constructed; the plasmid is transformed into W3110 escherichia coli to realize endogenous expression of the fusion protein, and the engineered outer membrane vesicles C-KLAK-OMVs with anti-tumor activity are successfully prepared. On the basis that the original morphology of the bacterial outer membrane vesicle is reserved, the anti-tumor activity of KLAK is also reserved, migration and invasion of tumor cells HGC27 and MDA-MB-231 can be remarkably inhibited, and apoptosis of the tumor cells HGC27 and MDA-MB-231 is promoted; meanwhile, the medicine loading potential is realized; in addition, the C-KLAK-OMVs also improves the problem of insufficient stability of the KLAK peptide in serum, and provides a new thought and reference for targeted therapy of tumors.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to a genetically engineered bacterial outer membrane vesicle and a preparation and application thereof. BACKGROUND

[0002] Gastric cancer is the fifth most common cancer and the third leading cause of cancer death worldwide. According to the statistics of the International Agency for Research on Cancer, by 2020, there were more than 1 million new cases of gastric cancer worldwide, and about 769,000 deaths. Risk factors for the disease include Helicobacter pylori infection, age, high salt intake, and a diet low in fruits and vegetables. Breast cancer accounts for about one-third of all malignant tumors in women, and its mortality rate is about 15% of the total number of diagnosed cases. Current treatments for gastric cancer and breast cancer include surgical intervention, radiotherapy, and taking chemotherapy drugs. They each have their own advantages and disadvantages, and are suitable for different types of patients. Traditional chemotherapy drugs have many drawbacks, including a lack of selectivity affecting healthy cells and undesirable secondary effects such as nausea, vomiting, and strong systemic secondary effects. Therefore, developing new drug delivery systems that are efficient, low in toxicity, and have both anticancer properties and biocompatibility has become an important direction in the study of tumor treatment.

[0003] Bacterial outer membrane vesicles (OMVs) are mainly membrane vesicles secreted by gram-negative bacteria. Their main components include bacterial lipopolysaccharide, outer membrane protein, and lipids. The types and yields of OMVs are affected by various factors such as growth conditions and external stimuli. Studies have found that OMVs from various bacteria can provide corresponding immune protection for the host by triggering different degrees of immune response. Escherichia coli, as a model organism, has mature genetic manipulation techniques, and its composition can be regulated by genetic modification (such as knocking out LPS to reduce toxicity, overexpressing tumor-targeting peptides to enhance specificity) or loading exogenous therapeutic molecules (such as antigens, antibody fragments). In addition, E. coli has low culture costs and mature fermentation techniques, and can produce OMVs on a large scale to meet the needs of clinical translation. Therefore, OMVs from E. coli have broad application prospects in the fields of tumor treatment, vaccine development, and drug nanocarriers due to their natural nanostructure, immune regulation function, engineering capability, and low-cost production.

[0004] KLAKLAK is a lysine-leucine-rich amphipathic alpha-helical peptide, which has significant selectivity to the anion membrane of prokaryotes, and can cause mitochondrial swelling and mitochondrial membrane damage when KLAKLAK is internalized into eukaryotic cells through appropriate targeting mechanisms, thereby leading to apoptosis. And KLAKLAK can activate the biochemical pathways related to apoptosis, including the activation of caspase family proteins and PARP (poly ADP ribose polymerase). In addition, KLAKLAK can also be used to carry and deliver genes or small molecules to enhance the anti-tumor effect. KLAKLAK has the following advantages in application: (1) it can selectively kill cancer cells and specifically induce apoptosis by targeting mitochondria or by modifying targeting molecules, tumor-specific antibodies, etc.; (2) it has a killing effect on various tumor cells (such as breast cancer, lung cancer, glioma, etc.); (3) it does not directly damage DNA, avoiding the induction of mutations or secondary cancers, and does not need to rely on the cell cycle (also effective on static cells); (4) in addition to the apoptosis effect, it retains the properties of antibacterial peptides, and cooperates with anti-infection treatment by inducing pathogen cell apoptosis or membrane damage; (5) it can be used as a drug delivery system (such as carrying chemotherapy drugs), achieving "apoptosis induction + chemotherapy / gene therapy" synergy; (6) it can be connected to targeting ligands (such as folate, RGD peptide, antibodies, etc.), improving its enrichment ability in tumor / lesion tissues, and thus promoting tumor apoptosis. However, KLAKLAK peptide also faces significant challenges in clinical application: on the one hand, its stability in the body is poor, and it is easily degraded by proteases in the plasma and tissues, resulting in a short half-life and difficulty in fully exerting its function; on the other hand, although it has a certain targeting property, non-specific binding is still difficult to completely avoid in the complex in vivo environment, which may have potential impact on normal tissues; in addition, in the complex tumor microenvironment, the mitochondrial apoptosis pathway induced by KLAKLAK alone may not be sufficient to completely eliminate tumor cells, and its ability to penetrate solid tumor barriers is also limited, often requiring combination with other treatment methods.

[0005] Based on the above background, the present application aims to combine OMVs derived from Escherichia coli with KLAKLAK peptide, and to construct a drug delivery system for delivering KLAKLAK using Escherichia coli OMVs as carriers, in order to improve the stability and targeting of KLAKLAK in vivo, and to provide a new strategy for the treatment of tumors that is efficient, low in toxicity and has good application prospects. SUMMARY

[0006] (I) Technical problems solved

[0007] In view of the deficiencies of the prior art, the present application provides a genetically engineered bacterial outer membrane vesicle and its preparation and application.

[0008] (II) Technical solutions

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

[0010] In a first aspect, the present application provides an E. coli outer membrane vesicle with anti-tumor effect, referred to as C-KLAK-OMVs, which displays a ClyA-KLAK fusion protein formed by a tumor-killing peptide KLAKLAK and a membrane protein cytolysin A (ClyA) on the outer membrane surface of E. coli; the amino acid sequence of the tumor-killing peptide KLAKLAK is shown in SEQ ID NO. 1, and the amino acid sequence of the ClyA-KLAK fusion protein is shown in SEQ ID NO. 2.

[0011] Specifically, the ClyA-KLAK fusion protein is formed by connecting the N-terminal of the 3×MYC tag protein through Linker 1 at the C-terminal of the ClyA protein, and then connecting the N-terminal of the tumor-killing peptide KLAKLAK through Linker 2 at the C-terminal of the 3×MYC protein, and the structure is ClyA-Linker 1-3×MYC-Linker2-KLAK; wherein 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 MYC tag protein is shown in SEQ ID NO. 5, the amino acid sequence of the Linker2 protein is shown in SEQ ID NO. 6, and the amino acid sequence of KLAKLAK is shown in SEQ ID NO. 1.

[0012] In a second aspect, the present application provides a ClyA-KLAK-pGEX-6P-1 recombinant plasmid expressing the ClyA-KLAK fusion protein, which is prepared according to the following steps:

[0013] (1) Constructing the ClyA-KLAK fusion protein

[0014] The sequences of Linker 1, 3×MYC, Linker 2 and KLAKLAK peptide proteins are inserted in sequence at the C-terminal of the ClyA protein of the natural E. coli W3110 strain to construct the fusion protein ClyA-Linker 1-3×MYC-Linker2-KLAK. To facilitate subsequent cloning operations, an NcoI site is introduced at the 5' end of the fusion protein, and an XhoI site is introduced at the 3' end, to construct a fusion protein with the structure of NcoI-ClyA-Linker 1-3×MYC-Linker 2-KLAK-XhoI. The nucleotide sequence is shown in SEQ ID NO. 7;

[0015] (2) Constructing the recombinant plasmid ClyA-KLAK-pGEX-6P-1

[0016] The Ncol-ClyA-Linker 1-3×MYC-Linker 2-KLAK-Xhol fusion protein gene was cloned into the multiple cloning site of the pGEX-6P-1 vector suitable for fusion protein expression, and the recombinant plasmid ClyA-KLAK-pGEX-6P-1 was successfully constructed, and the nucleotide sequence of the recombinant plasmid is shown as SEQ ID NO. 8.

[0017] In a third aspect, the present application provides a preparation method of C-KLAK-OMVs, comprising the following steps:

[0018] (1) The recombinant plasmid ClyA-KLAK-pGEX-6P-1 was transformed into W3110 E. coli competent cells to obtain a monoclonal strain stably expressing the ClyA-KLAK fusion protein, which was named C-KLAK strain;

[0019] (2) The C-KLAK strain was inoculated into LB medium containing ampicillin, and after shaking at 37℃ and 220 rpm for 4h, 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 make the final concentration of IPTG in the culture medium 1mM. Subsequently, the expression of foreign proteins was induced overnight at 21℃ and 200 rpm, and the C-KLAK bacterial solution was obtained;

[0020] (3) After centrifugation of the above bacterial solution at 4℃ and 5000×g for 15 min, the supernatant was collected, and then filtered with a 0.45μm filter membrane. The filtrate was concentrated 20 times using an ultrafiltration centrifuge tube with a relative molecular mass cutoff of 100kD. It was filtered again using a 0.22μm filter membrane, and then centrifuged at 150000×g for 3h. The supernatant was discarded, and the precipitate was collected as C-KLAK-OMVs.

[0021] In a fourth aspect, the present application provides the use of the C-KLAK-OMVs in the preparation of a drug for targeted treatment of tumors.

[0022] (III) Beneficial effects

[0023] The application provides a genetically engineered bacterial outer membrane vesicle and a preparation and application thereof, and the ClyA-KLAK fusion protein is constructed by combining the bacterial outer membrane protein ClyA from E. coli W3110 with the tumor-killing peptide KLAK through genetic engineering technology; the recombinant plasmid ClyA-KLAK-pGEX-6P-1 expressing the ClyA-KLAK fusion protein is constructed; the fusion protein is endogenously expressed by transforming the plasmid into the W3110 E. coli, and the engineered bacterial outer membrane vesicle C-KLAK-OMVs displaying the ClyA-KLAK fusion protein on the surface is successfully prepared. The vesicle retains the anti-tumor activity of KLAK on the basis of maintaining the original morphology of the bacterial outer membrane vesicle, can significantly inhibit the migration and invasion of tumor cells HGC27 and MDA-MB-231, and promote the apoptosis of the tumor cells; meanwhile, the vesicle has the potential of loading drugs, and in addition, the C-KLAK-OMVs also improve the problem of poor stability of the KLAK peptide in serum. The bacterial outer membrane vesicle is modified through genetic engineering, so that the bacterial outer membrane vesicle has the anti-tumor effect, and a new idea and reference are provided for the treatment of tumors. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 It is a structural schematic diagram of the fusion protein ClyA-KLAK.

[0025] Figure 2 It is a Sanger sequencing identification of the ClyA-KLAK-pGEX-6P-1 prokaryotic expression plasmid.

[0026] Figure 3 It is an agarose gel electrophoresis diagram of the recombinant plasmid ClyA-KLAK-pGEX-6P-1 prokaryotic expression plasmid.

[0027] Figure 4 It is the expression of the marker protein of the ClyA-KLAK fusion protein in C-KLAK E. coli.

[0028] Figure 5 It is a transmission electron microscope diagram of W3110-OMVs.

[0029] Figure 6 It is a transmission electron microscope diagram of C-KLAK-OMVs.

[0030] Figure 7 It is a particle size distribution diagram of W3110-OMVs.

[0031] Figure 8 It is a particle size distribution diagram of C-KLAK-OMVs.

[0032] Figure 9 It is the expression of the fusion protein on the surface of KLAK-OMVs detected by Western Blot method.

[0033] Figure 10 To detect the effect of C-KLAK-OMVs on the migration of gastric cancer cells HGC27 and breast cancer cells MDA-MB-231 by cell scratch experiment; independent sample t test, ****P<0.0001.

[0034] Figure 11 To detect the effect of C-KLAK-OMVs on the invasion of gastric cancer cells HGC27 by cell invasion experiment; independent sample t test, ***P<0.001, ****P<0.0001.

[0035] Figure 12 To detect the effect of C-KLAK-OMVs on the invasion of breast cancer cells MDA-MB-231 by cell invasion experiment; independent sample t test, ***P<0.001, ****P<0.0001.

[0036] Figure 13 To detect the effect of C-KLAK-OMVs on the apoptosis of gastric cancer cells HGC27 by cell apoptosis experiment; independent sample t test, **P<0.01, ***P<0.001.

[0037] Figure 14 To detect the effect of C-KLAK-OMVs on the apoptosis of breast cancer cells MDA-MB-231 by cell apoptosis experiment; independent sample t test, **P<0.01, ***P<0.001.

[0038] Figure 15 To detect the effect of KLAK monopeptide and C-KLAK-OMVs on the invasion ability of HGC27 gastric cancer cells after incubation with serum for 1h and 6h respectively by cell scratch experiment; independent sample t test, ****P<0.0001.

[0039] Figure 16 To detect the effect of C-KLAK-OMVs on the invasion ability of HGC27 gastric cancer cells after incubation with serum for 1h and 6h by cell invasion experiment; independent sample t test.

[0040] Figure 17 To detect the effect of KLAK monopeptide on the invasion ability of HGC27 gastric cancer cells after incubation with serum for 1h and 6h by cell invasion experiment; independent sample t test, ***P<0.001, ****P<0.0001.

[0041] Figure 18 To detect the effect of KLAK monopeptide and C-KLAK-OMVs on the apoptosis ability of HGC27 gastric cancer cells after incubation with serum for 1h and 6h respectively by cell apoptosis experiment; independent sample t test, ****P<0.0001. DETAILED DESCRIPTION

[0042] The technical solutions and advantages of the embodiments of the present application will be described clearly and completely 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. Based on the embodiments of 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.

[0043] Embodiment 1

[0044] Construction of recombinant plasmid ClyA-KLAK-pGEX-6P-1

[0045] 1 Construction of ClyA-KLAK fusion protein

[0046] The C-terminal end of the ClyA protein of the natural E. coli W3110 strain is sequentially inserted with a linker protein Linker 1, a MYC tag protein sequence (3xMYC), a linker protein Linker 2, and a KLAKLAK peptide protein sequence to construct a fusion protein ClyA-Linker 1-3xMYC-Linker 2-KLAK. To facilitate subsequent cloning operations, an Ncol site is introduced at the 5' end of the fusion protein, and an Xhol site is introduced at the 3' end, forming a fusion protein with the structure NcoI-ClyA-Linker1-3xMYC-Linker 2-KLAK-XhoI. The nucleotide sequence of the fusion protein is shown as SEQ ID NO. 7.

[0047] 2 Construction of recombinant plasmid ClyA-KLAK-pGEX-6P-1

[0048] The Ncol-ClyA-Linker 1-3xMYC-Linker 2-KLAK-Xhol fusion protein gene is cloned into the multiple cloning site on the pGEX-6P-1 vector suitable for fusion protein expression, and the recombinant plasmid ClyA-KLAK-pGEX-6P-1 is successfully constructed. The nucleotide sequence of the recombinant plasmid is shown as SEQ ID NO. 8.

[0049] Embodiment 2

[0050] Preparation and identification of C-KLAK-OMVs with anti-tumor effect

[0051] 1 Establishment of E. coli monoclonal strain stably expressing ClyA-KLAK fusion protein

[0052] The recombinant plasmid ClyA-KLAK-pGEX-6P-1 was transformed into the competent cells of E. coli W3110, and then the competent cells were incubated on ice for 30 min, and then quickly placed in a 42°C water bath for 60 s, and then placed back on ice for 2 min. Then 800 μL of LB medium without antibiotics was added, and the culture was incubated at 37°C and 200 rpm for 1 h. The bacterial solution was centrifuged at 3000 g for 2 min, and the supernatant was discarded. 50 μL of the bacterial solution was used to resuspend the bacterial pellet, and the resuspended bacterial solution was spread on an LB plate medium added with ampicillin and incubated at 37°C overnight. The next day, a single colony was picked, and the strain was named C-KLAK strain.

[0053] The single colony was picked for colony PCR primary screening, and the PCR product was analyzed by 1% agarose gel electrophoresis. The positive colonies were sent for sequencing to verify the correctness of the recombinant gene sequence. The results are shown in Figure 2 The sequencing results of the fusion gene are consistent with the theoretical sequence, indicating that the plasmid ClyA-KLAK-pGEX-6P-1 is successfully constructed. Then, the positive colonies with correct sequencing were selected for expansion culture, and the recombinant plasmid was extracted and identified by agarose gel electrophoresis. The results are shown in Figure 3 The band of the recombinant plasmid ClyA-KLAK-pGEX-6P-1 is located near 1000 bp of DNA DL 5000 Marker, which is consistent with the theoretical value of 1110 bp. To verify the expression of the fusion protein, the target strain (C-KLAK) and the wild type control (W3110) were induced with IPTG (final concentration of 1 mM) at 21°C overnight when the OD600 reached 0.5-0.6. The bacterial cells were collected, lysed, and subjected to SDS-PAGE protein electrophoresis, and then transferred to a PVDF membrane. Western blot analysis was performed using an anti-MYC tag antibody. The results are shown in Figure 4 The ClyA-KLAK fusion protein is expressed in the C-KLAK strain, and the wild type W3110 strain does not express, which is consistent with the expectation. The above results show that the recombinant plasmid ClyA-KLAK-pGEX-6P-1 is successfully constructed, and the E. coli single colony strain stably expressing the ClyA-KLAK fusion protein is successfully screened.

[0054] 2Extraction of E. coli outer membrane vesicles

[0055] The C-KLAK strain was inoculated into 500 mL LB medium containing ampicillin, and after shaking at 37°C, 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 make the final concentration of IPTG in the medium 1 mM. Then the expression of foreign proteins was induced overnight at 21°C, 200 rpm. The C-KLAK bacterial solution was obtained, and the W3110 strain was inoculated in a non-resistant medium and cultured overnight under the same conditions as a control to obtain the W3110 bacterial solution.

[0056] After centrifuging the above bacterial solution at 5000 x g for 15 min at 4°C, the supernatant was collected and filtered with a 0.45 μm filter membrane. The filtrate was concentrated 20 times with an ultrafiltration centrifuge tube with a relative molecular mass cutoff of 100 kD. The concentrated supernatant was filtered again with a 0.22 μm filter membrane, and then centrifuged at 150,000 x g for 3 h. The supernatant was carefully discarded, and resuspended with 200 μL PBS buffer to obtain C-KLAK-OMVs and W3110-OMVs.

[0057] 3 Morphological identification of bacterial outer membrane vesicles

[0058] The morphology and structure of W3110-OMVs and C-KLAK-OMVs were identified by transmission electron microscopy. The results are shown in Figure 5 、 6 The diameter of the C-KLAK-OMVs and W3110-OMVs obtained by ultracentrifugation ranged from 20 to 250 nm, had a clear double-layer membrane structure like a tea tray, were single distributed, had a clear background, and had few contaminants.

[0059] 4 Dynamic light scattering for particle size determination

[0060] The particle size of W3110-OMVs and C-KLAK-OMVs was detected by dynamic light scattering (DLS). The results are shown in Figure 7 、 8 The particle size of W3110-OMVs and C-KLAK-OMVs was concentrated around 116.5 nm and 65.53 nm, and the particle size distribution was single-peak normal distribution with a polydispersity coefficient PDI of about 0.433 and 0.340. The defined diameter of bacterial OMVs was 20-250 nm. The microvesicle particle size obtained in this experiment was within the range of bacterial OMVs, and the particle dispersion was good.

[0061] 5 Western Blot detection of OMVs marker proteins

[0062] The total protein concentration of OMVs was determined by BCA kit. The extracted W3110-OMVs and C-KLAK-OMVs were divided into three groups respectively: control group, protease K treatment group and SDS treatment group, and were labeled correspondingly. According to the protein concentration determination results, the corresponding volume of OMVs suspension was prepared according to the amount of 1 μg of protein for sample loading. Equal volume of 40 mM Tris HCl (pH = 8) and protease K (final concentration 0.1 mg / mL) was added to the OMVs suspension of the protease K treatment group and the SDS treatment group, and after 30 min of treatment at 37°C, protease inhibitor PMSF was added to make its final concentration 1 mM; then 1% SDS solution was added to the OMVs suspension of the SDS treatment group. The WB method was used to detect the marker membrane protein ompA and fusion protein tag MYC in bacterial OMVs to identify the expression position of the target protein in OMVs.

[0063] The experimental results are shown in Figure 9 As shown in the table, the wild type W3110-OMVs and C-KLAK-OMVs of the control group and the protease K treatment group both expressed the membrane protein ompA; when the OMVs were destroyed by SDS and treated by protease K digestion, the ompA protein was destroyed. The expression of MYC tag protein can be detected in untreated C-KLAK-OMVs, and the MYC tag protein in C-KLAK-OMVs can be digested by protease K under the condition of intact lipid membrane, which verifies the membrane display of ClyA-KLAK fusion protein on E. coli OMVs.

[0064] The above experimental results show that the wild type E. coli W3110 and E. coli outer membrane vesicles displaying ClyA-KLAK fusion protein are extracted by combining high-speed centrifugation, ultrafiltration and ultra-high-speed centrifugation. The bacterial supernatant is concentrated by using an ultrafiltration tube to enrich E. coli outer membrane vesicles, and then E. coli outer membrane vesicles are extracted by using ultra-high-speed centrifugation. The precipitate obtained by ultra-high-speed centrifugation is identified as bacterial outer membrane vesicles by particle size analysis, transmission electron microscopy and Western blot method, and the constructed ClyA-KLAK fusion protein is successfully displayed on the surface of E. coli outer membrane vesicles, which can be used for subsequent anti-tumor verification experiments.

[0065] Example 3

[0066] Tumor killing effect of C-KLAK-OMVs

[0067] 1 Cell scratch experiment

[0068] The gastric cancer cells HGC27 and breast cancer cells MDA-MB-231 were respectively inoculated at 5×10 5The density of 1 x 105cells / well was uniformly spread in a 6-well plate, and when the cell density reached more than 90%, a "cross" scratch was made on the monolayer cells using a 200 μL pipette gun perpendicular to the bottom of the well plate. To remove the interference in the field of view, the detached cell fragments were washed away with PBS buffer, and then 2 mL of RPMI-1640 complete culture medium containing 20% FBS was added to the HGC27 cells, 2 mL of DMEM complete culture medium containing 10% FBS was added to the MDA-MB-231 cells, and W3110-OMVs (Control group) or C-KLAK-OMVs were added at a concentration of 100 μg / mL, and PBS was added. The migration of the two types of cells was recorded by taking pictures at 0 h, 24 h, and 48 h after scratching, and the cell migration rates at 24 h and 48 h were calculated according to the following formula:

[0069] Cell migration rate = (0 h scratch distance - 24 / 48 h scratch distance) / 0 h scratch distance x 100%

[0070] The results are shown in Figure 10 In the HGC27 cells, the cell migration rates at 24 h and 48 h in the C-KLAK-OMVs group were significantly lower than those in the Control group (P < 0.0001); the same trend was observed in the MDA-MB-231 cells, and the cell migration rates at 24 h and 48 h in the C-KLAK-OMVs group were significantly lower than those in the Control group (P < 0.0001), indicating that C-KLAK-OMVs had a significant inhibitory effect on the migration of tumor cells.

[0071] 2 Cell invasion experiment

[0072] Gastric cancer cells HGC27 and breast cancer cells MDA-MB-231 were prepared into a concentration of 1 x 105cells / mL and 2 x 105cells / mL, respectively. 5 5 ​Cell suspension was prepared at 100 μg / mL. W3110-OMVs (Control group) or C-KLAK-OMVs were added to the cell suspension to achieve an OMV concentration of 100 μg / mL. 200 μL of the OMV-containing cell suspension was added to the upper chamber of a Transwell plate. In the lower chamber (24-well plate), 800 μL of RPMI-1640 complete medium containing 20% ​​FBS (for HGC27) and 800 μL of DMEM complete medium containing 10% FBS (for MDA-MB-231) were added, respectively. The 24-well plates were incubated at 37°C, 5% CO2 for 24 h or 48 h. The Transwell plates were removed, the culture medium was discarded, and any cells that had not yet penetrated the membrane were gently wiped away with a cotton swab. After washing with PBS, the cells were fixed with 800 μL of 4% paraformaldehyde for 30 min. After washing again with PBS, stain with 800 μL crystal violet solution for about 30 min, then wash the chamber with PBS. Take pictures under an inverted microscope, and select 3-5 high-power fields to count the transmembrane cells.

[0073] Experimental results are as follows Figure 11 , 12 As shown, by Figure 11 It was found that in HGC27 gastric cancer cells, compared with the Control group, the number of invasive cells in the C-KLAK-OMVs group was significantly reduced at both 24h and 48h (P<0.0001); Figure 12 Similarly, in breast cancer cells MDA-MB-231, the number of invasive cells in the C-KLAK-OMVs group was significantly reduced at 24h and 48h (P<0.0001), indicating that C-KLAK-OMVs has a significant inhibitory effect on tumor cell invasion.

[0074] 3. Apoptosis experiment

[0075] Gastric cancer cells HGC27 and breast cancer cells MDA-MB-231 were respectively treated with 5×10 5Cells were seeded at a density of 1 cell / well in 6-well plates and when the cell density reached about 50%, fresh medium was replaced and C-KLAK-OMVs (final concentration of 100 μg / mL) or W3110-OMVs (control group) were added, respectively. After 24 h or 48 h of co-culture, the cell culture supernatant was collected in 2 mL centrifuge tubes for later use. Cells were trypsinized without EDTA and added with the previously collected cell culture medium, and then gently blown to completely detach and disperse the adherent cells into a single-cell suspension. The cell suspension was transferred to a centrifuge tube and centrifuged at 1200 rpm for 3 min. The supernatant was discarded (about 50 μL of culture medium can be left to avoid the cells being sucked away). The cells were washed twice with 1 mL of pre-cooled PBS. Before use, 10x Binding Buffer was diluted with deionized water at a ratio of 1:9 to prepare 1x Binding Buffer. The cells were resuspended with 1 mL of 1x Binding Buffer, 10 μL of the cell suspension was taken for counting, and the concentration was adjusted to 1-5x10 6 / mL. 100 μL of the cell suspension was taken in a 5 mL flow tube, and the following control tubes were designed:

[0076] Blank control tube: use Control group cells without Annexin V / FITC and propidium iodide solution (PI) for adjusting the voltage;

[0077] Two single-dye tubes: use experimental group cells with only 5 μL of Annexin V / FITC or PI for adjusting compensation;

[0078] Detection tube: add 5 μL of Annexin V / FITC and 5 μL of PI, mix well, and incubate at room temperature for 15 min in the dark, then add 400 μL of 1x Binding Buffer to terminate staining and mix well. All samples were detected on a flow cytometer, and according to the fluorescence signals of Annexin V-FITC and PI, the cell population in the Q2 quadrant (Annexin V-FITC + / PI + , late apoptosis / necrosis cells) and the Q3 quadrant (Annexin V-FITC + / PI - , early apoptosis cells) were identified as apoptotic cells.

[0079] The experimental results are shown in Figure 13 , 14 Compared with the Control group, the proportion of early apoptosis (Q3 quadrant) and late apoptosis (Q2 quadrant) cells in the C-KLAK-OMVs treatment group showed a significant upward trend at 24 h and 48 h. Quantitative analysis showed that in HGC27 cells, Figure 13At 24 h, the total apoptosis rate of cells treated with C-KLAK-OMVs increased from 16.67 in the Control group to 19.86 (P<0.01), and at 48 h, the total apoptosis rate of cells treated with C-KLAK-OMVs increased from 16.475 in the Control group to 80.445 (P<0.001); a similar trend was observed in MDA-MB-231 cells. Figure 14 At 24 h, the apoptosis rate in the C-KLAK-OMVs-treated group increased from 6.61 in the Control group to 7.66 (P<0.01), and at 48 h, the total apoptosis rate in the C-KLAK-OMVs-treated group increased from 7.74 in the Control group to 9.656 (P<0.01). These experimental results indicate that C-KLAK-OMVs can significantly induce apoptosis in HGC27 and MDA-MB-231 cells.

[0080] Example 4

[0081] Comparison of serum stability of C-KLAK-OMVs and KLAK monopeptides

[0082] 1. Cell scratch assay

[0083] Gastric cancer cells HGC27 were treated with 5×10 5 Cells were evenly seeded in 6-well plates at a density of 90% or higher. Using a 200 μL pipette tip held vertically to the bottom of the well, a cross-shaped scratch was made on the monolayer of cells. To eliminate visual interference, detached cell debris was washed away with PBS buffer. Then, 2 mL of RPMI-1640 complete medium containing 20% ​​FBS was added, followed by the addition of C-KLAK-OMVs and KLAK peptides, which had been co-incubated with serum for 1 h or 6 h, at concentrations of 100 μg / mL. Cell migration was photographed and recorded at 0 h, 24 h, and 48 h post-scratching, and the cell migration rate at 24 h and 48 h was calculated using the following formula:

[0084] Cell migration rate = (0h scratch distance - 24 / 48h scratch distance) / 0h scratch distance × 100%

[0085] The results are as follows Figure 15As shown, there was no significant difference in the rate of wound healing (i.e. cell migration rate) at 24h and 48h between the C-KLAK-OMVs incubated with serum for 1h and 6h (P>0.05); the rate of wound healing (migration distance) at 24h and 48h was significantly higher in the KLAK peptide group incubated with serum for 6h than in the group incubated with serum for 1h (P<0.0001). The above test results show that both C-KLAK-OMVs and KLAK peptide can inhibit tumor cell migration, but the inhibitory effect of C-KLAK-OMVs on cell migration is significantly enhanced compared with KLAK peptide, indicating that C-KLAK-OMVs significantly improve the stability of KLAK peptide in a serum-containing environment.

[0086] 2 Cell invasion test

[0087] Gastric cancer cells HGC27 were prepared into a cell suspension with a concentration of 1 x 10 5 μg / mL. C-KLAK-OMVs and KLAK peptide obtained by incubating with serum for 1h and 6h were added to the cell suspension, respectively, so that the concentration of OMVs was 100 μg / mL. 200 μL of cell suspension containing OMVs was added to the upper chamber of the Transwell chamber, and 800 μL of RPMI-1640 complete medium containing 20% FBS was added to the lower chamber (24-well plate), and the 24-well plate was placed in a 37°C, 5% CO2 incubator for 24h or 48h. The Transwell chamber was removed, the medium was discarded, and the cells on the surface of the chamber membrane that had not passed through were gently wiped off with a cotton swab, and then the cells were fixed with 800 μL of 4% paraformaldehyde for 30 min after PBS washing. After washing with PBS again, the cells were stained with 800 μL of crystal violet solution for about 30 min, and then the chamber was washed with PBS. Photographs were taken under an inverted microscope, and 3-5 high-power fields were selected for counting the number of cells that had passed through the membrane.

[0088] The experimental results are shown in Table 2 and Table 3. Figure 16 、 17 As shown in Table 2 and Table 3, there was no significant difference in the number of HGC27 cells that had passed through the chamber at 24h and 48h between the C-KLAK-OMVs incubated with serum for 1h and 6h (P>0.05); but in the KLAK peptide group, the number of HGC27 cells that had passed through the chamber at 24h and 48h was significantly higher in the group incubated with serum for 6h than in the group incubated with serum for 1h (P<0.01). The above test results show that both C-KLAK-OMVs and KLAK peptide have the ability to inhibit tumor cell invasion, but the inhibitory effect of C-KLAK-OMVs on cell invasion is significantly higher than that of KLAK peptide, indicating that the stability of C-KLAK-OMVs in serum is significantly improved.

[0089] 3. Apoptosis assay

[0090] Gastric cancer cells HGC27 were treated with 5×10 5 Cells were evenly seeded at a density of 1 cell / well in 6-well plates. When the cell density reached approximately 50%, the medium was replaced with fresh culture medium (final concentration 100 μg / mL), and C-KLAK-OMVs and KLAK peptides, which had been co-incubated with serum for 1 h and 6 h respectively, were added. After co-culturing for 24 h, the cell culture supernatant was collected into 2 mL centrifuge tubes for later use. Cells were digested with trypsin without EDTA, and the previously collected cell culture medium was added. The cells were gently pipetted to completely detach the adherent cells and disperse them into a single-cell suspension. The cell suspension was transferred to centrifuge tubes and centrifuged at 1200 rpm for 3 min. The supernatant was discarded (approximately 50 μL of culture medium can be retained to avoid aspirating cells). The cells were washed twice with 1 mL of pre-chilled PBS. Before use, 10× Binding Buffer was diluted with deionized water at a ratio of 1:9 to prepare 1× Binding Buffer. Resuspend the cells in 1 mL of 1×Binding Buffer, take 10 μL of the cell suspension for counting, and adjust the concentration to 1-5×10⁻⁶. 6 / mL. Take 100μL of cell suspension into a 5mL flow cytometry tube and design the following control tube:

[0091] Blank control tubes: Using Control group cells, without Annexin V / FITC and propidium iodide solution (PI) to adjust the voltage;

[0092] Two single-staining tubes: using experimental group cells, only 5 μL Annexin V / FITC or PI was added to adjust compensation;

[0093] Detection tubes: Add 5 μL Annexin V / FITC and 5 μL PPI, mix well, and incubate at room temperature in the dark for 15 min. Then add 400 μL 1× Binding Buffer to stop staining and mix thoroughly by pipetting. All samples were analyzed using flow cytometry. Based on the fluorescence signals of Annexin V-FITC and PPI, samples located in quadrant Q2 (Annexin V-FITC) were selected. + / PI + Late apoptotic / necrotic cells) and Q3 quadrant (Annexin V-FITC) + / PI - Cell populations that are early apoptotic cells are identified as apoptotic cells.

[0094] Experimental results are as follows Figure 18As shown, the total apoptosis rate of cells co-incubated with serum for 1h in the KLAK monopeptide treatment group was 14.38%, which was significantly higher than 8.86% of cells co-incubated with serum for 6h (P<0.0001), indicating that KLAK monopeptide was easily degraded by proteases in the serum environment; while in the C-KLAK-OMVs treatment group, there was no significant difference in the apoptosis rate of cells co-incubated with serum for 1h or 6h (P>0.05). The above test results show that both C-KLAK-OMVs and KLAK monopeptide can promote tumor cell apoptosis, and the pro-apoptotic effect of C-KLAK-OMVs is better than that of KLAK monopeptide, indicating that C-KLAK-OMVs significantly enhance the stability of KLAK peptide in the serum environment, so that its apoptosis-inducing activity is not easily significantly weakened with the extension of serum incubation time.

[0095] KLAKLAK is a lysine-leucine-rich amphipathic alpha-helical peptide, which has significant selectivity for the anionic membrane of prokaryotes. When KLAKLAK is internalized into eukaryotic cells through appropriate targeting mechanisms, it can cause mitochondrial swelling and mitochondrial membrane damage, thereby leading to cell apoptosis. Moreover, KLAKLAK can activate the biochemical pathways related to apoptosis, including the activation of caspase family proteins and PARP (poly ADP ribose polymerase). In addition, KLAKLAK can also be used to carry and deliver genes or small molecules to enhance the anti-tumor effect. However, the stability of KLAKLAK peptide in vivo is poor, and it is easily degraded by proteases in plasma and tissues, resulting in a short half-life and difficulty in fully exerting its function, which limits its clinical application. The present application combines the bacterial outer membrane protein ClyA from E. coli W3110 with the tumor-killing peptide KLAKLAK through genetic engineering technology, constructs a recombinant plasmid ClyA-KLAK-pGEX-6P-1 expressing the fusion protein ClyA-KLAK; by transforming the plasmid into W3110 E. coli, the fusion protein is expressed endogenously, and the engineered bacterial outer membrane vesicle C-KLAK-OMVs displaying the fusion protein ClyA-KLAK on the surface is successfully prepared. The vesicle retains the original morphology of the bacterial outer membrane vesicle and also retains the anti-tumor activity of KLAK, can significantly inhibit the migration and invasion of tumor cells HGC27 and MDA-MB-231, and promote their apoptosis; at the same time, it has the potential to load drugs, and in addition, C-KLAK-OMVs also improves the problem of insufficient stability of KLAK peptide in serum, providing a new idea and reference for the treatment of tumors.

[0096] 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 with antitumor activity, characterized in that, The outer membrane vesicles of the *E. coli* exhibit a ClyA-KLAK fusion protein formed by the tumor-killing peptide KLAKLAK and ClyA; the amino acid sequence of the tumor-killing peptide KLAKLAK is shown in SEQ ID NO.1, and the amino acid sequence of the ClyA-KLAK fusion protein is shown in SEQ ID NO.

2.

2. A recombinant plasmid, ClyA-KLAK-pGEX-6P-1, expressing the ClyA-KLAK fusion protein, characterized in that, The recombinant plasmid was prepared according to the following steps: (1) Construction of ClyA-KLAK fusion protein A fusion protein with the structure Ncol-ClyA-Linker 1-3×MYC-Linker 2-KLAK-Xhol was constructed, and the nucleotide sequence of the fusion protein is shown in SEQ ID NO.7; (2) Construction of recombinant plasmid ClyA-KLAK-pGEX-6P-1 The Ncol-ClyA-Linker 1-3×MYC-Linker 2-KLAK-Xhol fusion protein gene was cloned into the multiple cloning site of the pGEX-6P-1 vector, which is suitable for fusion protein expression, and the recombinant plasmid ClyA-KLAK-pGEX-6P-1 was successfully constructed. The nucleotide sequence of the recombinant plasmid is shown in SEQ ID NO.

8.

3. The method for preparing Escherichia coli outer membrane vesicles with antitumor activity as described in claim 1, characterized in that, Includes the following steps: (1) The recombinant plasmid ClyA-KLAK-pGEX-6P-1 was transformed into W3110 Escherichia coli competent cells to obtain a single clone strain that stably expresses the ClyA-KLAK fusion protein, which was named C-KLAK strain. (2) The C-KLAK strain was inoculated into LB medium containing ampicillin and shaken at 37℃ and 220rpm for 4h. The OD value of the bacterial solution was measured every 20min. When the OD value reached 0.5-0.6, IPTG was added to make the final concentration of IPTG in the medium 1mM. Then, the exogenous protein expression was induced overnight at 21℃ and 200rpm to obtain the C-KLAK bacterial solution. (3) After centrifuging the above bacterial culture at 4℃ and 5000×g for 15 min, collect the supernatant and filter it with a 0.45μm filter membrane. Concentrate the filtrate 20 times with an ultrafiltration centrifuge tube with a molecular weight cutoff of 100kD. Filter it again with a 0.22μm filter membrane, and then centrifuge it at 150000×g for 3 h. Discard the supernatant and collect the precipitate, which is C-KLAK-OMVs.

4. The use of the Escherichia coli outer membrane vesicles as described in claim 1 in the preparation of a drug for targeted tumor therapy.

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