Bacterial outer membrane vesicles secreted by Escherichia coli for treating inflammatory bowel disease, preparation method and application thereof

Abstract

The present invention discloses a method for large-scale extraction of extracellular membrane vesicles (OMV / FGF2) secreted by Escherichia coli that has ampicillin resistance and can secrete bFGF protein after gene editing. The method cultured Escherichia coli in LB liquid containing 100ug / mL ampicillin, and used ultrasonic separation and filtration technology to effectively separate the extracellular membrane vesicles loaded with bFGF protein from Escherichia coli without Escherichia coli cells. The extraction method is simple and easy to operate, and the yield of extracellular membrane vesicles is greatly improved under the premise of retaining most of the effective biological components of bacterial outer membrane vesicles. At the same time, the method effectively removes the live bacterial cells of Escherichia coli, ensures the safety of the body, and greatly reduces the risk of bacteremia or scurvy induced by microorganisms entering the blood. In addition, the OMV / FGF2 extracted by the method was applied to colitis wounds, and it was found that it could significantly promote the healing of colitis wounds.

Description

Technical Field

[0001] The invention relates to the technical field of biomedical wound surface materials, in particular to an FGF engineered bacterial outer membrane vesicle secreted by Escherichia coli for treating inflammatory enteritis, and a preparation method and application thereof. Background Art

[0002] Basic fibroblast growth factor FGF2 (also known as FGF2) is a member of the fibroblast growth factor family. There are now 24 known FGF family members and four FGF cytokine receptors. FGF2 plays a multidirectional regulatory role in proliferation, differentiation, migration and survival in a variety of cell types. It has also been shown to be effective in ulcer, wound and epithelial healing, and is routinely used as an important component of human embryonic stem cell culture medium. FGF2 exhibits a strong angiogenic effect both in vivo and in vitro, and has the effect of stimulating smooth muscle cell growth, wound healing and tissue regeneration. The wound healing effect makes FGF2 a potential therapeutic agent with commercial importance.

[0003] At present, FGF2 has been purified from various bovine organs and tissues, including adenomas, brains, hypothalamus, thymus and kidneys. However, due to its extremely low content, it is almost impossible to obtain a large amount of FGF2 from animal tissues, so it is expensive to purify a small amount of FGF2. Experiments have shown that when FGF2 is directly expressed in various strains, about 110 mg of protein can be produced per liter of bacterial liquid, but after purification, the amount of protein that can be obtained per liter of bacterial liquid is about 20 mg. The main reason for its low yield is that FGF2 is expressed in the form of inclusion bodies in the bacterial system and has poor stability and is easily degraded. In addition, since FGF2 degrades rapidly at 37°C, long-term maintenance of growth factors in tissues or culture media is an ideal choice for protein therapy and stem cell culture. However, this culture method is hindered by the low thermal stability and limited half-life of the FGF2 molecule, and the output of FGF2 is extremely low. At the same time, the existing bacterial drug delivery technology also has certain drug safety risks. Excessive administration is a greater risk to patients, and too little administration may not have a therapeutic effect. Summary of the invention

[0004] The purpose of the present invention is to provide a bacterial outer membrane vesicle secreted by Escherichia coli, which is easy to achieve large-scale production, has high application safety, and is helpful for treating inflammatory bowel disease, as well as a preparation method and application thereof.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solution: a method for preparing bacterial outer membrane vesicles secreted by Escherichia coli for treating inflammatory bowel disease, comprising the following steps:

[0006] S1: Recovery and cultivation of E. coli: E. coli was cultured in sterile LB medium containing 100ug / mL ampicillin. The E. coli in the culture tube was placed in a 37°C bacterial incubator and cultured on a shaker at 200rpm for 24 hours. The culture was repeated for multiple generations until the bacterial characteristics were stable.

[0007] S2: Inoculation and expansion of Escherichia coli: The Escherichia coli with stable properties obtained in step S1 was inoculated into a sterile LB medium containing 100 ug / mL ampicillin, and placed in a 37°C bacterial incubator and cultured on a shaking table at 200 rpm for 24 h;

[0008] S3: Obtaining bacterial outer membrane vesicles of Escherichia coli: Collect the bacterial suspension cultured in step S2, ultrasonicate it in an ultrasonic cleaning machine for 20 minutes, and then centrifuge it at 4°C at a speed of 6000g for 20 minutes, then take the supernatant and centrifuge it again at 4°C at a speed of 6000g for 20 minutes, collect the supernatant after centrifugation, filter it through a syringe filter with a pore size of 0.45um and separate the Escherichia coli, after sterilization, centrifuge the filtrate with a 100kDA ultrafiltration tube for ultrafiltration, collect the liquid in the core of the ultrafiltration tube, and obtain the Escherichia coli bacterial outer membrane vesicle biological filtrate without live Escherichia coli cells, that is, the bacterial outer membrane vesicles of Escherichia coli;

[0009] S4: Obtaining sterile FGF engineered bacterial outer membrane vesicles: Centrifuge the biological filtrate obtained in step S3 at 150,000 g for 2 hours at 4°C, remove the supernatant and resuspend the precipitate with PBS, centrifuge again at 150,000 g for 2 hours at 4°C, remove the supernatant and resuspend the precipitate with PBS, and the resulting resuspension is the sterile FGF engineered bacterial outer membrane vesicles.

[0010] Furthermore, the frequency of the ultrasonic cleaning machine in step S3 is 100 kHz, and after obtaining the sterile FGF-engineered bacterial outer membrane vesicles in step S4, the bacterial outer membrane vesicles are packaged and stored at -80°C.

[0011] Furthermore, it also includes FGF engineered bacterial outer membrane vesicles obtained by the above method for preparing bacterial outer membrane vesicles secreted by Escherichia coli for treating inflammatory bowel disease.

[0012] The beneficial effects of the present invention are as follows: the present invention cultivates and secretes FGF engineered bacterial outer membrane vesicles through gene-edited Escherichia coli, and the bacterial secretion of FGF2 protein is beneficial to the large-scale production of bacterial outer membrane vesicles, ensures the biological activity of FGF2 protein while also improving the stability of FGF2 protein, and on the premise of retaining most of the effective biological components of bacterial outer membrane vesicles, greatly improves the yield of cell outer membrane vesicles, and can achieve large-scale mass production of FGF2 protein, solving the problem of extremely low yield and inability to mass produce due to low thermal stability and limited half-life of FGF2 protein in the prior art. At the same time, the present invention utilizes ultrasonic technology and centrifugal filtration technology to remove harmful bacterial components in the liquid, increase the safety of its application, greatly reduce the risk of bacteremia or scurvy induced by microorganisms entering the blood, avoid the problem of drug safety in the prior art, and the bacterial outer membrane vesicles secreted by intestinal Escherichia coli also have a favorable promoting effect and safety in the field of repair and regeneration of intestinal inflammatory wounds, providing a simple and effective application idea for research in the field of repair and regeneration of intestinal inflammatory wounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The figure is a characterization diagram of the outer membrane vesicle OMV / FGF2 of FGF-engineered bacteria. Among them, a is the NTA particle size analysis result of OMV / FGF2, b is the Zeta potential analysis result of OMV / FGF2, c is the ELISA analysis result of FGF2 concentration on OMV / FGF2, d is a representative transmission electron microscopy image of OMV / FGF2, among which (i) represents OMV secreted by E. coli DH5α, (ii) represents OMV / FGF2 secreted by E. coli FGF2, (iii) represents OMV bound by FGF2 primary antibody and colloidal gold secondary antibody, (iv) represents OMV / FGF2 bound by FGF2 primary antibody and colloidal gold secondary antibody, the scale bars of (i)-(iii) are 200nm, and the scale bar of (iv) is 500nm, e is the Western blot analysis result of FGF2 on OMV / FGF2, and f is a statistical graph of the gray value of the band.

[0014] Figure 2 Figure 4 is the cell biocompatibility of FGF-engineered bacterial outer membrane vesicles. Figures ac are the results of cytotoxicity tests of different concentrations of OMV / FGF2 on NIH 3T3 (a), HaCat (b), and IEC-6 (c) cells after a certain period of culture. The black dotted lines in ac represent the survival rate of the blank control group. d is the cell migration performance of NIH 3T3 cells under the same concentrations of FGF2, OMV, and OMV / FGF2. e is the migration rate statistics of NIH 3T3 cells, and the scale is 500 microns.

[0015] Figure 3 The in vivo biosafety of FGF engineered bacterial outer membrane vesicles. a is the hemolysis rate of blood cells measured after mixing different concentrations of OMV / FGF2 with blood cells for a period of time, b is a representative image after mixing different concentrations of OMV / FGF2 with blood cells for a period of time, and c is the relative concentration of endotoxin in different batches of OMV / FGF2.

[0016] Figure 4 is the oral stability of FGF engineered bacterial outer membrane vesicles. Where ab is the particle size and particle concentration of OMV / FGF2 measured by NTA at the specified time points after incubating equal amounts of OMV / FGF2 in PBS and SGF (pH 1.2) at 37 °C, and c is the distribution of OMV / FGF2 particle size at different time points.

[0017] Figure 5 Figure 2 is the in vitro and in vivo stability of FGF engineered bacterial outer membrane vesicles. A to C are the protein activities of OMV / FGF2 and FGF2 after storage at pH 7.4, 4°C (a), pH 7.4, 37°C (b), and pH 6.5, 37°C (c) for different time periods, d is a representative in vivo fluorescence intensity statistical graph of OMV / FGF2 labeled with Cy5 fluorescence in the gastrointestinal tract of mice after oral administration at different time periods, and e is a representative in vivo fluorescence image of OMV / FGF2 labeled with Cy5 fluorescence in the gastrointestinal tract of mice after oral administration at different time periods.

[0018] Figure 6 The process of cells binding and taking up FGF-engineered bacterial outer membrane vesicles. a is a representative laser confocal image of mouse colon tissue sections 4 hours after oral administration of fluorescently labeled vesicles, with a scale of 50 microns, and b is a representative fluorescence image of cells collected and taken under laser confocal imaging after co-culture of OMV / FGF2 vesicles with Cy5 fluorescence with intestinal cells for 4 hours, with a scale of 10 microns.

[0019] Figure 7 The effect of FGF-engineered bacterial outer membrane vesicles on the healing of inflammatory bowel disease in mice. a is a representative image of the mouse colon obtained on the 13th day after different treatments, b is a statistical graph of the weight of mice at different time points after different treatments, c is a statistical graph of the weight of mice at the 13th day after different treatments, d is a statistical graph of the colon length of mice at the 13th day after different treatments, e is a statistical graph of the disease activity index (DAI) of mice at different time points after different treatments, f is a representative H&E staining image of the colon tissue of mice in different experimental groups on the 13th day, and g is the histomorphological evaluation result of colon damage based on inflammatory cell infiltration (0-3), lesion depth (0-3), crypt destruction (0-4), and lesion range (1-4) as scoring criteria.

[0020] Figure 8 The effect of FGF-engineered bacterial outer membrane vesicles on the restoration of mouse colon barrier. a and c are immunohistochemical fluorescence images of tight junction-related proteins Occludin and ZO-1, respectively, and b and d are statistical graphs of the average fluorescence intensity of Occludin and ZO-1 in the colon.

[0021] Fig. 9 Diversity analysis of mouse colon microorganisms by FGF-engineered bacterial outer membrane vesicles. Where a is the species cumulative curve, and be is the alpha diversity analysis to evaluate sample richness and species diversity results.

[0022] Fig.10 It shows that FGF engineered bacterial outer membrane vesicles regulate intestinal microorganisms to promote inflammation healing. Among them, a is the distribution of intestinal microbial community structure at the phylum level, b is the distribution of intestinal microbial community structure at the genus level, c is the score map of differential species, d is the example map of differential species annotation branches, and eh are the differential species abundances of Lachnospirales, Butyricococcus, Burkholderia, and Red Stinkbugs.

[0023] Fig.11 This indicates that FGF engineered bacterial outer membrane vesicles reduce intestinal inflammation. a is a representative immunohistochemical image of TNF-α, IL-1β, and IL-6 in different treatment groups of tissue sections, and b is a statistical immunohistochemical image of TNF-α, IL-1β, and IL-6 in different treatment groups of tissue sections. DETAILED DESCRIPTION

[0024] In order to make the technical means, innovative features and functions implemented by the present invention easy to understand, the present invention will be further described below.

[0025] like Figure 1-11 As shown, the technical solution of the method for preparing bacterial outer membrane vesicles secreted by Escherichia coli for treating inflammatory bowel disease of the present invention comprises the following steps:

[0026] S1: Resuspend the lyophilized powder of genetically engineered E. coli in 10 mL of sterile LB medium containing 100 ug / mL ampicillin, place the resuspended E. coli in a culture tube, and then culture it in a 37°C bacterial incubator at 200 rpm for 24 h. Subculture the E. coli for multiple times at a ratio of 1% (v / v) until the properties are stable.

[0027] S2: Inoculation and expansion of Escherichia coli: The Escherichia coli with stable properties obtained in step S1 was inoculated into a sterile LB medium containing 100 ug / mL ampicillin, and placed in a 37°C bacterial incubator and cultured on a shaking table at 200 rpm for 24 h;

[0028] S3: Obtaining bacterial outer membrane vesicles of Escherichia coli: Collect the bacterial suspension cultured in step S2, ultrasonicate it in an ultrasonic cleaning machine (frequency is 100kHz) for 20 minutes, and then centrifuge it at 6000g for 20 minutes at 4°C, then take the supernatant and centrifuge it again at 6000g for 20 minutes at 4°C, collect the supernatant after centrifugation, filter it through a syringe filter with a pore size of 0.45um and separate the Escherichia coli, after sterilization, centrifuge the filtrate with a 100kDA ultrafiltration tube for ultrafiltration at 4000g for 15 minutes, discard the liquid in the outer tube after centrifugation, collect the liquid in the core of the ultrafiltration tube, and obtain the Escherichia coli bacterial outer membrane vesicle biological filtrate without live Escherichia coli cells, that is, the bacterial outer membrane vesicles of Escherichia coli;

[0029] S4: Obtain sterile FGF engineered bacterial outer membrane vesicles: Centrifuge the biological filtrate obtained in step S3 at 150,000 g for 2 hours at 4°C, remove the supernatant and resuspend the precipitate with PBS, centrifuge again at 150,000 g for 2 hours at 4°C, remove the supernatant and resuspend the precipitate with PBS, and filter again with a 0.45um syringe filter to ensure complete sterility. The resulting resuspension is the sterile engineered bacterial outer membrane vesicles loaded with bFGF protein. The bacterial outer membrane vesicles are packaged and stored at -80°C.

[0030] The embodiments of the present invention measure the particle size, Zeta potential and particle concentration of bacterial outer membrane vesicles by nanoparticle tracking analyzer (NTA). Figure 1 As shown in a, the analysis results show that the average particle size of FGF engineered bacterial outer membrane vesicles OMV / FGF2 is about 120 nm. Figure 1 As shown in b, the average Zeta potential is about -13.7mV. Figure 3 As shown in b, the particle concentration is as high as 2×10 11 / mL, in order to understand the protein content of FGF2 on the extracted OMV / FGF2, the protein content of the vesicles was measured by BCA, and the content of FGF2 protein was detected by enzyme-linked immunosorbent assay, such as Figure 1 As shown in Figure c, it was calculated that each 1 mg of bacterial outer membrane vesicles contained about 400 ng of FGF2 protein, and the surface of the bacterial outer membrane vesicles was detected using a transmission electron microscope (TEM), as shown in Figure 2. Figure 1 d(ii) and Figure 1d (i), the test results show that the FGF engineered bacterial outer membrane vesicles OMV / FGF2 negatively stained with phosphotungstic acid have similar morphology and diameter to the bacterial outer membrane vesicles OMV extracted from E. coli DH5α. Then, rabbit anti-FGF2 IgG antibody was used as the primary antibody to incubate the vesicles, and then goat anti-rabbit IgG with 10 nm colloidal gold was used as the secondary antibody. After TEM detection, Figure 1 d (iv), the results show that colloidal gold particles are attached to the surface of OMV / FGF2, while Figure 1 As shown in d (iii), the bacterial outer membrane vesicles OMV obtained by extraction from E. coli DH5α only have the image after negative staining with phosphotungstic acid, which proves that FGF2 protein is distributed on the surface of OMV / FGF2. The collected bacterial outer membrane vesicles were subjected to protein immunoblotting (WB) experiment, as shown in Figure 1 ef validated and demonstrated the high expression of FGF protein on OMV / FGF2 secreted by engineered bacteria.

[0031] In the embodiment of the present invention, the FGF engineered bacterial outer membrane vesicles prepared by the above method were experimentally analyzed, and the specific characterization diagrams and cell biocompatibility were as follows: Figure 1 and Figure 2 shown.

[0032] like Figure 3 As shown, in order to further verify the safety of FGF-engineered bacterial outer membrane vesicles and study their application scope and potential, FGF-engineered bacterial outer membrane vesicles were mixed with mouse blood cells and their hemolytic ability was tested. The results showed that OMV / FGF2 has good safety and will not cause hemolysis of mouse blood cells. At the same time, the endotoxin level of OMV / FGF2 was detected by ELISA kit, as shown in Figure 3 c. The experimenters found that when the concentration of the vesicles was at the level of 100 μg / mL, the endotoxin content remained at 4.86±1.23 ng / mL, that is, every 100 μg of vesicles contained about 5 ng of endotoxin, which met the requirements for oral preparations and confirmed the application potential of OMV / FGF2 in the oral field.

[0033] like Figure 4 As shown in Figure 2, in order to evaluate the stability of OMV / FGF2 in vitro and in vivo, OMV / FGF2 was incubated with artificial gastric juice (pH 1.2) at 37 °C for 4 h to simulate the process of vesicles staying in the stomach. After NTA statistical analysis, the results were as follows: Figure 4 As shown in Figures ac, there was no significant difference in the particle size and number of OMV / FGF2, indicating that the stability of bacterial outer membrane vesicles passing through the gastric lumen after oral ingestion was satisfactory.

[0034] like Figure 5-6As shown in the figure, in order to verify the protein activity of FGF2 protein on bacterial outer membrane vesicles under different conditions, FGF2 and OMV / FGF2 were incubated together under different conditions for 0, 6, 12, and 24 hours, and then their FGF2 protein concentrations were detected by ELISA at each time point and compared with their initial concentrations. The experiment found that the FGF2 protein was almost inactive after 24 hours of incubation, as shown in the figure. Figure 5 As shown in Figures ac, the OMV / FGF2 group still maintained the biological activity of FGF2 relatively well. At the same time, in order to verify the passage of OMV / FGF2 through the digestive tract in vivo, the experiment used Cy5-NHS with fluorescent groups and OMV / FGF2 to co-culture and obtain OMV / FGF2-Cy5 with fluorescent groups. C57BL / 6J male mice were randomly selected and gavaged with the same volume of OMV / FGF2-Cy5 at different time periods. Then, the mice were killed at different time points and the in vivo fluorescent imaging pictures of the gastrointestinal tract of the mice were taken, as shown in Figure 6. Figure 5 As shown in de, the experimental results showed that the engineered bacterial outer membrane vesicles OMV / FGF2 had passed through the stomach at about the second hour and began to be distributed in the colorectal area of ​​the mouse at the fourth hour. OMV / FGF2 was still significantly retained in the colon until 24 hours later. Then, the colon tissue of the mouse was taken out at the fourth hour, frozen, embedded, sectioned and stained with fluorescence, as shown in Figure 6 a. After observing the location of vesicles on the mouse colon, it was found that vesicles are commonly present on the intestinal surface and can be absorbed by intestinal cells. To verify this phenomenon, the experimenters continued to conduct experiments on ICE-6 cells, such as Figure 6 b. The experimenters found that cells can indeed absorb vesicles into the cell through endocytosis.

[0035] like Figure 7 As shown, in order to evaluate the effect of OMV / FGF2 on inflammatory bowel disease in mice, inflammatory bowel disease in mice was induced by dextran sulfate sodium (DSS), and OMV / FGF2 containing 100 μg of total protein was administered by oral gavage every day for 6 consecutive days starting from the 7th day. Here, the colitis mice treated with PBS, FGF2 and OMV were used as the control group. Figure 7 After feeding 2.5% DSS in drinking water every day for 7 consecutive days, mice in all groups except the healthy control group were observed to have significant weight loss, anal bleeding or loose stools, and increased DAI disease index, proving that the model was successfully established. Subsequently, the drug was administered by gavage every day for 6 consecutive days after the model was successfully established. Figure 7 b shows that the body weight of mice in the treatment group treated with OMV / FGF2 was comparable to that before modeling, and was significantly higher than that of the control groups of PBS, FGF2 and OMV. Figure 7a and d show that oral delivery of OMV / FGF2 effectively corrected the reduction in colon length in colitis mice. The colon tissues of mice were sectioned and stained with hematoxylin / eosin (H&E). Figure 7 f It can be observed that the pathological symptoms caused by leukocyte infiltration in the distal colon of mice treated with OMV / FGF2, such as mucosal edema and crypt swelling and destruction, were significantly reduced. Tissue sections were subjected to immunohistochemical analysis of inflammatory indicators, such as Figure 7 e. Through observation and statistics, it was found that OMV / FGF2 can reduce the accumulation and expression of inflammatory factors in the colon, thereby alleviating inflammation. These experimental results prove that oral OMV / FGF2 can improve the symptoms and pathological manifestations of DSS-induced colitis.

[0036] like Figure 8 As shown, compared with the individually scattered goblet cells with pathological cavities in the colitis mice in the PBS-administered group, the goblet cells in the distal colon of the OMV / FGF2-administered mice were significantly improved in number and function, resulting in the formation of a thicker mucus layer in the intestinal lumen. The remarkable efficacy of OMV / FGF2 in repairing the mucus barrier can be attributed to the internalization of bacterial outer membrane vesicles OMV / FGF2, the subsequent stimulation of goblet cells to produce mucus, and the OMV / FGF2-mediated increase in the abundance of beneficial bacterial species responsible for mucus production. Figure 8 b and 8d showed that the expression of occludin and ZO-1 in the distal colon epithelial cells of the mice treated with OMV / FGF2 was significantly increased, similar to that of the healthy control group, while the expression of other control groups was significantly lower than that of the OMV / FGF2 treatment group. The enhanced tight junctions activated by OMV / FGF2 can promote epithelial cell proliferation, thereby promoting mucosal repair. Therefore, it can be considered that with the gradual recovery of intestinal epithelial damage, more intestinal epithelia with normal morphology appeared in the OMV / FGF2 treatment group, from which more overlapping and complete fluorescence signals were observed. OMV / FGF2 plays a key role in strengthening the integrity of the intestinal barrier by regulating tight junctions.

[0037] like Figure 9-10 To evaluate the effect of OMV / FGF2 on intestinal microbiota, mice were induced to have inflammatory bowel disease by drinking dextran sulfate sodium (DSS) for 7 consecutive days. Then, OMV / FGF2 containing 100 μg of total protein was administered by oral gavage every day for 6 consecutive days. The composition of intestinal microbiota of mice was analyzed by extracting colon contents and measuring 16S ribosomal RNA gene sequencing, as shown in Figure 2. Fig. 9 The results showed that compared with PBS-treated mice, the OMV / FGF2-treated group showed significant improvements in both the richness and diversity of the intestinal microbiota. Fig.10g It can be observed that the proportion of Burkholderia genus was high in the DSS model group, but low in the healthy control group and experimental treatment group. Fig.10 a, The proportion of Proteobacteria increased in the DSS model group, while the relative abundance of bacteria belonging to the Proteobacteria decreased in the OMV / FGF2-treated group. Proteobacteria is the largest phylum containing many pathogens and is considered to be a microbial signature of dysbiosis in the intestinal microbiota. These structures suggest that OMV / FGF2 can effectively reverse intestinal microbial imbalance by upregulating beneficial bacteria, especially Bacteroidetes, while reducing opportunistic pathogens.

[0038] like Fig.11 As shown in a, the proportion of TNF-α in the colon tissue immunohistochemical staining of the healthy control group mice was similar to that of the experimental treatment group. Fig.11 As shown in b, the TNF-α levels of the model mice constructed by DSS, as well as the vesicle intervention group and the protein intervention group were significantly higher than those of the healthy control group. The proportion of IL-1β in the colon tissue immunohistochemical staining of the healthy control group mice was similar to that of the experimental treatment group, while the IL-1β levels of the model mice constructed by DSS were significantly higher than those of the healthy control group, with significant differences (P < 0.0001). The IL-6 content in the colon tissue of the healthy control group mice was low, similar to the data of the experimental treatment group, while the IL-6 content of the model group mice was high, with significant statistical differences, because it can be considered that OMV / FGF2 can By upregulating beneficial microbial flora, avoiding the massive reproduction of intestinal opportunistic pathogens and promoting the polarization of T cells to an anti-inflammatory phenotype, the recruitment of inflammatory cell infiltration in the colon tissue is reduced, thereby alleviating inflammatory pathological changes. Ultimately, it can be considered that the FGF engineered bacterial outer membrane vesicles prepared by the above preparation method contribute to the restoration of tight junctions of the colon barrier of mice, can help repair intestinal inflammatory wounds of mice, improve colitis inflammation of mice, and play a certain role in improving the intestinal microecology of mice. It can be considered that the gene-edited cell outer membrane vesicles obtained in the present invention contribute to the repair of intestinal inflammatory wounds on the basis of ensuring the safety of intestinal wounds.

[0039] Although the present invention has been disclosed as above in terms of specific embodiments, they are not intended to limit the present invention. Any technician in this field may make slight changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the scope of the attached claims.

Claims

1. Use of bacterial outer membrane vesicles secreted by Escherichia coli in preparing a drug for treating colitis, wherein the bacterial outer membrane vesicles are prepared by the following steps: S1: Recovery and cultivation of E. coli: E. coli was cultured in sterile LB medium containing 100ug / mL ampicillin. The E. coli in the culture tube was placed in a 37°C bacterial incubator and cultured on a shaker at 200rpm for 24 hours. The culture was repeated for multiple generations until the bacterial characteristics were stable. S2: Inoculation and expansion of Escherichia coli: The Escherichia coli with stable properties obtained in step S1 was inoculated into a sterile LB medium containing 100 ug / mL ampicillin, and placed in a 37°C bacterial incubator and cultured on a shaking table at 200 rpm for 24 h; S3: Obtaining bacterial outer membrane vesicles of Escherichia coli: Collect the bacterial suspension cultured in step S2, ultrasonicate it in an ultrasonic cleaning machine for 20 minutes, and then centrifuge it at 4°C at a speed of 6000g for 20 minutes, then take the supernatant and centrifuge it again at 4°C at a speed of 6000g for 20 minutes, collect the supernatant after centrifugation, filter it through a syringe filter with a pore size of 0.45um and separate the Escherichia coli, after sterilization, centrifuge the filtrate with a 100kDA ultrafiltration tube for ultrafiltration, collect the liquid in the core of the ultrafiltration tube, and obtain the Escherichia coli bacterial outer membrane vesicle biological filtrate without live Escherichia coli cells, that is, the bacterial outer membrane vesicles of Escherichia coli; S4: Obtain sterile FGF engineered bacterial outer membrane vesicles: Centrifuge the biological filtrate obtained in step S3 at 150,000 g at 4°C for 2 hours, remove the supernatant and resuspend the precipitate with PBS, centrifuge again at 150,000 g at 4°C for 2 hours, remove the supernatant and resuspend the precipitate with PBS, and the resulting resuspension is the sterile FGF engineered bacterial outer membrane vesicles.

2. The use of a bacterial outer membrane vesicle secreted by Escherichia coli according to claim 1 in the preparation of a drug for treating colitis, characterized in that: The frequency of the ultrasonic cleaning machine in step S3 is 100 kHz. After obtaining the sterile FGF engineered bacterial outer membrane vesicles in step S4, the bacterial outer membrane vesicles are packaged and stored at -80°C.

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