Engineering probiotics for targeted therapy of colitis as well as construction method and application of engineering probiotics

Abstract

The invention discloses engineering probiotics for targeted therapy of colitis and a construction method and application thereof.The construction method of the engineering probiotics for targeted therapy of colitis comprises the following steps that 1, PelB and optimized Fc and IL-4 genes are connected with homologous arms through PCR, seamless cloning is conducted, and then the PelB and optimized Fc and IL-4 genes are inserted into pBAD33 plasmids to construct recombinant plasmids; step 2, transferring the plasmids obtained in the step 1 into competent E.coliDH5alpha through a heat shock method, carrying out sequencing verification after bacterial colony PCR verification, so as to determine whether the recombinant plasmids are successfully constructed or not; and step 3, under the condition that the recombinant plasmid is successfully constructed, transferring the recombinant plasmid into competent EcN by utilizing a heat shock method, screening by adopting a chloramphenicol-resistant LB solid plate, culturing a bacterial colony obtained by screening in an LB liquid culture medium containing chloramphenicol, and centrifugally collecting and storing. The invention realizes the characteristics of targeting an inflammation site and treating inflammation.

Description

Technical Field

[0001] The present invention relates to the field of engineering bacteria, and in particular to an engineering probiotic for targeted treatment of colitis and a construction method and application thereof. Background Art

[0002] Ulcerative colitis (UC) is an incompletely understood intestinal inflammatory disease characterized by chronic autoimmune inflammation of the colonic mucosa to the submucosal tissue. The disease is clinically manifested as extensive mucosal inflammation of the colorectum. Patients may experience symptoms such as diarrhea, abdominal pain, and weight loss, which can have a serious impact on the quality of life in severe cases. The pathogenesis of UC is still unclear, but studies have shown that UC is associated with intestinal mucosal barrier dysfunction and intestinal flora disorders, followed by a sharp increase in the level of inflammatory factors. The delicate balance of pro-inflammatory and anti-inflammatory cytokines throughout the gastrointestinal tract is essential for healthy intestinal barrier function and tissue homeostasis. Changes in this balance, especially the overproduction of pro-inflammatory cytokines, can lead to active inflammation, cell infiltration, and loss of integrity and function of the intestinal barrier. Although there are a series of conventional treatments, such as aminosalicylic acid drugs, corticosteroids and immunosuppressants, these drugs have limited efficacy and cannot solve the root causes of UC, such as gastrointestinal immune response disorders, intestinal mucosal barrier dysfunction and intestinal microbiome disorders. Long-term use may be accompanied by serious side effects and drug dependence, and targeted treatment cannot be achieved. At the same time, traditional probiotics have a single therapeutic effect, and their anti-inflammatory properties are not prominent. They cannot secrete functional proteins to achieve precise treatment, and cannot effectively regulate inflammation and promote tissue repair. Therefore, there is an urgent need to study new solutions to enhance the anti-inflammatory properties of probiotics and improve the function of the intestinal barrier, thereby improving their therapeutic effects and enabling oral probiotic therapy to play a greater role in disease treatment. Summary of the invention

[0003] The main purpose of the present invention is to provide an engineered probiotic for targeted treatment of colitis and a construction method and application thereof, aiming to solve the technical problem that the existing oral probiotics have poor use effects.

[0004] To achieve the above-mentioned purpose, the present invention provides an engineered probiotic for the targeted treatment of colitis. The engineered probiotic has the Latin name of Escherichia coli Nissle 1917FI, which is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration, with a deposit number of CGMCC NO.31539, a deposit date of August 5, 2024, and an address of the depository unit: Beijing, China.

[0005] In addition, to achieve the above object, the present invention also provides a method for constructing the above-mentioned engineered probiotics for targeted treatment of colitis, characterized in that the method comprises the following steps:

[0006] Step 1, PelB and the optimized Fc and IL-4 genes were connected to homology arms by PCR, and inserted into the pBAD33 plasmid after seamless cloning to construct a recombinant plasmid;

[0007] Step 2, the plasmid obtained in step 1 is transformed into competent E. coli DH5α by heat shock method, and after colony PCR verification, sequencing verification is performed to determine whether the recombinant plasmid is successfully constructed;

[0008] Step 3, under the condition that the recombinant plasmid is successfully constructed, the recombinant plasmid is transferred into the competent EcN by the heat shock method, and the chloramphenicol-resistant LB solid plate is used for screening. The screened colonies are cultured in LB liquid culture medium containing chloramphenicol, and collected and preserved by centrifugation.

[0009] Optionally, the recombinant plasmid is Pbad33-pelB-FC-IL-4, and the gene sequence of the recombinant plasmid is shown in SEQ ID NO.13.

[0010] Optionally, the optimized Fc gene sequence is shown as SEQ ID NO.7, and the optimized IL-4 gene sequence is shown as SEQ ID NO.9.

[0011] In addition, to achieve the above-mentioned purpose, the present invention also provides a pharmaceutical composition, which includes the above-mentioned engineered probiotics for targeted treatment of colitis.

[0012] Optionally, the pharmaceutical composition further comprises a pharmaceutically or pharmacologically acceptable carrier.

[0013] In addition, to achieve the above object, the present invention also provides a kit, which includes the above-mentioned engineered probiotics for targeted treatment of colitis or the above-mentioned pharmaceutical composition.

[0014] In addition, to achieve the above objectives, the present invention also provides the use of the above-mentioned engineered probiotics for targeted treatment of colitis, the above-mentioned pharmaceutical composition or the above-mentioned kit in the preparation of intestinal flora regulating drugs or drugs or health products for preventing intestinal inflammation and damage.

[0015] Optionally, the dosage form of the medicine or health product is tablets, capsules, soft capsules, granules, pills, oral liquids, emulsions or dry suspensions.

[0016] Beneficial effects:

[0017] The engineered probiotics of the present invention adopt optimized Fc and IL-4 genes to connect homology arms through PCR to synthesize pelB-FC-IL-4 linearized fragments, and insert into pBAD33 plasmid after seamless cloning to obtain a recombinant plasmid, and construct a final expression vector to achieve transformation EcN, so as to obtain a new engineered probiotic for targeted treatment of colitis. The engineered probiotics can produce the target protein in situ in a sustainable manner under the condition of normal bacterial growth, and the produced target protein does not need to form inclusion bodies but is directly secreted outside the cell. At the same time, the secretory protein of the engineered bacteria has the characteristics of targeting the inflammatory site and treating inflammation, and can be used as a drug for targeted treatment of colitis, preparation of intestinal flora regulating drugs, and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The results of colony PCR identification of E. coli EcN carrying plasmids pBAD33 empty vector and pBAD33-pelB-FC-IL4; Figure 2 This is the plasmid map of the E. coli expression system of IL-4; Figure 3 Western Blotting results of E. coli EcN protein expressed by plasmids pBAD33 empty vector and pBAD33-pelB-FC-IL4; Figure 4 This is the SEM electron microscope image of FI EcN; Figure 5 The body weight changes of colitis mice in different groups; Figure 6 The colon lengths of mice in different groups; Figure 7 HE staining images of colon tissues of mice in different groups; Figure 8 The colon tissues of different groups of mice were scored histologically; Fig. 9 is the content of inflammatory factor IL-6 in the intestinal tissues of mice in different groups; Fig.10 is the content of inflammatory factor TNF-α in the intestinal tissues of mice in different groups; Fig.11 The content of inflammatory factor IL-1β in the intestinal tissue of mice in different groups; Fig.12 The content of inflammatory factor IL-10 in intestinal tissue of mice in different groups. Fig.13 is the observed OTUs index in the intestinal tissues of mice in different groups; Fig.14 The Venn diagram of OTU content distribution in intestinal tissues of mice in different groups; Fig.15 is the Chao1 diversity index in the intestinal tissues of mice in different groups; Fig.16 is the Shannon diversity index in the intestinal tissues of mice in different groups; Fig.17 This is a heat map of the clustering of species abundance at the family level in the intestinal tissues of mice in different groups; Fig.18 Bubble diagram of species distribution at genus level in intestinal tissues of mice in different groups; Fig.19is the relative content of Akkermansia in the intestinal tissues of mice in different groups; Fig. 20 is the relative content of Lachnospiraceae_NK4A136 in the intestinal tissues of mice in different groups; Fig.21 is the relative content of Lactobacillus in the intestinal tissues of mice in different groups; Fig. 22 is the relative content of Desulfovibrio in the intestinal tissues of mice in different groups; Fig.23 A simplified schematic diagram of the establishment of the ulcerative colitis model. DETAILED DESCRIPTION

[0019] It should be understood that the specific embodiments described herein are only used to explain the present invention, and are not used to limit the present invention.

[0020] Example 1

[0021] Experimental animals

[0022] 24 female ICR mice aged 7-8 weeks were purchased from Hunan Slake Jingda Experimental Animal Co., Ltd., production license number: SCXK (Xiang) 2016-0002, and were uniformly raised in the experimental animal center of our college, with a temperature of 23℃±2℃, humidity of 55%±5%, 12h day and night cycle lighting, caged, and free access to food and water. All animal experiments met the requirements of the Experimental Animal Ethics Review Committee of our school.

[0023] Experimental reagents

[0024] LB medium (g / L): 5 g yeast extract; 5 g sodium chloride; 10 g tryptone; 1000 mL distilled water; pH 7.0. Table 1 shows the main reagents used in the experiment, and Table 2 shows the primer sequences used.

[0025] Table 1 Main experimental reagents

[0026]

[0027]

[0028] Table 2 shows the primer sequences used

[0029]

[0030] Experimental methods

[0031] Strains, plasmids and recombinant gene sequences

[0032] The strains of E. coli DH5α, strain of E. coli Nissle1917, strain pTD103luxl-EcN, and plasmid pBAD33 used in this experiment were stored in the research group. The gene sequence of plasmid pBAD33 is shown in SEQ ID NO.4.

[0033] PelB was obtained from the National Center for Biotechnology Information (NCBI) website, and its sequence is shown as SEQ ID NO.5.

[0034] The Fc, IL-4 and 6×His gene fragments were obtained from the NCBI website and optimized and synthesized by Changsha Abiwei Biotechnology Co., Ltd. The gene sequences before and after optimization are shown in Table 3 below.

[0035] Table 3-Gene sequences of Fc, IL-4 and 6×His gene fragments before and after optimization

[0036]

[0037] Construction method of engineered bacteria FI EcN

[0038] Step 1, connect the homology arms of PelB and the optimized Fc and IL-4 genes by PCR, and insert them into the pBAD33 plasmid after seamless cloning to construct a recombinant plasmid. Specifically, the Fc and IL-4 genes are optimized in advance, and then the optimized Fc and IL-4 genes are connected to the homology arms by PCR (primers Fc-IL-4-F1 in Table 2 are used), and inserted into the pBAD33 plasmid after seamless cloning. Among them, Fc can enable the engineered probiotics to target macrophages and stimulate immune response. And the steps of seamless cloning are according to Follow the instructions for the Seamless Cloning Kit.

[0039] Step 2: The plasmid obtained in step 1 is transformed into competent E. coli DH5α by heat shock method, and after colony PCR verification, sequencing verification is performed to determine whether the recombinant plasmid is successfully constructed.

[0040] Step 3, under the condition that the recombinant plasmid is successfully constructed, the recombinant plasmid is transferred into the competent EcN by the heat shock method, and the chloramphenicol-resistant LB solid plate is used for screening, the screened colonies are cultured in an LB liquid culture medium containing chloramphenicol, and collected and preserved by centrifugation, and the FI EcN bacterial liquid is sequenced and verified to obtain an engineered probiotic for the targeted treatment of colitis. The Latin name of the engineered probiotic is Escherichia coli Nissle 1917FI, which is deposited in the General Microbiology Center of the China Microbiological Culture Management Committee with a deposit number of CGMCC NO.31539.

[0041] Ability to express IL-4 protein

[0042] (1) Collect the supernatant. After culturing the FI EcN for 3.5 hours, add 100 μL of arabinose (34 mg / mL) and culture for another 13-14 hours. Centrifuge at 6500 rpm for 10 minutes, collect the supernatant and store it in a 4°C refrigerator.

[0043] (2) The obtained bacterial culture supernatant was mixed with 5× protein loading buffer and denatured in a metal bath at 100°C for 10 min. Load the sample, transfer to the membrane after electrophoresis, and then fix in TBS containing 3.5% BSA for 2.5 hours. Incubate with primary antibody anti-6×His (Thermo Fisher Scientific) in a 4°C refrigerator overnight. Then incubate with peroxidase-labeled goat anti-mouse secondary antibody for one and a half hours, and finally develop and observe the bands.

[0044] (3) After the bacterial supernatant was concentrated in an ultrafiltration centrifuge tube, a nickel affinity chromatography column (HisNi-NTA, 5 mL) was used for protein purification. The sample was loaded, and after the column was balanced, different concentrations of imidazole (250 mM, 300 mM, 350 mM, 400 mM) were used for elution, and the eluate was collected. Finally, the amount of IL-4 protein in the eluate with different concentrations of imidazole was detected using a His-ELISA kit.

[0045] Furthermore, in step 2, the step of determining whether the recombinant plasmid is successfully constructed is to transform the constructed plasmid into the competent E. coli, and use Fc-IL-4-F2 and Fc-IL-4-F3 in Table 2 as primers, and the colonies obtained by the above transformation as templates to PCR amplify the target gene fragment, and identify whether the plasmid is successfully transferred by colony PCR identification. The above PCR product is subjected to agarose gel electrophoresis. Primer 2 (Fc-IL-4-F2) can see a band of about 1603 bp in size, and primer 3 (Fc-IL-4-F3) can see a band of about 1402 bp in size, as shown in FIG. Figure 1As shown, it indicates that the E. coli pBAD33-pelB-IL-4 expressing interleukin-4 was successfully constructed, and the inserted sequence is shown in SEQ ID NO.12.

[0046] Further, the recombinant plasmid construction diagram, such as Figure 2 As shown, and the sequence of the recombinant plasmid is shown as SEQ ID NO.13.

[0047] Qualitative characterization of induced expression and secretion of interleukin-4 protein

[0048] The secretion capacity of this recombinant E. coli strain was evaluated by detecting the expression of extracellular IL-4. After the recombinant E. coli was cultured in LB medium for 24 hours, the protein in the supernatant of the recombinant E. coli culture medium was collected. The expression of extracellular IL-4 was detected by Western blotting. The results are shown in Figure 2. Figure 3 The results showed that the expression of IL-4 was detected, which further confirmed that the Escherichia coli expressing IL-4 was successfully constructed. At the same time, the constructed IL-4 target protein was fused with a 6×His tag at the C-terminus, which can be purified by Ni-affinity chromatography, which greatly simplifies the purification process, and a high-purity target protein can be obtained by one-step purification, which is a commonly used purification method in protein research. Therefore, the eluate of different concentrations of imidazole solution was used to obtain the protein content of IL-4 secreted expression by ELISA. As shown in Table 4, the expression concentrations of strain FI EcN in the eluate of different imidazole concentrations were 12.75, 14.33, 16.57 and 19.02 ng / mL, respectively.

[0049] Table 4 IL-4 protein concentration in eluents with different imidazole concentrations

[0050]

[0051] And the obtained FI EcN was characterized by SEM electron microscope, and the Figure 4 The structure shown.

[0052] Furthermore, in order to illustrate the therapeutic effect of the prepared FI EcN on intestinal inflammation and damage in UC mice, the following is a specific experimental description.

[0053] Example 2

[0054] Experimental animals

[0055] 40 female ICR mice aged 7-8 weeks were purchased from Hunan Slake Jingda Experimental Animal Co., Ltd., production license number: SCXK (Xiang) 2016-0002, and were uniformly raised in the local experimental animal center, with a temperature of 23℃±2℃, a humidity of 55%±5%, a 12h day and night cycle lighting, caged, and free access to food and water. All animal experiments were in accordance with the requirements of the local experimental animal ethics review committee.

[0056] Experimental instruments

[0057] Table 5 shows the main instruments used in the experiment, and Table 6 shows the main reagents used in the experiment.

[0058] Table 5 Main instruments used

[0059]

[0060] Table 6 Main reagents used

[0061]

[0062] Experimental methods

[0063] Biocompatibility

[0064] 7-8 week old ICR female mice were gavaged with 0.2 mL FI EcN every other day for 7 days, and the control group was gavaged with 0.2 mL PBS for a total of 4 times. The body weight of mice was monitored every day. The mice were killed on the 7th day, and their hearts, livers, spleens, lungs, and kidneys were removed and fixed in tissue fixative for 24-36 hours, dehydrated with ethanol gradient, embedded in paraffin, and made into 5 μm sections, followed by H&E staining, observation under a microscope, and photography.

[0065] Establishment of a mouse model of ulcerative colitis

[0066] Female ICR mice aged 7-8 weeks were randomly divided into 3 groups of 8 mice, namely, control group (blank group), dextran sulfate sodium DSS group, and FI EcN group. The ulcerative colitis model was established. Fig.23 As shown in the figure, the specific steps are as follows: After the mice have adapted for 7 days, the control group drinks water normally, and the other groups drink 3% DSS (36-50kDa) freely for one week. 10g / L arabinose was added to the drinking water of each group of mice 3 days before the first gavage until sampling. After 14 days, each preparation group was gavaged every other day for a total of 4 times. The control group and the DSS group were gavaged with 200μL PBS (pH7.4) every other day, and the FI EcN group was gavaged with 200μL FI EcN (1×10 10 cfu / mL), and the body weight of mice was monitored every day during the experiment.

[0067] Recording of mouse biochemical indicators and sample collection

[0068] During the experiment, the weight of the mice was recorded daily. After the experiment, the length of the colon of each mouse was recorded and photographed.

[0069] Pathological analysis

[0070] The colon samples were washed twice with saline, immersed in 1 mL of tissue fixative solution containing 4% paraformaldehyde, and the tissues were embedded in paraffin. 10 μm paraffin sections were used for H&E staining observation, and the H&E staining results were analyzed and evaluated. H&E staining scoring criteria: The total histological score is expressed as E+I, with a total score of 16 points. The specific scoring criteria are shown in Table 7.

[0071] Table 7 DSS colitis histological grading scheme

[0072]

[0073] Inflammatory factor detection

[0074] The colon samples were homogenized and the contents of IL-6, TNF-α, IL-1β, and IL-10 were determined according to the instructions of the ELISA kit.

[0075] Statistical analysis

[0076] Unless otherwise stated, all experimental results were repeated at least three times, and all data were analyzed by GraphPad Prism8.0. All quantitative data are expressed as mean±SD. The significance was evaluated by unpaired Student's t-test and one-way and two-way analysis of variance (ANOVA), and p<0.05 was considered to be significantly different.

[0077] Experimental Results

[0078] (1) Effects of different treatment regimens on pathological indicators of mice

[0079] An important parameter for monitoring the severity of colitis is the weight of the mice. During treatment, the weight of the mice was monitored regularly. Figure 5 As shown in the figure, the weight of mice in the DSS group showed a significant downward trend, while the weight of mice in the FI EcN group recovered significantly. Figure 6 As shown, the colon length of mice in the DSS group was significantly shortened, and the colon length of the FI EcN-treated group was 7.56 cm, which was significantly higher than that of the DSS group (6.20 cm), indicating that FI EcN has a good ability to treat colitis.

[0080] (2) Effects of different treatment regimens on mouse histopathology

[0081] H&E staining was used to observe the specific morphology of colon tissue sections. Compared with the Control group, the colon of mice in the DSS group showed obvious inflammatory response, manifested by a large number of neutrophil infiltration, crypt dissolution and disappearance, and the colon mucosa and goblet cells were almost completely destroyed ( Figure 7 ), colon histopathological scores were significantly increased. The FI EcN group restored the structural integrity of the colon to some extent. Histological evaluation of colon sections revealed that the average score in the DSS group was 7.66. FI EcN treatment significantly reduced the colon histopathological scores of UC mice ( Figure 8 ). In summary, FI EcN can effectively alleviate the level of colon inflammation and improve colon tissue damage in mice with ulcerative colitis.

[0082] (3) Effects of different treatment regimens on the levels of inflammatory factors in mice

[0083] In order to understand the effect of FI EcN on the immune stress indexes of mice, the content of inflammatory factors in the colon of each group of mice can be measured. The ELISA method was used to detect the changes in the content of inflammatory factors IL-6, TNF-α, IL-1β, IL-4, IL-10, and MPO in the intestinal tissue of mice. Specifically, Figure 9-12 As shown, in the Control group, the content of IL-6 was 0.3415 pg / mg, the content of TNF-α was 0.5912 pg / mg, the content of IL-1β was 0.7953 pg / mg, the content of IL-4 was 0.8117 pg / mg, and the content of IL-10 was 2.384 pg / mg. In the DSS group, the content of IL-6 was 16.19 pg / mg, the content of TNF-α was 19.33 pg / mg, the content of IL-1β was 12.78 pg / mg, the content of IL-4 was 0.08658 pg / mg, and the content of IL-10 was 1.009 pg / mg. In the FI EcN group, the content of IL-6 was 0.9013pg / mg, the content of TNF-α was 0.6812pg / mg, the content of IL-1β was 7.146pg / mg, and the content of IL-10 was 5.509pg / mg. Compared with the DSS group, the content of anti-inflammatory factor IL-10 in the FI EcN group increased by 2.31 times, while the content of pro-inflammatory factors IL-6, TNF-α and IL-1β decreased by 94.41%, 96.46% and 44.11%, respectively. It can be seen that when the immune system in the colon mucosa is activated for a long time, it will increase the release of various pro-inflammatory factors (such as interleukin IL-6, TNF-α and IL-1β), while the release of anti-inflammatory factors (such as IL-10 and IL-4) will be inhibited.

[0084] Based on the fact that IL-6 is a multifunctional cytokine that can promote the occurrence and maintenance of inflammatory responses, and is also involved in regulating the activation and migration of immune cells. TNF-α is a potent proinflammatory cytokine that is mainly produced by monocytes and macrophages. Excessive or long-term production of TNF-α is associated with the inflammatory process and the pathogenesis of various diseases. IL-1β, as an inflammatory mediator, can cause vasodilation, local exudation, and leukocyte activation, thereby exacerbating the degree of inflammatory response. Furthermore, the excessive release of these three factors is closely related to the aggravation and persistence of intestinal inflammation. In contrast, IL-10 is an anti-inflammatory factor that mainly plays a role in regulating and inhibiting immune responses. It can inhibit the production of proinflammatory factors and the activation of immune cells, regulate the intensity and duration of inflammatory responses, and help maintain immune balance and tissue homeostasis. Therefore, IL-4 can effectively promote the transformation of macrophage M1 phenotype to M2 phenotype, increase anti-inflammatory factors, and reduce proinflammatory immune cells in inflammatory sites. According to Figure 9-12 The experimental results shown in the figure show that compared with the mice in the DSS group, FI EcN treatment can effectively increase the content of anti-inflammatory factors in the mouse colon and reduce the content of pro-inflammatory factors. IL-4 also shows therapeutic effects in various inflammatory disease models, mainly through inhibiting the release of inflammatory mediators, regulating immune responses, promoting tissue repair, and regulating cell polarization. It can reduce the degree of inflammatory response, regulate the body's immune response, thereby promoting the repair and regeneration of damaged tissues, and help alleviate the pathological process and clinical symptoms of inflammatory diseases. Therefore, FI EcN is quite effective in maintaining the level of inflammatory factors in UC mice.

[0085] Based on the above examples, it can be seen that the body weight and colon length of mice treated with DSS are significantly reduced, and the HE pathological sections show that there are a lot of inflammatory infiltrations in the colon tissue, and the colon histological score is significantly reduced, which indicates that the mouse ulcerative colitis model is successfully established. And after the treatment intervention of the preparation group, the therapeutic effect of the FI EcN group on the mouse phenotype, the mouse weight, colon length and histopathological sections are very similar to those of the Control group. This shows that FI EcN has a significant therapeutic effect on mouse ulcerative colitis, and its therapeutic effect may involve multiple mechanisms such as inhibiting inflammatory response and promoting tissue repair. And the DSS-induced mouse ulcerative colitis model is widely used to simulate the pathological process of human UC, and the pathological characteristics obtained in the above examples include intestinal bleeding, diarrhea, weight loss and colon mucosal infiltration of immune cells (such as lymphocytes, macrophages and neutrophils), which are consistent with the clinical manifestations of human UC, and thus provide a theoretical basis for the subsequent application in the treatment of human UC.

[0086] In addition, the observed OTUs index in the intestinal tissues of mice in different groups can be further determined. Fig.13As shown, the average OTU number in the DSS group was 350, which was significantly lower than that in the Conrol group, while the number of OTUs in the FI EcN was significantly higher than that in the DSS group, with a small difference from the Control group, indicating that the FI EcN treatment had a positive effect on the number of OTUs. Among them, Control, n = 5; DSS, n = 5; FI EcN, n = 5) Each point represents a treated mouse (n, number of mice).

[0087] The Venn diagram was used to determine the number of unique and common OTUs in different groups to intuitively present the similarities and differences in the OTU composition of each experimental group. Fig.14 The OTU Venn diagram is shown, in which the Control group had 1726 unique OTUs, the DSS group had 815 unique OTUs, and the FI EcN group had 977 unique OTUs. The results showed that oral administration of FI EcN can significantly increase the species richness of intestinal flora.

[0088] As well as diversity analysis, the alpha diversity of species in the sample is usually evaluated using Chao, Simpson and Shannon indices. Among them, the Chao1 index can reflect the community richness, and the Shannon index is used to reflect the community diversity. Specifically, Fig.15 The Chao1 index results are shown. The Chao1 index can reflect the richness of the community. We observed that the Chao1 index in the DSS group decreased significantly. After the FI EcN intervention, the Chao1 index showed an upward trend. There was no significant difference between the Chao1 index and the Control group. The results showed that FI EcN can effectively improve the richness of the intestinal flora. (Control, n = 5; DSS, n = 5; FI EcN, n = 5) Each point represents a treated mouse (n, number of mice).

[0089] as well as Fig.16 The figure shows the Shannon index of the intestinal tissues of mice in different groups. The Shannon index of the DSS group was significantly lower than that of the FI EcN group. The above results show that FI EcN can effectively improve the diversity of intestinal flora in colitis mice. (Control, n = 5; DSS, n = 5; FI EcN, n = 5) Each point represents a treated mouse (n, number of mice).

[0090] The families and genera of the bacterial species in the intestinal tissues of different groups of mice were further determined. Fig.17Shown is a heat map of species abundance clustering at the family level. As can be seen from the figure, FI EcN treatment significantly increased the relative abundance of Akkermansia, Lachnospiraceae_NK4A136 and Lactobacillus in colitis. As an obligate anaerobic bacterium, Akkermansia can produce butyrate to reduce the secretion of key inflammatory factors in inflammatory bowel disease, such as tumor necrosis factor-α, interferon-γ and IL-1β. In addition, it increased the secretion of anti-inflammatory factors such as IL-10 in vitro and in vivo, and increased mucus production in vitro. Lachnospiraceae_NK4A136 can promote colonization resistance to intestinal pathogens by producing short-chain fatty acids. Lactobacillus can enhance intestinal barrier function by increasing mucus production, stimulating the release of antimicrobial peptides and the production of secretory immunoglobulin A, increasing the tight junction integrity of intestinal epithelial cells and providing resistance to pathogens. At the same time, FI EcN treatment significantly reduced the relative abundance of Desulfovibrionaceae in colitis. Desulfovibrionaceae can produce lipopolysaccharide, severely damage the intestinal barrier, and is a virulent pathogen that aggravates colitis. These results indicate that FI EcN exhibits significant intestinal flora regulation ability, and oral probiotic delivery therapy plays an excellent role in treating dysbiosis in colitis mice. (Control, n = 4; DSS, n = 4; FI EcN, n = 4) Each point represents a treated mouse (n, number of mice).

[0091] As well as Fig.18 Bubble plot of species distribution at the genus level is shown. The relative richness of the microbial community at the genus level can be determined by community bubble plot analysis. SCFA is a key metabolite that maintains the homeostasis of the intestinal environment and has an important anti-inflammatory effect. The genus Muribaculaceae can indirectly promote the fermentation of fiber to produce SCFAs. This bacterium increased after FI EcN treatment and was significantly reduced in the DSS group. In addition, FI EcN treatment also increased the relative abundance of Akkermansia, a beneficial intestinal bacterium that gradually decreases with the development of enteritis and is significantly increased after FI EcN treatment. In conclusion, in DSS-induced colitis mice, FI EcN can effectively increase the abundance of beneficial bacteria, reduce pathogenic bacteria, improve the richness and diversity of intestinal microbiota, regulate the composition of intestinal microbial flora, and help enhance the therapeutic effect on colitis. (Control, n = 5; DSS, n = 5; FI EcN, n = 5) Each point represents a treated mouse (n, number of mice).

[0092] And determine the relative abundance (relative abundance%) of different families in different groups, such as Fig.19 Shown is the relative abundance of Akkermansia, Fig. 20 is the relative abundance of Lachnospiraceae_NK4A136 and Fig.21 is the relative abundance of Lactobacillus, and it can be seen from the figure that FI EcN treatment significantly increased the relative abundance of Akkermansia, Lachnospiraceae_NK4A136 and Lactobacillus in colitis. (Control, n = 5; DSS, n = 5; FI EcN, n = 5) Each point represents a treated mouse (n, number of mice).

[0093] And, if Fig. 22 The relative abundance of Desulfovibrionaceae is shown, among which FI EcN treatment significantly reduced the relative abundance of Desulfovibrionaceae in colitis. The principle is that Desulfovibrionaceae can produce lipopolysaccharide, which seriously damages the intestinal barrier and is a virulent pathogen that aggravates colitis. The above results show that FI EcN exhibits significant intestinal flora regulation ability and plays an excellent role in treating dysbiosis in colitis mice. (Control, n = 5; DSS, n = 5; FI EcN, n = 5) Each point represents a treated mouse (n, number of mice).

[0094] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or system including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or system. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or system including the element.

[0095] The serial numbers of the above embodiments of the present invention are only for description and do not represent the advantages or disadvantages of the embodiments.

Claims

1. An engineered probiotic for targeted treatment of colitis, characterized in that: The Latin name of the engineered probiotic is Escherichia coli Nissle 1917FI, and it is deposited in the General Microbiological Center of China Microbiological Culture Management Committee with a deposit number of CGMCCNO.31539.

2. A method for constructing an engineered probiotic for targeted treatment of colitis as claimed in claim 1, characterized in that: The method comprises the following steps: Step 1, PelB and the optimized Fc and IL-4 genes were connected to homology arms by PCR, and inserted into the pBAD33 plasmid after seamless cloning to construct a recombinant plasmid; Step 2, the plasmid obtained in step 1 is transformed into competent E. coli DH5α by heat shock method, and after colony PCR verification, sequencing verification is performed to determine whether the recombinant plasmid is successfully constructed; Step 3, under the condition that the recombinant plasmid is successfully constructed, the recombinant plasmid is transferred into the competent EcN by the heat shock method, and the chloramphenicol-resistant LB solid plate is used for screening. The screened colonies are cultured in LB liquid culture medium containing chloramphenicol, and collected and preserved by centrifugation.

3. The method for constructing an engineered probiotic for targeted treatment of colitis according to claim 2, characterized in that: The recombinant plasmid is Pbad33-pelB-FC-IL-4, and the gene sequence of the recombinant plasmid is shown in SEQ ID NO.

13.

4. The method for constructing an engineered probiotic for targeted treatment of colitis according to claim 2, characterized in that: The optimized Fc gene sequence is shown in SEQ ID NO.7, and the optimized IL-4 gene sequence is shown in SEQ ID NO.

9.

5. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the engineered probiotics for targeted treatment of colitis according to claim 1.

6. The pharmaceutical composition according to claim 5, characterized in that The pharmaceutical composition also includes a pharmaceutically or pharmacologically acceptable carrier.

7. A kit, characterized in that: The kit comprises the engineered probiotics for targeted treatment of colitis according to claim 1 or the pharmaceutical composition according to any one of claims 5-6.

8. Use of the engineered probiotics for targeted treatment of colitis according to claim 1, the pharmaceutical composition according to any one of claims 5 to 6, or the kit according to claim 7 in the preparation of drugs for regulating intestinal flora or drugs or health products for preventing intestinal inflammation and damage.

9. The use according to claim 8, characterized in that: The dosage form of the medicine or health product is tablet, capsule, soft capsule, granule, pill, oral liquid, emulsion or dry suspension.

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