A mucosal repair engineered probiotic and its application

Abstract

This invention belongs to the field of genetic engineering technology, specifically relating to a mucosal repair engineered probiotic and its applications. The mucosal repair engineered probiotic constructed in this invention... Escherichia coli The Nissle 1917 expression vector EcN-pBbB8K-CsgA-LFCA-TFF3 links lactoferrin peptide LFCA and trefoil factor 3 TFF3 with EcN-CsgA, enhancing the engineered bacteria's ability to repair Caco-2 intestinal epithelial cells and promoting the proliferation and aggregation of intestinal epithelial cells. This provides an important foundation for the engineered bacteria to establish continuous colonization and cell-cell interaction in the digestive tract environment.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a mucosal repair engineered probiotic and its application. Background Technology

[0002] Salmonella typhimurium ( Salmonella typhimurium As a foodborne pathogen, spirochetes are one of the main causes of diarrhea-related intestinal diseases in various animals and humans. They can increase the number of bacteria colonizing the intestines, leading to colitis. A key step in their infection cycle is invading intestinal epithelial cells. Once inside the body, these bacteria rapidly grow in the intestines using oxygen, and the proliferating bacteria are released into the intestinal lumen, exacerbating the infection. Currently, antibiotics are one of the main treatments for bacterial diseases, but emerging bacterial resistance seriously threatens public health security. Therefore, there is an urgent need to find effective antibiotic alternatives.

[0003] Probiotics have been used to enhance immunity and maintain the balance of the gut microbiota ecosystem, and are considered a powerful alternative therapy for preventing pathogen infections. Escherichia coli Nissle 1917, or EcN for short, is the only known non-pathogenic Escherichia coli strain and is a probiotic. Clinical results have shown that it can promote the growth of beneficial bacteria in the gut, inhibit the excessive proliferation of pathogenic bacteria, enhance the body's antibacterial ability, and reduce damage to the intestinal mucosa. Engineered probiotics based on EcN have been applied in the field of intestinal disease prevention and control.

[0004] However, oral probiotics are limited in their survival and colonization in the gut by the gastrointestinal environment. Damage to intestinal epithelial cells following disease exposes the intestinal endothelium to potentially harmful substances such as bacteria and their products. Rapid restoration of intestinal mucosal and epithelial integrity is therefore crucial for disease treatment. Complete regeneration depends on the slow proliferation and differentiation of stem cells. In contrast, recovery can occur within hours of injury and depends on the migration of epithelial cells from surrounding areas to the wound site. Therefore, constructing engineered bacteria capable of repairing the intestinal mucosa and epithelium is essential for improving the survival and colonization of probiotics in the gut, thereby enabling the clinical application of engineered microbial therapy.

[0005] Currently, in research on the treatment of intestinal diseases, [the following is being studied / developed]: S. typhimurium There is still no solution for treating bacterial intestinal diseases by preparing engineered probiotics to repair and repair the slow healing rate of the intestinal epithelial mucosa and the untimely local mucosal immunity caused by infection. Summary of the Invention

[0006] To overcome the shortcomings of the prior art, this invention provides a mucosal repair engineered probiotic and its application. This bacterium contains the recombinant plasmid pBbB8K-CsgA-LFCA-TFF3. This engineered bacterium can rapidly recruit surrounding cells, repair damaged epithelial tissue, and enhance the expression of tight junction proteins ZO-1, Claudin-1, and Occludin. Simultaneously, it can promote the maturation and differentiation of DCs, B cells, and Treg cells, induce immunoglobulin secretion, inhibit the activation of the p65 NF-κB signaling pathway, increase the expression of anti-inflammatory factors, and decrease the expression of pro-inflammatory factors, thereby regulating local mucosal immune responses and maintaining gut microbiota homeostasis. The engineered bacterium constructed in this application solves the problem... S. typhimurium The slow healing rate of epithelial mucosa and delayed local mucosal immunity caused by infection provide theoretical support for the research of vaccines for animal diarrheal diseases. The mucosal repair probiotics prepared in this invention can significantly inhibit bacterial enteritis in mice, providing an effective treatment for inflammatory bowel disease and other gastrointestinal diseases.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] In a first aspect, the present invention provides a mucosal repair engineered probiotic containing a CsgA-LFCA-TFF3 fusion gene. The CsgA-LFCA-TFF3 fusion gene is formed by the tandem formation of the CsgA gene, the lactoferrin LFCA gene, and the enterotrilobite TFF3 gene from the EcN strain.

[0009] Amyloid proteins are defined as amyloid filaments, aggregates of proteins, and are commonly used as protein components of the extracellular matrix in bacterial biofilms. One of the most well-studied functional amyloid proteins is friable filaments, an extracellular filament produced by *E. coli*. Friable filaments are important for adhesion to inert surfaces and for the properties of host cells, biofilm formation, and other communities. Its major subunit protein is CsgA. Friable filament production is also crucial for initial adhesion to biotic and abiotic surfaces and is associated with environmental resistance and pathogenesis. Trefoil factor 3 (TFF3) is a secreted protein; mucin-associated trefoil factor (TFF) peptides are an integral part of gastrointestinal cell protection and mucosal repair. Structural analysis shows that the formation of stable trefoil structures via disulfide bonds provides protease resistance. Antimicrobial peptides (AMPs) have emerged as a potential new strategy for the prevention and treatment of bacterial infections. Antimicrobial peptides (AMPs) have become a novel class of drug candidates for treating infectious diseases. These short peptides, typically containing 10 to 500 amino acids, are an important component of the innate immune system and form the first line of defense against infection. However, there are no reports of expressing the three genes by fusion in existing technologies.

[0010] As an optional embodiment of the present invention, the nucleotide sequence of CsgA is shown in SEQ ID NO.1, the nucleotide sequence of lactoferrin LFCA is shown in SEQ ID NO.2, and the nucleotide sequence of intestinal trefoil factor TFF3 is shown in SEQ ID NO.3.

[0011] As an optional embodiment of the present invention, the nucleotide sequence of the CsgA-LFCA-TFF3 fusion gene is as shown in SEQ ID NO.4; or the CsgA-LFCA-TFF3 fusion gene is another nucleotide sequence that can express the same amino acid sequence as SEQ ID NO.4 through codon degeneracy.

[0012] In a preferred embodiment of the present invention, the fusion gene further contains an enzyme cleavage site, wherein the enzyme cleavage site is... Bgl II and Xho I site.

[0013] As an optional embodiment of the present invention, a linker sequence is added between genes CsgA and LFCA. The linker sequence is as follows:

[0014] GGTGGAGGAGGCTCTGGTGGAGGCGGTAGCGGAGGCGGAGGGTCG (SEQ ID NO. 5).

[0015] In a preferred embodiment of the present invention, a linker sequence was added between the genes LFCA and TFF3 and optimized. The optimized sequence is: Linker C: GGCGGTGGCGGTGGCGGT (SEQ ID NO.6).

[0016] Different linker sequences have a significant impact on the replication of recombinant vectors containing fusion genes in the host. This invention demonstrates through experiments optimizing linker sequences that the linker combination of nucleotide sequences such as SEQ ID NO.5 and SEQ ID NO.6 has the best expression effect in host cells.

[0017] As an optional embodiment of the present invention, the probiotics are... Escherichia coli Nissle1917 was the starting strain.

[0018] A second aspect of the present invention provides a fusion gene formed by the tandem of the CsgA gene, the lactoferrin LFCA gene, and the trefoil factor TFF3 gene.

[0019] As an optional embodiment of the present invention, the fusion gene is composed of CsgA-Linker-LFCA-Linker C-TFF3 in series.

[0020] As an optional embodiment of the present invention, the fusion gene sequence is shown in SEQ ID NO.4.

[0021] A third aspect of the present invention provides a fusion protein encoded by the fusion gene CsgA-LFCA-TFF3 described in the second aspect, the amino acid sequence of which is shown below.

[0022] MKLLKVAAIAAIVFSGSALAGVVPQYGGGGNHGGGGNNSGPNSELNIYQYGGGNSALALQTDARNSDLTITQHGGGNGADVGQGSDDSSIDLTQRGFGNSATLDQWNGKNSEMTVKQFGGGNGAAVDQTASNSSVNVTQVGFGNNATAH QYGGGGSGGGGSGGGGSFKCRRWQWRMKKLGAPSITCVRRAFGGGSSVDGKEDLIWKLLSKAQEKFGKNKSRTATATAGGGGGGEEYVGLSANQCAVPAKDRVDCGYPHVTPKECNNNRGCCFDSRIPGVPWCFKPLQEAECTF(SEQID NO.7)

[0023] A fourth aspect of the present invention provides an expression vector comprising the fusion gene described in the second aspect.

[0024] As an optional embodiment of the present invention, the expression vector is obtained by ligating a fusion gene to a vector pBbB8K, wherein the ligation method is enzyme digestion ligation; the enzyme digestion site is... Bgl II and Xho I.

[0025] As an optional embodiment of the present invention, the recombinant expression vector is obtained by effectively linking the above-mentioned polynucleotide molecules to the expression vector. The expression vector is any one or more of viral vectors, plasmids, bacteriophages, phage particles, sclerosing particles, F sclerosing particles, or artificial chromosomes. The viral vector may include adenovirus vectors, retrovirus vectors, or adeno-associated virus vectors. The artificial chromosomes include bacterial artificial chromosomes (BAC), phage P1-derived vectors (PAC), yeast artificial chromosomes (YAC), or mammalian artificial chromosomes (MAC).

[0026] A fifth aspect of the present invention provides a pharmaceutical composition, pharmaceutical preparation, microbial agent, or feed comprising the mucosal repair engineered probiotics described in the first aspect or their fermentation products or metabolites, or the fusion protein described in the third aspect.

[0027] A sixth aspect of the invention provides the use of the adhesion-enhancing engineered probiotics described in the first aspect, the fusion gene described in the second aspect, or the fusion protein described in the third aspect in the preparation of medicaments, vaccines, bacterial agents, or feeds for the prevention and / or treatment of antibacterial enteritis; wherein the antibacterial enteritis is Salmonella typhimurium infection.

[0028] A seventh aspect of the invention provides the use of the mucosal repair engineered probiotics described in the first aspect, the fusion gene described in the second aspect, the fusion protein described in the third aspect, the recombinant plasmid described in the fourth aspect, or the pharmaceutical composition, pharmaceutical preparation, microbial agent, or feed described in the fifth aspect in the preparation of any of the following products:

[0029] 1) Prepare Caco-2 repair agent for intestinal epithelial cells and / or prepare Salmonella typhimurium K88 inhibitor;

[0030] 2) Prepare products that activate immune cells;

[0031] 3) Prepare products that maintain the integrity of the intestinal barrier, resist pathogen infection, and improve intestinal inflammation.

[0032] The above one or more technical solutions have the following beneficial effects:

[0033] 1. The mucosal repair engineered probiotics constructed in this invention Escherichia coli The Nissle 1917 expression vector EcN-pBbB8K-CsgA-LFCA-TFF3, through optimized linker sequences, connects EcN-CsgA, lactoferrin peptide LFCA, and trefoil factor 3 TFF3. This enhances the engineered bacteria's ability to repair Caco-2 intestinal epithelial cells and promotes the proliferation and aggregation of intestinal epithelial cells, providing an important foundation for the engineered bacteria to establish sustained colonization and cell-cell interactions in the digestive tract environment.

[0034] 2. By detecting the expression of tight junction proteins in intestinal epithelial cells, the results showed that under pathogenic bacterial infection, the engineered strain EcN-pBbB8K-CsgA-LFCA-TFF3 could increase the expression levels of tight junction proteins Occludin and ZO-1, and reduce the phosphorylation level of p65 protein induced by ETEC K88 invasion of cells. Compared with the wild-type strain, it could inhibit the activation of the NF-κB signaling pathway.

[0035] 3. Using the engineered bacterium EcN-pBbB8K-CsgA-LFCA-TFF3 as a delivery vector, it can be orally inoculated into animals. Utilizing its rapid repair effect on intestinal epithelial cells and its competitive inhibition of pathogenic bacteria, it can effectively enhance the interaction between the engineered bacteria and immune-related cells, strengthen the activation of antigen-presenting cells (dendritic cells), and promote the proliferation of Treg and B lymphocytes. Furthermore, by increasing sIgA and IgG levels and decreasing TNF-α and IL-1β levels, it enhances specific immune responses and anti-inflammatory reactions. The mucosal immune response induced by the engineered bacteria can protect mice against... S. typhimurium Infection can improve intestinal tissue pathological damage by regulating the structure of the gut microbiota. This study found that engineered bacteria can increase the content of the short-chain fatty acid caproic acid, a metabolite of the gut microbiota. It is speculated that the intervention of engineered bacteria regulates the abundance of short-chain fatty acid-producing metabolic bacteria, thereby increasing caproic acid production.

[0036] 4. Using the engineered bacteria described in this application as the substrate strain, protective antigens, health-promoting genes, or drug genes of animal or human pathogenic bacteria can be expressed to prepare novel functional engineered probiotics. These probiotics can be formulated into oral probiotic preparations, making them "microbial factories" for producing therapeutically active substances in humans or animals. This avoids the problems of traditional treatments relying on drug intervention, focusing on regulating inflammatory pathways, and making epithelial mucosal repair and regeneration a slow process. These novel engineered probiotics, as oral preparations, possess multiple functions including gene expression, continuous colonization, and probiotic effects. They have broad application prospects and significant social and economic benefits in the food, feed, pharmaceutical, and health industries.

[0037] Advantages of additional aspects of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0038] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0039] Figure 1 This is a schematic diagram of testing the relative expression level of the TFF3 gene with different linker sequences in Embodiment 1 of the present invention.

[0040] Figure 2 The growth curve detection results of strain ECN-pBbB8K-CsgA-LFCA-TFF3 in Example 1 of this invention are shown.

[0041] Figure 3 The results are from the quantitative binding assay of Congo red for EcN-pBbB8K-CsgA-LFCA-TFF3.

[0042] Figure 4 The results are from scanning electron microscopy analysis of EcN-pBbB8K-CsgA-LFCA-TFF3.

[0043] Figure 5 The results of Western blot analysis of recombinant protein from strain EcN-pBbB8K-CsgA-LFCA-TFF3 are shown.

[0044] Figure 6 The results of immunofluorescence detection for strain EcN-pBbB8K-CsgA-LFCA-TFF3 are shown.

[0045] Figure 7 The results of the detection of the invasive and proliferative capacity of Caco-2 cells by EcN-pBbB8K-CsgA-LFCA-TFF3 are shown in Figure A, where A represents the effect of EcN-pBbB8K-CsgA-LFCA-TFF3 on the invasive capacity of Caco-2 cells, and B represents the effect of EcN-pBbB8K-CsgA-LFCA-TFF3 on the proliferative capacity of Caco-2 cells.

[0046] Figure 8 The effect of EcN-pBbB8K-CsgA-LFCA-TFF3 on the expression of tight junction proteins in Caco-2 cells is shown in Figure 1. In Figure 2, A represents the expression of Occludin protein in Caco-2 cells detected by Western blot, B represents the expression of Claudin-1 protein in Caco-2 cells detected by Western blot, and C represents the expression of ZO-1 protein in Caco-2 cells detected by Western blot.

[0047] Figure 9 The effect of EcN-pBbB8K-CsgA-LFCA-TFF3 on NF-κB pathway activation in Caco-2 cells is shown in Figure A, where Western blot analysis shows the p65 protein expression in Caco-2 cells, and Western blot analysis shows the p-p65 protein expression in Caco-2 cells.

[0048] Figure 10The results of RT-PCR detection of EcN-pBbB8K-CsgA-LFCA-TFF3 in Caco-2 cells are as follows: A is the result of RT-PCR detection of ZO-1 gene expression in Caco-2 cells; B is the result of RT-PCR detection of Claudin-1 gene expression in Caco-2 cells; C is the result of RT-PCR detection of Occludin gene expression in Caco-2 cells; D is the result of RT-PCR detection of p65 gene expression in Caco-2 cells; and E is the result of RT-PCR detection of IL-17 gene expression in Caco-2 cells.

[0049] Figure 11 This study investigated the effect of EcN-pBbB8K-CsgA-LFCA-TFF3 on dendritic cell activation in SP, PPs, and MLN. In the data, A represents the number of dendritic cells in the spleen of mice in each group; B represents the number of dendritic cells in Pell's nodes of mice in each group; C represents the number of dendritic cells in the mesenteric lymph nodes of mice in each group; and A1 represents the number of CD11c cells in the spleen of mice detected by flow cytometry. + CD86 + The mean fluorescence signal intensity of DCs cells, B1 represents the CD11c concentration in mouse mesenteric lymph nodes detected by flow cytometry. + CD86 + The mean fluorescence signal intensity of DCs cells, C1 represents the CD11c concentration in mouse Pearson's nodes detected by flow cytometry. + CD86 + The mean fluorescence signal intensity of DCs cells, A2 represents the CD11c concentration in mouse spleen detected by flow cytometry. + CD80 + The mean fluorescence signal intensity of DCs cells, B2 represents CD11c in mouse mesenteric lymph nodes. + CD80 + The mean fluorescence signal intensity of DCs cells, C2 is the CD11c in mouse Pell's node. + CD80 + The average fluorescence signal intensity of DCs cells.

[0050] Figure 12 The effect of EcN-pBbB8K-CsgA-LFCA-TFF3 on the proliferation of B lymphocytes in SP and MLN, where A represents B220 in mouse spleen. + IgA + B cell count, where B represents B220 in mouse mesenteric lymph nodes. + IgA + B cell count.

[0051] Figure 13The effect of EcN-pBbB8K-CsgA-LFCA-TFF3 on the proliferation of Treg cells in SP and MLN cells, where A represents CD4+ in mouse spleen. + CD25 + FOXP3 + T cell count, B represents CD4+ in mouse mesenteric lymph nodes. + CD25 + FOXP3 + T cell count.

[0052] Figure 14 The results show the detection of sIgA in mouse feces and IgG in serum.

[0053] Figure 15 The results of ELISA detection of IL-10, TNF-α and IL-1β in mouse serum are shown. In the figure, A represents the expression level of IL-10 in the serum of each group of mice, B represents the expression level of TNF-α in the serum of each group of mice, and C represents the expression level of IL-1β in the serum of each group of mice.

[0054] Figure 16 The results show the transcriptional levels of IL-10, TNF-α, and IL-1β genes in mouse colon tissue. In this table, A represents the IL-10 gene expression level in each group of mouse colon tissue detected by RT-PCR, B represents the TNF-α gene expression level in each group of mouse colon tissue detected by RT-PCR, and C represents the IL-1β gene expression level in each group of mouse colon tissue detected by RT-PCR.

[0055] Figure 17 The study investigated the effects of engineered bacteria on the transcriptional levels of genes related to the intestinal innate immune signaling pathway. A represents the expression level of IL-33 gene in the colonic tissue of mice in each group, detected by RT-PCR; B represents the expression level of IL-22 gene in the colonic tissue of mice in each group, detected by RT-PCR; C represents the expression level of IL-17 gene in the colonic tissue of mice in each group, detected by RT-PCR; and D represents the expression level of p65 gene in the colonic tissue of mice in each group, detected by RT-PCR.

[0056] Figure 18 The study investigated the effect of engineered bacteria on the transcriptional levels of intestinal tight junction protein-related genes. In this study, A represents the expression level of the ZO-1 gene in the colon tissue of mice in each group, detected by RT-PCR, and B represents the expression level of the Occludin gene in the colon tissue of mice in each group, detected by RT-PCR.

[0057] Figure 19 The results showed the survival rate and weight changes of mice.

[0058] Figure 20 The results of histopathological examination of the duodenal tissue of mice in each group are shown.

[0059] Figure 21 The results of histopathological examination of the spleen tissue of mice in each group are shown.

[0060] Figure 22 The results show the number of goblet cells in the small intestine of mice in each group.

[0061] Figure 23 The classification composition of the gut microbiota of mice in each group is shown below. A represents the taxonomic composition analysis at the genus level, B represents the heatmap of species composition at the genus level, C represents the taxonomic composition analysis at the phylum level, and D represents the heatmap of species composition at the phylum level.

[0062] Figure 24 The results show the detection results of the mouse metabolite Hexanoic acid content. Detailed Implementation

[0063] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0064] The present invention will now be further illustrated with specific examples. These examples are for illustrative purposes only and do not limit the scope of the invention. Unless otherwise specified, experimental conditions not explicitly stated in the examples are generally performed under conventional conditions or as recommended by the reagent company. Unless otherwise specified, all reagents and consumables used in the following examples are commercially available.

[0065] The main materials and equipment used in this invention are as follows:

[0066] 1. Strains, plasmids, and cell lines

[0067] Escherichia coli Nissle 1917 strain, S. typhimurium ATCC 25241 strain, Escherichia coli MC1061 strain and Caco-2 cell line were purchased from BNCC (PMID:29155272; DOI:10.1016 / j.fsi.2017.10.043); pBbB8K plasmid was purchased from Changchun Huijin Baili Biotechnology Co., Ltd.; pEASY-Blunt vector was purchased from Beijing TransGen Biotech Co., Ltd.; lactoferrin peptide LFCA gene was synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0068] 2. Main reagents

[0069] Restriction endonucleases Bgl II. Xho I, Nco I and EcoRV and T4 DNA ligase were purchased from TransGen Biotech Ltd.; high-purity plasmid miniprep kit was purchased from Beijing Kangwei Century Biotechnology Co., Ltd.; DNA gel extraction kit was purchased from Omega Bio-Tek Inc.; 10×H buffer and DNA Marker (DL5000, DL10000) were purchased from Takara Bio Inc.; fetal bovine serum, trypsin (containing EDTA), anti-fluorescence attenuation mounting medium, MEM cell culture medium, kanamycin, and chloramphenicol were purchased from Beijing Solarbio Science & Technology Co., Ltd.; developing solution, DMSO, and BCA protein concentration assay kit were purchased from Beyotime Biotechnology Co., Ltd.; HRP-labeled rabbit anti-His-Tag IgG was purchased from Bioss; Rat Anti-Mouse CD11c, Rat Anti-Mouse CD80, Rat Anti-Mouse CD86, Rat Anti-Mouse CD45R / B220, Rat Anti-Mouse CD3, Rat Anti-Mouse IgA, Rat Anti-Mouse CD4, and Rat Anti-Mouse... CD8 antibody was purchased from BD Biosciences.

[0070] Example 1: Construction and Validation of Mucosal Repair Engineered Probiotic EcN / pBbB8K-CsgA-LFCA-TFF3

[0071] 1. Primer design and synthesis

[0072] Obtain using NCBI database E. coli The reading frames of Nissle 1917-CsgA (GenBank: HAY2139435.1) and pUC57-TFF3 (GenBank: NC_000021.9) and their upstream and downstream gene sequences were obtained. The CsgA gene of strain EcN was obtained by PCR cloning. The primer sequences are shown in Table 1.

[0073] Table 1 PCR primer sequences

[0074]

[0075] 2. Construction and identification of the recombinant strain MC1061 / pBbB8K-CsgA-LFCA-TFF3

[0076] 2.1 Optimization of linker connection sequence

[0077] Different linker sequences were synthesized, and the CsgA (SEQ ID NO.1), LFCA (SEQ ID NO.2), and TFF3 (SEQ ID NO.3) genes were fused using SOE-PCR to obtain the fusion fragment CsgA-LFCA-TFF3. The linker sequence between the CsgA and LFCA genes is shown in SEQ ID NO.5. Three sets of flexible linker peptides were designed to link the CsgA-LFCA gene fragment (SEQ ID NO.8) with the TFF3 (SEQ ID NO.3) gene, and the optimal linker sequence was screened. Linker A sequence: GGGGSGGGGSGGGGS, Linker B sequence: EAAAKEAAAK, Linker C sequence: GGCGGTGGCGGTGGCGGT. The recombinant CsgA-LFCA-linker-TFF3 fragment was cloned into the pET-28a(+) vector and transformed into BL21(DE3) competent cells. Positive clones were verified by sequencing, and the optimal linker sequence was determined to be GGCGGTGGCGGTGGCGGT using RT-PCR. Results are as follows: Figure 1 As shown.

[0078] 2.2 Construction and Identification of Recombinant Vectors

[0079] Gene fragments of CsgA (coiled fiber), LFCA (lactoferrin peptide), and TFF3 (tFF3) were amplified by PCR. Using the linker sequences selected in section 2.1, the LFCA (SEQ ID NO.2) and TFF3 (SEQ ID NO.3) genes were fused using SOE-PCR to obtain the fusion fragment LFCA-TFF3 (SEQ ID NO.9). Similarly, the CsgA (SEQ ID NO.1), LFCA (SEQ ID NO.2), and TFF3 (SEQ ID NO.3) genes were fused using SOE-PCR to obtain the fusion fragment CsgA-LFCA-TFF3 (SEQ ID NO.4). Based on these results, designs containing… Bgl II and Xho Primers at site I were used to perform PCR amplification of the CsgA, LFCA-TFF3, and CsgA-LFCA-TFF3 fusion genes to introduce them into the genome. Bgl II and Xho I site, application Bgl II and XhoAfter digestion of the pBbB8K plasmid and the amplified fragment with enzyme I, the amplified fragment was purified and recovered. The amplified fragment was cloned into the pBbB8K plasmid using T4 ligase to obtain recombinant plasmids pBbB8K-CsgA, pBbB8K-LFCA-TFF3, and pBbB8K-CsgA-LFCA-TFF3. These plasmids were transformed into competent E. coli MC1061 cells, and positive clones were screened. The recombinant vectors pBbB8K-CsgA, pBbB8K-LFCA-TFF3, and pBbB8K-CsgA-LFCA-TFF3 with correct sequences were obtained by PCR, double enzyme digestion, and DNA sequencing.

[0080] 3. Construction and identification of engineered bacteria

[0081] 3.1 Chemical transformation of recombinant plasmids to E. coli Nissle 1917 receptive state

[0082] The recombinant vectors pBbB8K-CsgA, pBbB8K-LFCA-TFF3, and pBbB8K-CsgA-LFCA-TFF were transformed into [a specific vector] via chemical transformation. E. coli Recombinant strains EcN-pBbB8K-CsgA, EcN-pBbB8K-LT (abbreviation of recombinant strain EcN / pBbB8K-LFCA-TFF3, hereinafter the same) and EcN-pBbB8K-CLT (abbreviation of recombinant strain EcN / pBbB8K-CsgA-LFCA-TFF3, hereinafter the same) were obtained from Nissle 1917 competent cells by PCR, double enzyme digestion reaction and DNA sequencing.

[0083] 3.2 Determination of growth curve of engineered bacteria

[0084] The recombinant engineered bacteria were inoculated into 100 mL of fresh LB broth at a 1% inoculation rate and cultured on a shaker at 37°C and 185 rpm. OD values ​​were measured every hour using a spectrophotometer. 600 The absorbance at nm was continuously monitored for 12 hours, and the growth curve of the strain was plotted. The results showed that the growth rate of the engineered strain was basically the same as that of the wild-type strain, with no significant difference, indicating that the expression of the target protein had no effect on the growth of the host strain. Figure 2 ).

[0085] 3.3 Quantitative Binding Analysis of Congo Red

[0086] The engineered bacteria were inoculated at a ratio of 1.5% into LB medium (containing 100 μg / mL Kan) for activation. The activated strains EcN-wt, EcN-pBbB8K-CsgA, EcN-pBbB8K-LT, and EcN-pBbB8K-CLT were then inoculated at a ratio of 1% into their respective LB media (containing 100 μg / mL Kan). For EcN-pBbB8K-CsgA, EcN-pBbB8K-LT, and EcN-pBbB8K-CLT, an additional 0.05% L-(+) arabinose was added as an induction condition to induce protein expression. The induced cultures were cultured overnight at 37°C in a shaking incubator to ensure sufficient protein expression. The following day, 1 mL of bacterial culture was centrifuged at 8000 rpm for 10 min, then resuspended in 0.025 mM Congo red PBS solution for 10 min, centrifuged at 14000 rpm for 10 min, and the supernatant was transferred to a 96-well plate with three replicates for each strain. The OD value of the supernatant was measured using a microplate reader. 490 The absorbance at nm was used to calculate the yield of the calibrated crimped fibers. This was done by subtracting the measured absorbance from the absorbance measured in 0.025 mM Congo red PBS solution, and then using the OD of the original bacterial culture. 600 Calibration is performed using nm. For example... Figure 3 As shown, the results indicate that under these conditions, the number of fused EcN-pBbB8K-CsgA and the other two EcN-pBbB8K-CLT and EcN-pBbB8K-LT fusions expressed and assembled into coiled fibers was significantly higher than that of the control vector EcN-pBbB8K-GFP expressing GFP.

[0087] 3.4 Scanning Electron Microscopy Identification

[0088] Scanning electron microscopy results of engineered bacteria are as follows Figure 4 As shown, scanning electron microscopy results confirm that the engineered bacteria EcN-pBbB8K-CsgA, EcN-pBbB8K-LT, and EcN-pBbB8K-CLT can assemble into nanofiber structures of natural crimped fibers. Among them, EcN-pBbB8K-CLT can aggregate in large quantities under the action of crimped fibers to form a dense "PATCH".

[0089] 3.5 Protein Expression Identification by Western Blot

[0090] Recombinant strains of EcN-pBbB8K-CsgA, EcN-pBbB8K-LT, and EcN-pBbB8K-CLT were inoculated into LB medium (containing 100 μg / mL Kan) and cultured overnight at 37°C. The three strains were then re-inoculated at a 1% ratio into their respective LB media (containing 100 μg / mL Kan; 0.05% L-(+) arabinose) and cultured overnight at 37°C and 200 rpm. The supernatant was discarded after centrifugation, and the cultures were washed twice with sterile PBS and resuspended. 800 μL of the resuspended culture was mixed with 200 μL of 5×SDS PAGE Buffer by pipetting and aspirating, and boiled for 10 min. After being placed at room temperature, the cultures were centrifuged at 12,000 rpm for 1 min, and the supernatant was collected for protein electrophoresis.

[0091] The results are as follows Figure 5 As shown, the results indicate that the target bands appeared at 30.2 kDa, 17 kDa, and 15.2 kDa, which is in line with expectations, indicating that the recombinant plasmid can correctly express the fusion protein.

[0092] 3.6 Immunofluorescence detection

[0093] Centrifuge the cultured bacterial suspension at 4000 rpm for 10 min, discard the supernatant, wash twice with sterile PBS buffer, and resuspend in 4 mL PBS. Take 10 μL of the bacterial suspension, spread it evenly on a glass slide, and let it air dry around an alcohol lamp. Then, add 30 μL of anti-His primary antibody to the specimen, place it in a dark box, and incubate at 37°C for 30 min. Rinse in PBS for 10 min, remove the slide, and blot dry the surrounding liquid with absorbent paper, being careful not to let it dry completely. Add 30 μL of fluorescent secondary antibody to the specimen and incubate at 37°C for 30 min. Wash three times with PBS running water for 5 min each time, add an anti-fluorescence quencher, and observe under an inverted fluorescence microscope.

[0094] like Figure 6 As shown, fluorescence microscopy revealed obvious green fluorescence signals in EcN-pBbB8K-CsgA, EcN-pBbB8K-LT, and EcN-pBbB8K-CLT, while no obvious fluorescence signal was observed in the empty vector EcN. This further proves that the recombinant protein can be anchored and correctly expressed on the bacterial surface.

[0095] Example 2: Detection of the regulatory effect of adhesion-enhanced engineered probiotics on intestinal epithelial cell function

[0096] 1. Trypan blue staining test

[0097] Cells were loaded at a rate of 1×10 5Seeds were planted into 24-well plates at a density of 5 × 10⁶ cells / well, with 1 mL of complete culture medium added to each well. The plates were then incubated until confluent. The cell density after confluence was approximately 5 × 10⁶ cells / well. 5 Cells were counted per well, and the complete culture medium was discarded. The cells were washed three times with pre-warmed PBS. The engineered bacteria / Salmonella were mixed at a ratio of 5 × 10⁶ cells / well at an infection multiple (MOI) of 100:1. 7 Cells were diluted to a concentration of 1 / mL and suspended in water. 200 μL of diluted bacterial suspension and 800 μL of MEM (without antibiotics) were inoculated into each well and co-cultured with Caco-2 cells for 2, 4, 6, and 8 h. After co-culture, the culture medium was discarded, and the cells were gently washed once with pre-warmed PBS. Trypsin was added to digest the cells, and the single-cell suspension was diluted 10-fold and mixed with trypan blue solution at a 9:1 ratio. The mixture was then added to a red blood cell counting chamber, and the number of viable cells was immediately measured to calculate the viability. Cells in the blank wells served as controls. Each group had three replicates, and the experiment was repeated three times. S.typhimurium After treatment, the in vitro proliferation of Caco-2 cells was significantly inhibited at 8 h, and this inhibitory effect gradually increased with time. Conversely, the growth inhibition of Caco-2 cells was significantly reduced after 8 h of treatment with engineered bacteria EcN-pBbB8K-CsgA, EcN-pBbB8K-LT, and EcN-pBbB8K-CLT. This also demonstrates that the optimal interaction time between cells and bacteria is within 6 h, without causing cell damage.

[0098] 2. Cell invasion assay

[0099] Caco-2 cells that have been passaged to generations 5-15 will be used at a density of 10 cells per well. 5 Bacterial cells were seeded at a density of 1000 μL in 24-well plates and cultured to 90% confluence in 500 μL of standard cell culture medium. The bacterial culture was centrifuged to collect the pellet, washed with PBS, and diluted with MEM containing 1% fetal bovine serum to a concentration of 5 × 10⁻⁶ cells / well. 7 Before adding bacteria (200 μL), Caco-2 cells were washed twice with PBS to remove antibiotics. The bacteria and Caco-2 cells were co-incubated for 2 h, and the culture medium was removed. The Caco-2 cells were then washed twice with 500 μL PBS, and MEM containing 100 μg / mL gentamicin (containing 1% fetal bovine serum) was added. After 1 h of culture, the culture medium was removed, and 500 μL of 1% Triton X-100 was added. The cells were incubated with 1% Triton X-100 at 37°C for 20-30 min. After incubation, the cells were mixed by pipetting. The mixture was serially diluted and dropped onto a kanamycin plate, and the colony-forming units (CFU) of bacteria invading Caco-2 cells were counted. After cell lysis, the plate counts were as follows: Figure 7 As shown in A, compared with the positive control group S.typhimuriumCompared with the EcN-pBbB8K-CsgA group (0.00192%), the EcN-pBbB8K-LT group (0.00155%) and the EcN-pBbB8K-CLT group (0.00006%) showed lower levels of invasiveness to cells, and the EcN group (0.00379%) also did not have an invasive effect on intestinal epithelial cells.

[0100] 3. Cell proliferation assay

[0101] After cell counting, seed 5000 cells / well into 96-well plates, adding 200 μL of complete culture medium to each well. Perform 6 replicates for each strain. Incubate in a cell culture incubator; cell adhesion takes approximately 4 hours after seeding. Centrifuge the engineered bacteria / Salmonella at 12000 rpm for 2 min, collect 10 μL of the supernatant, filter through a membrane, and seed into the corresponding wells. Incubate the plates for an appropriate time (e.g., 6, 12, 24, or 48 h). Add freshly prepared medium containing 10% CCK-8. Incubate for 0.5–4 h, measuring absorbance at 450 nm at 0.5, 1, 2, and 4 h. Select the optimal time point and plot a bar graph. Calculate cell viability = [(experimental wells) - (blank wells)] / [(control wells) - (blank wells)] × 100. Detection showed that cell viability reached its peak at 2 h. Figure 7 As shown in B, the cell proliferation rate of the engineered bacteria EcN-pBbB8K-CLT treatment group was 1.4375%, which was significantly higher than that of the control group (1.0375%).

[0102] 4. Detection of protein expression in cells by engineered bacteria

[0103] Caco-2 cells were divided into 2×10 5 Caco-2 cells were seeded at a concentration of [number] cells / mL in six-well plates, suspended in place with 2 mL of complete culture medium per well, and incubated in a cell culture incubator until the cells reached confluence. The complete culture medium was discarded, and the cells were washed three times with pre-warmed PBS. Caco-2 cells were then treated with engineered bacteria / Salmonella at a fold increase in infection (MOI) of 100:1, with 2 mL of incomplete culture medium (without penicillin-streptomycin) added to each well. After 3 h of incubation, cells were collected, and total protein was extracted. The total protein concentration was determined using the BCA method, and the expression of tight junction proteins and key proteins involved in NF-κB signaling pathway activation was detected using Western blot. Results are as follows: Figure 8-9 As shown, the engineered bacteria can enhance the expression levels of Occludin, Ccludin-1 and ZO-1 proteins in cells, indicating that they can improve the intestinal epithelial cell barrier function. At the same time, the engineered bacteria can reduce the phosphorylation level of p65 protein in cells, indicating that they can inhibit the activation of the NF-κB signaling pathway.

[0104] 5. Real-time quantitative PCR detection of cellular protein expression

[0105] (1) Induction of cellular protein expression and extraction of total RNA

[0106] After preparing the monolayer of cells in a six-well plate as described above, inoculate with engineered bacteria / Salmonella and co-culture at 37°C in 5% CO2 for 3 h. Discard the supernatant, wash three times with PBS, and collect 2×10⁶ cells / well plate. 6 Transfer cells to a 1.5 mL centrifuge tube. Following the nucleic acid extraction or purification instructions, add 100 μL of lysis buffer R1, vortex for 30 s, and incubate at room temperature for 1 min. Then add 600 μL of lysis buffer R2, invert thoroughly to mix, and incubate at room temperature for 5 min (do not centrifuge at this point). Aspirate the supernatant into a purification column fitted with a receiving tube, centrifuge at 12000 rpm for 30 s, discard the liquid in the receiving tube, add 600 μL of washing buffer to the purification column, centrifuge at 12000 rpm for 30 s, discard the liquid, repeat the washing once, centrifuge at 1000 rpm for 1 min, and transfer the purification column to a new centrifuge tube. Add 30 μL of elution buffer to the center, incubate at room temperature for 1 min, and centrifuge at 12000 rpm for 1 min to recover total RNA from the cells. Determine RNA concentration using NanoDrop, and perform 0.8% agarose gel electrophoresis on the product. Observe the gel under an imaging system to see if it contains intact RNA fragments.

[0107] (2) RT-PCR detection

[0108] 1) Reverse transcription of total cellular RNA

[0109] Based on the determined concentration, each sample was quantified to 1000 ng, and cDNA was synthesized using the EasyScript® One-Step gDNA Removal and cDNA Synthesis SuperMix kit. The reaction conditions were: 42℃ for 15 min, followed by inactivation at 85℃ for 5 s.

[0110] The results are as follows Figure 10 As shown, the RT-PCR results were similar to the Western blot results, and compared with the positive control... S.typhimurium Compared with the control group, EcN-pBbB8K-CsgA significantly increased the gene levels of Claudin-1, Occludin, and ZO-1 in Caco-2 cells, and downregulated the expression levels of p65 and IL-17.

[0111] Example 3: Study on the mechanism of probiotics promoting mucosal tissue healing

[0112] 1. Engineered Probiotic Immunization Program

[0113] Mice were randomly divided into the following nine groups: a. control group (PBS buffer), b. EcN-wt group, c. EcN-CsgA group, d. EcN-LT, e. EcN-CLT, fD-EcN, gD-EcN-CsgA, hD-EcN-LT, and iD-EcN-CLT. The ae group, with 12 mice per group, was randomly housed in two separate cages. A dirty cage sharing group (fi group), with 9 mice per group, was also established.

[0114] The immunization program was as follows: After 7 days of acclimatization, the AE group was immunized with probiotics orally according to Table 3-2. Mice were fasted for 2-4 hours before immunization and resumed normal feeding 1 hour after immunization. Immunization began on day 1 of the formal experiment, with each immunization lasting 3 consecutive days, spaced one week apart, for a total of 3 immunizations. After each immunization, the FI group received cages and excrement from the AE group. The bedding for all groups was changed every three days, and the mice's condition was observed daily, with supplemental feeding as needed.

[0115] 2. Detection of immune-related cell proliferation and differentiation

[0116] Mouse spleens (SP), mesenteric lymph nodes (MLNs), and Pell's lymph nodes (PPs) were aseptically harvested to prepare single-cell suspensions. CD11c-percp-Cy5.5, CD80-PE, and CD86-FITC antibodies were added and incubated at 4°C in the dark for 30 min to detect dendritic cell activation. CD45R / B220-APC and IgA-FITC antibodies were added and incubated at 4°C in the dark for 30 min to detect B lymphocyte proliferation. CD3-APC-Cy7, CD4-APC, CD25-PE, and FOXP3-FITC antibodies were added and incubated at 4°C in the dark for 30 min to detect Treg cell proliferation. Results are as follows: Figure 11 As shown, the PPs and CD11c in MLN of mice in the engineered strain EcN / pBbB8K-CsgA-LFCA-TFF3 group were compared. + The number of dendritic cells (DCs) was significantly higher than that of the control group. After oral immunization of mice with the engineered bacteria, dendritic cells in SP, MLN and PP were activated, which facilitated antigen presentation and accelerated the recognition and uptake of pathogens and their antigens.

[0117] like Figure 12-13 As shown, the engineered bacteria promoted the growth of CD45R in mouse PPs. + IgA + B lymphocyte proliferation and promoted the exchange of PPs and CD3 in the spleen. + CD4 + Treg cell proliferation was significantly higher than that of the control group.

[0118] 3. Detection of immunoglobulin and cytokine levels

[0119] The levels of sIgA in feces and IgG, TNF-α, IL-1β, and IL-10 in serum of immunized mice were detected using ELISA. Results are as follows: Figure 14-15 As shown, the engineered strain EcN / pBbB8K-CLT significantly increased the fecal sIgA content and serum IgG level in mice, while decreasing the serum TNF-α and IL-1β content.

[0120] Total RNA and serum RNA were extracted from mouse small intestinal tissue and reverse transcribed into cDNA. The expression levels of TNF-α, IL-1β, IL-10, ZO-1, Occludin, IL-22, IL-33, IL-17, and p65 genes were detected by RT-PCR. Results are as follows: Figure 16-18 As shown, the engineered bacteria significantly downregulated the expression levels of TNF-α, IL-1β, p65, and IL-17 genes, and upregulated the expression levels of IL-22, IL-33, IL-10, ZO-1, and Occludin genes.

[0121] 4. Detection of survival rate and weight change in challenged mice

[0122] The challenge protocol is as follows: Five days after the end of immunization, administer Salmonella Typhimurium. S. typhimurium ATCC25241 (2×10 7 Mice were infected by intraperitoneal injection of CFU / mouse. They were fed normally, and their health status was monitored after infection, with changes in body weight recorded. Results are as follows: Figure 19 As shown, the survival rate of mice in the control group (Ctrl) was the lowest at 50%, while the survival rates of mice in the EcN and EcN-pBbB8K-LT groups were both 83.3%, and the survival rates of mice in the EcN-pBbB8K-CsgA and EcN-pBbB8K-CLT groups were both 100%.

[0123] 5. Histopathological examination

[0124] The duodenum, jejunum, ileum, colon, rectum, and spleen of mice before and after viral challenge were collected and fixed with 4% paraformaldehyde for 24 hours. The sections were then placed in embedding casks for subsequent dehydration and clearing. Paraffin infiltration was performed at 58 ℃ for 30 min each with paraffin 1, paraffin 2, and paraffin 3. Immediately after paraffin infiltration, the sections were permeabilized and embedded using an embedding machine. The section thickness was 4 μm. The sections were spread in a 42 ℃ water bath and then placed in an 80 ℃ dry heat oven for 1 hour. After drying, the sections were subjected to xylene for 8 minutes and different concentrations of alcohol gradients for 1 minute each, followed by two washes with water. Hematoxylin staining was performed for 5 minutes, followed by two washes with water. Differentiation with 1% hydrochloric acid alcohol was performed for 5 seconds, followed by two washes with water. Eosin staining was performed for 2 minutes, followed by two washes with water. The sections were then mounted with neutral resin and examined under a microscope. Results are as follows: Figure 20-21 As shown, histopathological examination results revealed that the engineered bacteria reduced the degree of colonic crypt destruction and colonic area damage in mice, alleviated the inflammatory infiltration caused by Salmonella typhimurium, improved intestinal mucosal structure, and helped prevent or alleviate [the condition]. S. typhimurium The intestinal tissue pathological damage was caused. Observation of histopathological sections of mouse spleen tissue revealed significant hemorrhage in the spleens of the control group and D-EcN group mice. In the EcN group mice, the white and red pulp structures of the spleen were intact, with partial congestion of small blood vessels but no significant hemorrhage. Compared to the control group, EcN group, and D-EcN group, the spleen tissue of the engineered bacteria immunization group and the visceral cage group was healthy and showed no lesions.

[0125] After challenge with Salmonella typhimurium, 5 μm paraffin sections were obtained from the intestinal goblet cells of mice and dried. These sections were then stained using a periodic acid-Schiff (PAS) staining kit. After equilibration to room temperature 30 minutes prior to staining, the following steps were performed: a. Add 100 μL of periodic acid solution to each slide, incubate in a dark chamber for 10 min, then immerse in distilled water and place on a shaker for 5 min; b. Add 100 μL of Schiff reagent, incubate in a dark chamber at 37°C for 30 min, then wash with distilled water for 5 min; c. Add 100 μL of hematoxylin reagent to each slide, incubate at room temperature for 30 s, then wash three times with distilled water for 3 s each time, observing under a microscope until the excess stain is removed; d. Immerse in 90% ethanol and anhydrous ethanol for 2 minutes each. min. Immerse twice in xylene, 5 min each time; e. After the slide dries, add an anti-fluorescence quenching agent and mount the slide; f. Observe the staining of goblet cells under an inverted microscope and count the number of goblet cells on the three villi to create a table. The results are as follows: Figure 22 As shown, immunization increased the number of goblet cells in the colon and small intestine of mice in each group, enabling the intestinal tissue to play an important role in resisting internal and external invasions of the intestinal mucosa.

[0126] 7. Effects of engineered probiotics on the gut microbiota of mice

[0127] After the final immunization, fecal samples were collected from three mice in each group for 16S rRNA microbial diversity assay. Genomic DNA was extracted from the feces, and the V3-4 hypervariable region of the bacterial 16S rRNA gene was amplified by PCR. Libraries were constructed using the TruSeq® DNA PCR-Free Sample Preparation Kit. The constructed libraries were quantified by Qubit and Q-PCR. After the libraries were deemed acceptable, paired-end sequencing of the community DNA fragments was performed using the Illumina platform. Results are as follows: Figure 23 As shown, Bacteroidetes and Firmicutes dominate the gut microbiota of the engineered bacteria group mice. At the species level, the abundance of Heterobacter, Lactobacillus, and Bifidobacterium genera is relatively high, while the abundance of harmful bacteria (Desulfovibrio) and conditionally pathogenic bacteria (Pseudomonas aeruginosa) is reduced. This indicates that the engineered bacteria EcN-pBbB8K-CLT can effectively increase the abundance of probiotics and reduce the proportion of pathogenic bacteria in the mouse gut. By improving the richness and diversity of the gut microbiota, it enhances the host animal's anti-infection effect against ETEC K88.

[0128] For the analysis of short-chain fatty acids in metabolites, the sample was thawed on ice, and an appropriate amount was placed into a 2 mL centrifuge tube. 50 μL of 20% phosphate was added for resuspending, and 500 μM 4-methylvaleric acid was added as an internal standard. The mixture was shaken for 2 min. Then, it was centrifuged at 14000 g for 20 min, and the supernatant was transferred to a sample vial for GC-MS detection. The injection volume was 1 μL, the split ratio was 10:1, and a split injection method was used. The separation process used an Agilent DB-FFAP capillary column (30 m × 250 μm × 0.25 μm), and the gas chromatography system was coupled with a 5977B MSD mass spectrometer (Agilent). The peak area and retention time were extracted using MSD ChemStation software. Standard curves were plotted, and the content of short-chain fatty acids in the sample was calculated. The results are as follows: Figure 24 As shown, we found that engineered bacteria can increase the content of the short-chain fatty acid caproic acid, a metabolite of the gut microbiota. We speculate that the intervention of engineered bacteria modulates the abundance of short-chain fatty acid-producing metabolic bacteria, thereby increasing the production of caproic acid.

[0129] While the specific embodiments of the present invention have been described above in conjunction with the accompanying drawings, this is not intended to limit the scope of protection of the present invention. Those skilled in the art should understand that various modifications or variations that can be made by those skilled in the art without creative effort based on the technical solutions of the present invention are still within the scope of protection of the present invention.

Claims

1. A mucosa-repairing engineered probiotic, characterized in that, The mucosa-repairing engineered probiotic bacteria containing the CsgA-LFCA-TFF3 fusion gene, the sequence of the CsgA-LFCA-TFF3 fusion gene is shown as SEQ ID NO. 4, is prepared by the following steps: E. coli Nissle 1917 is the starting strain.

2. The mucosa-repairing engineered probiotic bacterium of claim 1, wherein, The nucleotide sequence of the CsgA is shown as SEQ ID NO. 1, the nucleotide sequence of the lactoferrin LFCA is shown as SEQ ID NO. 2, and the nucleotide sequence of the intestinal trefoil factor TFF3 is shown as SEQ ID NO.

3.

3. A pharmaceutical composition or inoculant or feed, characterized in that, The mucosal repair type of engineered probiotics according to any one of claims 1-2.

4. Pharmaceutical preparation, characterized in that, The mucosal repair type of engineered probiotics according to any one of claims 1-2.

5. Use of the mucosal repair type of engineered probiotics according to any one of claims 1-2 in the preparation of a pharmaceutical composition, a bacterial agent or a feed for resisting bacterial enteritis, the bacterial enteritis being bacterial enteritis caused by Salmonella typhimurium infection.

6. Use of the pharmaceutical preparation according to claim 4 in the preparation of a pharmaceutical composition, a bacterial agent or a feed for resisting bacterial enteritis, the bacterial enteritis being bacterial enteritis caused by Salmonella typhimurium infection.

7. Use of the mucosal repair type of engineered probiotics according to any one of claims 1-2 or the pharmaceutical composition or the bacterial agent or the feed according to claim 3 in any one of the following: 1) preparation of a Caco-2 intestinal epithelial cell repair agent and / or preparation of a Salmonella typhimurium K88 inhibitor; 2) preparation of a product for activating immune cells; 3) preparation of a product for maintaining the integrity of the intestinal barrier against pathogenic bacteria infection and improving intestinal inflammation; the pathogenic bacteria being Salmonella typhimurium.

8. Use of the pharmaceutical preparation according to claim 4 in any one of the following: 1) preparation of a Caco-2 intestinal epithelial cell repair agent and / or preparation of a Salmonella typhimurium K88 inhibitor; 2) preparation of a product for activating immune cells; 3) preparation of a product for maintaining the integrity of the intestinal barrier against pathogenic bacteria infection and improving intestinal inflammation; the pathogenic bacteria being Salmonella typhimurium.

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