Mucous membrane repair type engineering probiotics and application thereof

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to mucous membrane repair type engineering probiotics and application thereof. According to the constructed mucous membrane repair type engineering probiotic Escherichia coli Nissle 1917 expression vector EcN-pBbB8K-CsgA-LFCA-TFF3, lactoferrin peptide LFCA and a trefoil factor 3TFF3 are connected with EcN-CsgA, so that the repair capacity of the engineering bacterium on intestinal epithelial cells Caco-2 is enhanced, the proliferation and aggregation capacity of the intestinal epithelial cells can be promoted, and the mucous membrane repair type engineering probiotic Escherichia coli Nissle 1917 expression vector EcN-pBbB8K-CsgA-LFCA-TFF3 can be used for repairing the intestinal epithelial cells. And an important basis is provided for continuous colonization and cell interaction of engineering bacteria in a digestive tract environment.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and in particular relates to a mucosal repairing engineered probiotic and an application thereof. Background Art

[0002] Salmonella typhimurium ( Salmonella typhimurium ) is a foodborne pathogen and one of the main pathogens causing diarrhea-related intestinal diseases in many animals and humans. It can increase the number of bacterial colonization in the intestine and thus lead to colitis. The key link in its infection cycle is invasion of intestinal epithelial cells. Once entering the body, the bacteria use oxygen to grow rapidly in the intestine, and the proliferated bacteria are released into the intestinal cavity, exacerbating bacterial infection. Currently, antibiotics are one of the main treatments for bacterial diseases, but the emerging problem of bacterial resistance seriously threatens public health and safety. Therefore, there is an urgent need to find effective alternatives to antibiotics.

[0003] Probiotics have been used to enhance immunity and maintain the balance of the intestinal microbial ecosystem and are considered a powerful alternative therapy for preventing pathogen infections. Escherichia coli Nissle 1917, or E. coli (ECN), is the only non-pathogenic E. coli discovered so far and is a probiotic. Clinical results have shown that it can promote the growth of beneficial intestinal bacteria, inhibit the overgrowth of pathogens, enhance the body's antibacterial capacity, and reduce intestinal mucosal damage. Engineered probiotics based on E. coli are being used in the prevention and treatment of intestinal diseases.

[0004] However, oral probiotics are affected by the gastrointestinal environment and limit their survival and colonization in the intestine. After the disease occurs, intestinal epithelial cells are damaged, exposing the intestinal endothelium to potentially harmful substances, such as bacteria and their products. Rapid restoration of intestinal mucosal and epithelial integrity is particularly important for treating diseases. Complete regeneration depends on the slow proliferation and differentiation of stem cells. In contrast, recovery can occur within a few hours after injury and depends on the migration of epithelial cells from the surrounding area to the wound site. Therefore, constructing engineered bacteria that can repair intestinal mucosa and epithelium is crucial to improving the survival and colonization of probiotics in the intestine and realizing the clinical application of engineered microbial therapy.

[0005] Currently, in the research on the treatment of intestinal diseases, S. typhimurium There is no solution to the problems of slow healing of intestinal epithelial mucosa and untimely local mucosal immunity caused by infection through the preparation of engineered probiotics to repair and then fight bacterial intestinal diseases. Summary of the Invention

[0006] In order to overcome the deficiencies of the above-mentioned prior art, the present invention provides a mucosal repair engineered probiotic and its application, which contains a recombinant plasmid pBbB8K-CsgA-LFCA-TFF3. The engineered bacteria can quickly recruit surrounding cells, repair damaged epithelial tissue, and enhance the expression of tight junction proteins ZO-1, Claudin-1, and Occludin. At the same time, it can promote the maturation and differentiation of DCs, B and Treg cells, induce the secretion of immunoglobulins, inhibit the activation of the p65 NF-κB signaling pathway, increase the expression of anti-inflammatory factors, and reduce the expression of pro-inflammatory factors, thereby achieving the effect of regulating local mucosal immune response and maintaining bacterial flora homeostasis. The engineered bacteria constructed in this application solves the problem of S. typhimurium Problems such as slow epithelial mucosal healing and delayed local mucosal immunization caused by infection provide theoretical support for the development of vaccines for animal diarrheal diseases. The mucosal-repairing probiotics prepared by 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 object, the present invention provides the following technical solutions: The first aspect of the present invention provides a mucosal-repairing engineered probiotic containing a CsgA-LFCA-TFF3 fusion gene. The CsgA-LFCA-TFF3 fusion gene is formed by concatenating the CsgA gene, the lactoferrin LFCA gene, and the intestinal trefoil factor TFF3 gene of the EcN strain.

[0008] Amyloids are defined as aggregates of proteins that form starch-like fibrils, often forming protein components of the extracellular matrix in bacterial biofilms. Currently, one of the best-studied functional amyloid proteins is curlilin, an extracellular fibril produced by Escherichia coli. Curlilin fibrils are important for adhesion to inert surfaces, host cells, biofilm formation, and other community properties. Its primary subunit is CsgA. Curlilin fibril production is also crucial for initial adhesion to both biotic and abiotic surfaces and has been implicated in environmental resistance and pathogenesis. Trefoil factor 3 (TFF3) is a secreted protein. Mucin-associated trefoil factor (TFF) peptides are integral to gastrointestinal cytoprotection and mucosal repair. Structural analysis has revealed that the formation of a stable trefoil structure, linked to disulfide bonds, confers protease resistance. Antimicrobial peptides (AMPs) have emerged as a potential new strategy for preventing and treating bacterial infections. AMPs have emerged as a novel class of drug candidates for the treatment of infectious diseases. These short peptides, typically containing 10 to 500 amino acids, are important components of the innate immune system and serve as the first line of defense against infection. However, there is no report on the fusion expression of three genes in the prior art.

[0009] As an optional embodiment of the present invention, the nucleotide sequence of CsgA is shown as SEQ ID NO.1, the nucleotide sequence of lactoferrin LFCA is shown as SEQ ID NO.2, and the nucleotide sequence of intestinal trefoil factor TFF3 is shown as SEQ ID NO.3.

[0010] 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 decomposition.

[0011] As a preferred embodiment of the present invention, the fusion gene further contains an enzyme cleavage site, which is Bgl Ⅱ and Xho I site.

[0012] As an optional embodiment of the present invention, a linker sequence is added between genes CsgA and LFCA. The linker sequence is: GGTGGAGGAGGCTCTGGTGGAGGCGGTAGCGGAGGCGGAGGGTCG (SEQ ID NO. 5).

[0013] As a preferred embodiment of the present invention, a linker sequence was added between genes LFCA and TFF3 and optimized. The optimized sequence is: Linker C: GGCGGTGGCGGTGGCGGT (SEQ ID NO. 6).

[0014] Different linker sequences have a significant effect on the replication of recombinant vectors containing fusion genes in the host. The present invention confirms through experiments on linker sequence optimization that the linker combination with nucleotide sequences such as SEQ ID NO.5 and SEQ ID NO.6 has the best expression effect in host cells.

[0015] As an optional embodiment of the present invention, the probiotics are Escherichia coli Nissle1917 was used as the starting strain.

[0016] In a second aspect, the present invention provides a fusion gene comprising a CsgA gene, a lactoferrin LFCA gene, and an intestinal trefoil factor 3 gene connected in series.

[0017] As an optional embodiment of the present invention, the fusion gene is composed of CsgA-Linker-LFCA-Linker C-TFF3 in series.

[0018] As an optional embodiment of the present invention, the fusion gene sequence is shown as SEQ ID NO.4.

[0019] In a third aspect, 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 the fusion protein is shown below.

[0020] MKLLKVAAIAAIVFSGSALAGVVPQYGGGGNHGGGGNNSGPNSELNIYQYGGGNSALALQTDARNSDLTITQHGGGNGADVGQGSDDSSIDLTQRGFGNSATLDQWNGKNSEMTVKQFGGGNGAAVDQTASNSSVNVTQVGFGNNATAH QYGGGGSGGGGSGGGGSFKCRRWQWRMKKLGAPSITCVRRAFGGGSSVDGKEDLIWKLLSKAQEKFGKNKSRTATATAGGGGGGEEYVGLSANQCAVPAKDRVDCGYPHVTPKECNNNRGCCFDSRIPGVPWCFKPLQEAECTF(SEQID NO.7) The fourth aspect of the present invention provides an expression vector comprising the fusion gene described in the second aspect.

[0021] As an optional embodiment of the present invention, the expression vector is obtained by connecting the fusion gene and the vector pBbB8K, and the connection method is enzyme cleavage connection; the enzyme cleavage site is Bgl Ⅱ and Xho I.

[0022] As an optional embodiment of the present invention, the recombinant expression vector is obtained by effectively linking the above-mentioned polynucleotide molecule to an expression vector, and the expression vector is any one or more of a viral vector, a plasmid, a phage, a phagemid, a cosmid, an F cosmid, a phage or an artificial chromosome; the viral vector may include an adenoviral vector, a retroviral vector or an adeno-associated viral vector, and the artificial chromosome includes a bacterial artificial chromosome (BAC), a phage P1-derived vector (PAC), a yeast artificial chromosome (YAC) or a mammalian artificial chromosome (MAC).

[0023] The fifth aspect of the present invention provides a pharmaceutical composition, pharmaceutical preparation, bacterial agent or feed, which includes the mucosal repair engineered probiotics or its fermentation products or metabolites described in the first aspect or the fusion protein described in the third aspect.

[0024] The sixth aspect of the present invention provides the use of the adhesion-enhanced engineered probiotic 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 drugs, vaccines, bacterial agents or feeds for preventing and / or treating antibacterial enteritis; the antibacterial enteritis is Salmonella typhimurium infection.

[0025] The seventh aspect of the present invention provides the use of the mucosal repair engineered probiotic 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, bacterial agent or feed described in the fifth aspect in the preparation of any of the following products: 1) Preparation of intestinal epithelial cell Caco-2 repair agents and / or preparation of Salmonella typhimurium K88 inhibitors; 2) Preparation of products for activating immune cells; 3) Prepare products that maintain the integrity of the intestinal barrier to resist pathogen infection and improve intestinal inflammation.

[0026] One or more of the above technical solutions have the following beneficial effects: 1. The mucosal repair engineered probiotics constructed by the present invention Escherichia coli The Nissle 1917 expression vector EcN-pBbB8K-CsgA-LFCA-TFF3, by optimizing the linker sequence, connects EcN-CsgA, lactoferrin peptide LFCA and trefoil factor 3 TFF3, thereby enhancing the ability of the engineered bacteria to repair intestinal epithelial cells Caco-2 and promoting the proliferation and aggregation of intestinal epithelial cells, providing an important basis for the engineered bacteria to form sustained colonization and cellular interactions in the digestive tract environment.

[0027] 2. By detecting the expression of tight junction proteins in intestinal epithelial cells, the results showed that under the condition of pathogenic bacteria infection, the engineered bacteria EcN-pBbB8K-CsgA-LFCA-TFF3 can increase the expression levels of tight junction proteins Occludin and ZO-1, and reduce the phosphorylation level of p65 protein caused by ETEC K88 invasion of cells. Compared with the wild-type strain, it can inhibit the activation of the NF-κB signaling pathway.

[0028] 3. The engineered bacteria EcN-pBbB8K-CsgA-LFCA-TFF3 was used as a delivery vector and could be orally inoculated into animals. By utilizing its rapid repair effect on intestinal epithelial cells and competitive inhibition of pathogenic bacteria, it could 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. It could also enhance specific immune responses and anti-inflammatory responses by increasing sIgA and IgG levels and reducing TNF-α and IL-1β levels. The mucosal immune response induced by the engineered bacteria could protect mice from S. typhimurium Infection can improve intestinal tissue pathological damage by regulating the structure of the intestinal flora. This study found that engineered bacteria can increase the content of the short-chain fatty acid hexanoic acid in the intestinal microbial community. It is speculated that the intervention of engineered bacteria regulates the abundance of metabolic bacteria that produce short-chain fatty acids and increases the production of hexanoic acid.

[0029] 4. The engineered bacteria described in this application are used as chassis strains to express protective antigens, health or drug genes of animal or human pathogens to prepare new functional engineered probiotics. They are made into microecological oral preparations, making them a "microbial factory" that produces therapeutic active substances in humans or animals, avoiding the problems of traditional treatment regimens that rely on drug intervention, focus on regulating inflammatory pathways, and make epithelial mucosal repair and regeneration healing a slow process. As oral preparations, new engineered probiotics have multiple functions such as gene expression, continuous colonization and prebiotic effects. They are used in industrial fields such as food, feed, medicine, and health care, and have broad application prospects and significant social and economic benefits.

[0030] Advantages of additional aspects of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0032] Figure 1 This is a schematic diagram of the relative expression levels of the TFF3 gene tested with different connection sequences in Example 1 of the present invention.

[0033] Figure 2 This is the growth curve test result of the ECN-pBbB8K-CsgA-LFCA-TFF3 strain in Example 1 of the present invention.

[0034] Figure 3 This is the result of the EcN-pBbB8K-CsgA-LFCA-TFF3 Congo red quantitative binding assay.

[0035] Figure 4 The results of scanning electron microscopy detection of EcN-pBbB8K-CsgA-LFCA-TFF3.

[0036] Figure 5 This is the western blot detection result of the recombinant protein of the EcN-pBbB8K-CsgA-LFCA-TFF3 strain.

[0037] Figure 6These are the results of immunofluorescence detection of the EcN-pBbB8K-CsgA-LFCA-TFF3 strain.

[0038] Figure 7 These are the results of the detection of the invasion and proliferation ability of Caco-2 cells by EcN-pBbB8K-CsgA-LFCA-TFF3, where A is the effect of EcN-pBbB8K-CsgA-LFCA-TFF3 on the invasion ability of Caco-2 cells, and B is the effect of EcN-pBbB8K-CsgA-LFCA-TFF3 on the proliferation ability of Caco-2 cells.

[0039] Figure 8 Figure 3 shows the effect of EcN-pBbB8K-CsgA-LFCA-TFF3 on the expression of tight junction proteins in Caco-2 cells. A shows the expression of Occludin protein in Caco-2 cells detected by Western blot, B shows the expression of Claudin-1 protein in Caco-2 cells detected by Western blot, and C shows the expression of ZO-1 protein in Caco-2 cells detected by Western blot.

[0040] Figure 9 Figure 3 shows the effect of EcN-pBbB8K-CsgA-LFCA-TFF3 on the activation of NF-κB pathway in Caco-2 cells. A shows the expression of p65 protein in Caco-2 cells detected by Western blot. B shows the expression of p-p65 protein in Caco-2 cells detected by Western blot.

[0041] Figure 10 Figure 3 is the RT-PCR detection result of EcN-pBbB8K-CsgA-LFCA-TFF3 in Caco-2 cells. A is the RT-PCR detection result of ZO-1 gene expression in Caco-2 cells. B is the RT-PCR detection result of Claudin-1 gene expression in Caco-2 cells. C is the RT-PCR detection result of Occludin gene expression in Caco-2 cells. D is the RT-PCR detection result of p65 gene expression in Caco-2 cells. E is the RT-PCR detection result of IL-17 gene expression in Caco-2 cells.

[0042] Figure 11Figure 3 is the effect of EcN-pBbB8K-CsgA-LFCA-TFF3 on the activation of dendritic cells in SP, PPs and MLN, where A is the detection result of the number of dendritic cells in the spleen of each group of mice, B is the detection result of the number of dendritic cells in Peyer's patches of each group of mice, C is the detection result of the number of dendritic cells in the mesenteric lymph nodes of each group of mice, and A1 is the detection result of CD11c in the spleen of mice by flow cytometry + CD86 + B1 is the average fluorescence signal intensity of DCs cells, B2 is the flow cytometry detection of CD11c in mouse mesenteric lymph nodes + CD86 + The average fluorescence signal intensity of DCs cells, C1 is the flow cytometry detection of CD11c in Peyer's patches of mice + CD86 + A2 is the average fluorescence signal intensity of DCs cells, and A2 is the flow cytometry detection of CD11c in mouse spleen + CD80 + B2 is the average fluorescence signal intensity of DCs cells, B3 is the average fluorescence signal intensity of DCs cells in the mesenteric lymph nodes of mice, B4 is the average fluorescence signal intensity of DCs cells in the mesenteric lymph nodes of mice, + CD80 + The average fluorescence signal intensity of DCs cells, C2 is the CD11c in Peyer's patches of mice + CD80 + The mean fluorescence signal intensity of DCs cells.

[0043] Figure 12 Figure 2 is the effect of EcN-pBbB8K-CsgA-LFCA-TFF3 on the proliferation of B lymphocytes in SP and MLN, where A is B220 in the spleen of mice + IgA + The number of B cells, B is B220 in the mesenteric lymph nodes of mice + IgA + B cell count.

[0044] Figure 13 Figure 2 is the effect of EcN-pBbB8K-CsgA-LFCA-TFF3 on the proliferation of Treg cells in SP and MLN, where A is the CD4 + CD25 + FOXP3 + T cell count, B is the number of CD4 + CD25 + FOXP3 + T cell count.

[0045] Figure 14 These are the test results of sIgA in mouse feces and IgG in serum.

[0046] Figure 15 These are the results of ELISA test for IL-10, TNF-α, and IL-1β in mouse serum, where A is the expression level of IL-10 in the serum of each group of mice, B is the expression level of TNF-α in the serum of each group of mice, and C is the expression level of IL-1β in the serum of each group of mice.

[0047] Figure 16 These are the detection results of IL-10, TNF-α and IL-1β gene transcription levels in the colon tissues of mice, among which, A is the IL-10 gene expression level in the colon tissues of each group of mice detected by RT-PCR, B is the TNF-α gene expression level in the colon tissues of each group of mice detected by RT-PCR, and C is the IL-1β gene expression level in the colon tissues of each group of mice detected by RT-PCR.

[0048] Figure 17 Figure 3 is the effect of engineered bacteria on the transcription levels of genes related to the intestinal innate immune signaling pathway, among which, A is the RT-PCR detection of the IL-33 gene expression level in the colon tissue of each group of mice, B is the RT-PCR detection of the IL-22 gene expression level in the colon tissue of each group of mice, C is the RT-PCR detection of the IL-17 gene expression level in the colon tissue of each group of mice, and D is the RT-PCR detection of the p65 gene expression level in the colon tissue of each group of mice.

[0049] Figure 18 Figure 3 is the effect of engineered bacteria on the transcription level of intestinal tight junction protein-related genes, where A is the RT-PCR detection of ZO-1 gene expression level in the colon tissue of each group of mice, and B is the RT-PCR detection of Occludin gene expression level in the colon tissue of each group of mice.

[0050] Figure 19 The survival rate and body weight changes of mice.

[0051] Figure 20 The figures are the histopathological test results of the duodenum of mice in each group.

[0052] Figure 21 The spleen tissue pathological test results of mice in each group.

[0053] Figure 22 Figure 3 shows the detection results of the number of small intestinal goblet cells in each group of mice.

[0054] Figure 23 The taxonomic composition of intestinal microorganisms of each group of mice, among which A is the taxonomic composition analysis at the genus level, B is the species composition heat map at the genus level, C is the taxonomic composition analysis at the phylum level, and D is the species composition heat map at the phylum level.

[0055] Figure 24 This is the detection result of the metabolite Hexanoic acid content in mice. DETAILED DESCRIPTION

[0056] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0057] The present invention will now be further described with reference to specific examples. The following examples are intended only to illustrate the present invention and are not intended to limit its contents. Experimental conditions not specified in the examples are generally based on conventional conditions or those recommended by the reagent company. Reagents and consumables used in the following examples are commercially available unless otherwise specified.

[0058] The main materials and equipment used in the present invention are as follows: 1. Strains, plasmids, and cell lines Escherichia coli Nissle 1917 strain, S. typhimurium ATCC 25241 strain, Escherichia coli The MC1061 strain and Caco-2 cell line were purchased from BNCC (PMID: 29155272; DOI: 10.1016 / j.fsi.2017.10.043). The pBbB8K plasmid was purchased from Changchun Huijin Baili Biotechnology Co., Ltd.; the pEASY-Blunt vector was purchased from Beijing Quanshijin Biotechnology Co., Ltd.; and the lactoferrin peptide LFCA gene was synthesized by Sangon Biotechnology (Shanghai) Co., Ltd.

[0059] 2. Main Reagents Restriction enzymes Bgl Ⅱ. Xho I. Nco I and EcoRV and T4 DNA ligase were purchased from Quanshijin Biotechnology Co., Ltd.; a high-purity plasmid miniprep kit was purchased from Beijing Kangwei Century Biotechnology Co., Ltd.; a DNA gel recovery kit was purchased from Omega Bio-Tek, USA; 10×H buffer and DNA markers (DL5000 and DL10000) were purchased from Takara Biotechnology Co., Ltd.; fetal bovine serum, trypsin (containing EDTA), anti-fluorescence decay mounting medium, MEM cell culture medium, kanamycin, and chloramphenicol were purchased from Beijing Solebold Technology Co., Ltd.; developer, 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.

[0060] Example 1 Construction and verification of mucosal repair engineered probiotic EcN / pBbB8K-CsgA-LFCA-TFF3 1. Primer Design and Synthesis Obtained using NCBI database E. coli The reading frames and upstream and downstream gene sequence information of Nissle 1917-CsgA (GenBank: HAY2139435.1) and pUC57-TFF3 (GenBank: NC_000021.9) were obtained. The CsgA gene of the EcN strain was obtained by PCR cloning. The primer sequences are shown in Table 1.

[0061] Table 1 PCR primer sequences

[0062] 2. Construction and identification of the MC1061 / pBbB8K-CsgA-LFCA-TFF3 recombinant strain 2.1 Optimization of linker sequence 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 generate 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 (linkers) were designed to connect 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, and Linker C sequence: GGCGGTGGCGGTGGCGGT. The CsgA-LFCA-linker-TFF3 recombinant 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 confirmed to be GGCGGTGGCGGTGGCGGT by RT-PCR. Figure 1 shown.

[0063] 2.2 Construction and identification of recombinant vectors The curly fiber CsgA, lactoferrin peptide LFCA and TFF3 gene fragments were amplified by PCR respectively. The linker sequences obtained by screening in 2.1 were used to fuse the LFCA (SEQ ID NO.2) and TFF3 (SEQ ID NO.3) genes by SOE-PCR to obtain the fusion fragment LFCA-TFF3 (SEQ ID NO.9). The CsgA (SEQ ID NO.1), LFCA (SEQ ID NO.2) and TFF3 (SEQ ID NO.3) genes were fused by SOE-PCR to obtain the fusion fragment CsgA-LFCA-TFF3 (SEQ ID NO.4). Bgl Ⅱ and Xho I site primers were used to amplify CsgA, LFCA-TFF3, and CsgA-LFCA-TFF3 fusion genes to introduce Bgl Ⅱ and Xho I site, application Bgl Ⅱ and XhoThe pBbB8K plasmid and the amplified fragments were digested with enzyme I and purified and recovered. The amplified fragments were cloned into the pBbB8K plasmid using T4 ligase to obtain recombinant plasmids pBbB8K-CsgA, pBbB8K-LFCA-TFF3, and pBbB8K-CsgA-LFCA-TFF3, respectively. These recombinant plasmids were transformed into competent Escherichia coli MC1061 to screen for positive clones. 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.

[0064] 3. Construction and identification of engineered bacteria 3.1 Chemical transformation of recombinant plasmid E. coli Nissle 1917 competent state The recombinant vectors pBbB8K-CsgA, pBbB8K-LFCA-TFF3, and pBbB8K-CsgA-LFCA-TFF were transformed into E. coli In the Nissle 1917 competent cell, recombinant strains EcN-pBbB8K-CsgA, EcN-pBbB8K-LT (abbreviation of the recombinant strain EcN / pBbB8K-LFCA-TFF3, the same below), and EcN-pBbB8K-CLT (abbreviation of the recombinant strain EcN / pBbB8K-CsgA-LFCA-TFF3, the same below) were obtained by PCR, double enzyme digestion reaction, and DNA sequencing.

[0065] 3.2 Determination of the growth curve of engineered bacteria The engineered bacteria after gene recombinant were inoculated into 100 mL of fresh LB liquid medium at a 1% inoculum volume and cultured at 37°C with a shaker at 185 rpm. Using a spectrophotometer, take an appropriate amount of bacterial solution every hour to measure the OD value. 600 The absorbance at nm was continuously monitored for 12 hours and the strain growth curve was drawn. The results showed that the growth rate of the engineered bacteria was basically the same as that of the wild strain, with no significant difference, indicating that the expression of the target protein had no effect on the growth of the host bacteria ( Figure 2 ).

[0066] 3.3 Congo red quantitative binding analysis The engineered bacteria were activated by inoculating 1.5% of the culture medium into LB medium (containing 100 μg / mL Kan). The activated strains EcN-wt, EcN-pBbB8K-CsgA, EcN-pBbB8K-LT, and EcN-pBbB8K-CLT were inoculated at 1% of the culture medium into the corresponding LB medium (containing 100 μg / mL Kan). Protein expression was induced by the addition of 0.05% L-(+) arabinose to the EcN-pBbB8K-CsgA, EcN-pBbB8K-LT, and EcN-pBbB8K-CLT probiotics. The induced cultures were incubated overnight in a shaking incubator at 37°C to ensure sufficient protein expression. The next day, 1 mL of bacterial culture was taken, centrifuged at 8000 rpm for 10 min, and then suspended in 0.025 mM Congo red PBS solution, resuspended for 10 min, and centrifuged at 14000 rpm for 10 min. The supernatant was collected and plated in a 96-well plate with three replicates for each strain. The supernatant was measured at OD using a microplate reader. 490 The absorbance at nm was used to calculate the yield of the corrected curly fibers. The absorbance measured in 0.025 mM Congo red PBS solution was subtracted from the absorbance measured and the OD value of the original bacterial culture was used. 600 nm is calibrated. Figure 3 As shown, the results showed that under these conditions, EcN-pBbB8K-CsgA and the other two EcN-pBbB8K-CLT and EcN-pBbB8K-LT fusions were expressed and assembled into coiled fibers in significantly higher amounts than the control vector EcN-pBbB8K-GFP expressing GFP.

[0067] 3.4 Scanning electron microscopy The results of scanning electron microscopy of engineered bacteria are as follows Figure 4 As shown, scanning electron microscopy results confirmed that the EcN-pBbB8K-CsgA, EcN-pBbB8K-LT and EcN-pBbB8K-CLT engineered bacteria can assemble into nanofiber structures of natural curly fibers, among which EcN-pBbB8K-CLT can aggregate in large quantities under the action of curly fibers to form a dense "PATCH".

[0068] 3.5 Protein expression identification by Western blot 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. Each of the three strains was re-inoculated at a 1% ratio into the corresponding LB medium (containing 100 μg / mL Kan and 0.05% L-(+)arabinose) and cultured overnight at 37°C at 200 rpm. The supernatant was discarded by centrifugation, washed twice with sterile PBS, and resuspended for later use. 800 μL of the resuspended bacterial suspension was pipetted and mixed with 200 μL of 5× SDS PAGE buffer. The suspension was boiled for 10 minutes. After standing at room temperature, the suspension was centrifuged at 12,000 rpm for 1 minute. The supernatant was collected and subjected to protein electrophoresis.

[0069] The results are as follows Figure 5 As shown in the figure, the results showed that the target bands appeared at 30.2 kDa, 17 kDa, and 15.2 kDa, which were consistent with expectations, indicating that the recombinant plasmid can correctly express the fusion protein.

[0070] 3.6 Immunofluorescence detection The cultured bacterial suspension was centrifuged at 4000 rpm for 10 minutes, the supernatant discarded, and the cells were washed twice with sterile PBS buffer and resuspended in 4 mL of PBS. 10 μL of the bacterial suspension was evenly applied to a glass slide and allowed to dry near an alcohol lamp. Subsequently, 30 μL of anti-His primary antibody was added to the specimen, which was placed in a dark box and incubated at 37°C for 30 minutes. After rinsing in PBS for 10 minutes, the slide was removed and the surrounding liquid was blotted with absorbent paper, making sure not to dry the slide. 30 μL of fluorescent secondary antibody was added to the specimen, which was incubated at 37°C for 30 minutes in a dark box. The slide was then washed three times with running PBS for 5 minutes each time. A fluorescence quencher was added, and the specimen was observed under an inverted fluorescence microscope.

[0071] like Figure 6 As shown in the figure, by fluorescence microscopy, obvious green fluorescence signals were observed in EcN-pBbB8K-CsgA, EcN-pBbB8K-LT and EcN-pBbB8K-CLT, while no obvious fluorescence signal was observed in the empty vector bacteria EcN, further proving that the recombinant protein can be anchored and expressed correctly on the bacterial surface.

[0072] Example 2 Detection of the regulatory effect of adhesion-enhanced engineered probiotics on intestinal epithelial cell function 1. Trypan blue staining test The cells were plated at 1×10 5 The cells were seeded into 24-well plates at a density of 10 cells / well, and 1 mL of complete culture medium was added to each well. The plates were placed in a cell culture incubator and cultured until confluent. The cell density after the cells filled the 24-well plates was approximately 5×10 5The complete culture medium was discarded and the cells were washed three times with pre-warmed PBS. The engineered bacteria / Salmonella were inoculated at a multiplicity of infection (MOI) of 100:1 at a rate of 5 × 10 7 The cells were diluted to a suspension at 100 μL / mL. 200 μL of the diluted bacterial suspension and 800 μL of MEM (without the double-antibody) were inoculated into each well and co-cultured with Caco-2 cells for 2, 4, 6, and 8 hours. The culture medium was discarded and the cells were gently washed with pre-warmed PBS. The cells were trypsinized, and the single-cell suspension was diluted 10-fold and mixed with trypan blue dye in a 9:1 ratio. The suspension was then added to a red blood cell counting chamber, and the number of viable cells was immediately determined and the survival rate was calculated. The cells in the blank well served as a control. Three replicates were used for each group, and the experiment was repeated three times. S.typhimurium After treatment, Caco-2 cell proliferation in vitro was significantly inhibited at 8 hours, and this inhibitory effect gradually increased with time. In contrast, after 8 hours of treatment with the engineered bacteria EcN-pBbB8K-CsgA, EcN-pBbB8K-LT, and EcN-pBbB8K-CLT, the growth inhibition of Caco-2 cells was significantly reduced. This also demonstrates that the optimal interaction time between cells and bacteria is within 6 hours, without causing cell damage.

[0073] 2. Cell Invasion Assay Caco-2 cells at passage 5-15 were plated at 10 5 The cells were seeded at a density of 10 cells / mL in a 24-well plate and cultured in 500 μL of regular cell culture medium until 90% confluency was achieved. The bacterial culture was centrifuged to collect the pellet, washed with PBS, and diluted to a concentration of 5×10 cells / mL in MEM containing 1% fetal bovine serum. 7 / mL, before adding bacteria (200 μL), wash the Caco-2 cells twice with PBS to remove the antibiotics. The bacteria were incubated with Caco-2 cells for 2 h, and the culture medium was removed. The Caco-2 cells were then washed twice with 500 μL PBS, and then MEM (containing 1% fetal bovine serum) containing 100 μg / mL gentamicin was added. After 1 h of incubation, 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 the incubation, the mixture was pipetted and mixed. The mixture was serially diluted and dropped on a kanamycin plate to count the colony-forming units (CFU) of bacteria invading the Caco-2 cells. After lysing the cells, the plate was counted as shown in the figure. Figure 7 As shown in A, compared with the positive control group S.typhimuriumCompared with the EcN-pBbB8K-CsgA (0.00192%), EcN-pBbB8K-LT (0.00155%) and EcN-pBbB8K-CLT (0.00006%) groups, the cell invasion effects were at a lower level. Meanwhile, the EcN group (0.00379%) had no invasion effect on intestinal epithelial cells.

[0074] 3. Cell Proliferation Assay After cell counting, according to the appropriate number of cells plated, 5000 cells / well were inoculated into 96-well plates, and complete culture medium was added to each well to 200 μL. Six replicates were performed for each strain. Place in a cell culture incubator for culture, and it takes about 4 hours for the cells to adhere after inoculation. Centrifuge the engineered bacteria / Salmonella at 12000 rmp / 2min, take 10 μL of the supernatant, filter 0.22 μL through the membrane, and inoculate into the corresponding wells. Incubate the culture plate in the incubator for an appropriate period of time (for example, 6, 12, 24 or 48 hours). Add culture medium containing 10% CCK-8 (prepared immediately for use) in the form of liquid replacement. Culture for 0.5-4 hours, and measure the absorbance at 450 nm at 0.5, 1, 2 and 4 hours respectively. Select the most appropriate time point and draw a bar graph. Calculate cell viability = [(test well) - (blank well)] / [(control well) - (blank well)] × 100. It was found that the cell activity reached the best in the 2nd hour, such as 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%).

[0075] 4. Detection of protein expression in cells by engineered bacteria Caco-2 cells were plated at 2×10 5 The cells were inoculated into a six-well plate at a concentration of 100 / mL, suspended in 2 mL of complete medium per well, and cultured in a cell culture incubator until the cells were confluent. The complete medium was discarded, and the cells were washed three times with pre-warmed PBS. Caco-2 cells were treated with engineered bacteria / Salmonella at an infection multiplicity (MOI) of 100:1, and 2 mL of incomplete medium (without penicillin-streptomycin) was added to each well. The cells were cultured for 3 hours, the cells were collected and the total protein was extracted. The total protein concentration of the cells was determined by the BCA method, and the expression of tight junction proteins and key proteins involved in the activation of the NF-κB signaling pathway was detected by western blot. The results are shown in Figure 2. Figure 8-9 As shown in the results, the engineered bacteria can enhance the expression levels of Occludin, Ccludin-1 and ZO-1 proteins in cells, indicating that it can improve the barrier function of intestinal epithelial cells. At the same time, the engineered bacteria can reduce the phosphorylation level of cellular p65 protein, indicating that it can inhibit the activation of the NF-κB signaling pathway.

[0076] 5. Real-time fluorescence quantitative detection of cell protein expression (1) Induction of cell protein expression and extraction of total RNA After laying a monolayer of cells in a six-well plate according to the above method, the engineered bacteria / Salmonella were inoculated and co-cultured in a 5% CO2, 37℃ incubator for 3 h, the supernatant was discarded, and the cells were washed three times with PBS, and 2×10 6 Transfer 100 μL of lysis buffer R1 to a 1.5 mL centrifuge tube. Follow the instructions for nucleic acid extraction or purification. Add 100 μL of lysis buffer R1, vortex to mix for 30 s, and let stand at room temperature for 1 min. Then add 600 μL of lysis buffer R2, invert and mix thoroughly, and let stand at room temperature for 5 min. Do not centrifuge at this point. Pipette the supernatant into a purification column with a tube attached and centrifuge at 12,000 rpm for 30 s. Discard the tube. Add 600 μL of wash buffer to the purification column and centrifuge at 12,000 rpm for 30 s. Discard the liquid in the tube. Repeat the wash cycle. Centrifuge at 1000 rpm for 1 min. Transfer the purification column to a new centrifuge tube. Add 30 μL of elution buffer to the center of the tube. Let stand at room temperature for 1 min, and centrifuge at 12,000 rpm for 1 min to recover total cellular RNA. Determine RNA concentration using a NanoDrop and examine the product by electrophoresis on a 0.8% agarose gel. Place the gel under an imaging system to verify the integrity of the RNA fragments.

[0077] (2) RT-PCR detection 1) Reverse transcription of total cellular RNA Each sample was quantified to 1000 ng based on the measured concentration, and cDNA was synthesized using the EasyScript® One-Step gDNA Removal and cDNA Synthesis SuperMix Kit. The reaction conditions were: 42°C for 15 min and heat inactivation at 85°C for 5 s.

[0078] The results are as follows Figure 10 As shown, the RT-PCR results were similar to those of Western blot and compared with the positive control S.typhimurium Compared with the EcN-pBbB8K-CsgA group, the gene levels of Claudin-1, Occludin, and ZO-1 in Caco-2 cells were significantly increased, and the expression levels of p65 and IL-17 were downregulated.

[0079] Example 3 Study on the Mechanism of Engineered Probiotics in Promoting Mucosal Tissue Healing 1. Engineered Probiotic Immunization Program Mice were randomly divided into the following nine groups: a. control (PBS buffer), b. EcN-wt group, c. EcN-CsgA group, d. EcN-LT, e. EcN-CLT, fD-EcN, gD-EcN-CsgA, hD-EcN-LT, iD-EcN-CLT. Groups ae and e consisted of 12 mice each, randomly divided into two cages. A dirty cage-sharing group (fi group) consisted of 9 mice each.

[0080] The immunization protocol was as follows: After 7 days of acclimation, the AE group was orally inoculated with probiotics according to Table 3-2. The mice were fasted for 2-4 hours before immunization and returned to normal diet 1 hour after immunization. Immunizations were repeated for 3 consecutive days, with weekly intervals, for a total of 3 immunizations. The FI group received the cages and excreta of the AE group after each immunization. All groups had their bedding changed every 3 days, and the mice's condition was observed daily and their diet replenished as needed.

[0081] 2. Detection of Immune-Related Cell Proliferation and Differentiation Mouse spleens (SP), mesenteric lymph nodes (MLNs), and Peyer's patches (PPs) were sterilely collected and single-cell suspensions were prepared. CD11c-percp-Cy5.5, CD80-PE, and CD86-FITC antibodies were added and incubated at 4°C in the dark for 30 minutes to detect dendritic cell activation; CD45R / B220-APC and IgA-FITC antibodies were added and incubated at 4°C in the dark for 30 minutes 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 minutes to detect Treg cell proliferation. Figure 11 As shown, CD11c in PPs and MLN of mice in the engineered bacteria EcN / pBbB8K-CsgA-LFCA-TFF3 group + The number of DCs cells was significantly higher than that in the control group. After oral immunization of mice with the engineered bacteria, the activation of dendritic cells in SP, MLN and PPs was stimulated, which was beneficial for antigen presentation and accelerated the recognition and uptake of pathogenic bacteria and their antigens.

[0082] like Figure 12-13 As shown, the engineered bacteria promoted the expression of CD45R in mouse PPs. + IgA + B lymphocyte proliferation and promoted the expression of CD3 + CD4 + The proliferation of Treg cells was significantly higher than that of the control group.

[0083] 3. Immunoglobulin and cytokine level detection ELISA was used to detect the levels of sIgA in feces and IgG, TNF-α, IL-1β, and IL-10 in serum of immunized mice. Figure 14-15 As shown in the results, the engineered bacteria EcN / pBbB8K-CLT can significantly increase the fecal sIgA content and serum IgG level of mice, and reduce the serum TNF-α and IL-1β levels.

[0084] Total RNA from mouse small intestinal tissue and serum RNA was extracted and reverse transcribed into cDNA. RT-PCR was used to detect the expression levels of TNF-α, IL-1β, IL-10, ZO-1, Occludin, IL-22, IL-33, IL-17, and p65 genes. Figure 16-18 As shown in the figure, the engineered bacteria significantly downregulated the expression 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.

[0085] 4. Survival rate and weight change detection of challenged mice The challenge plan is as follows: 5 days after the end of immunization, use Salmonella typhimurium S. typhimurium ATCC25241 (2×10 7 CFU / mouse) were injected intraperitoneally into the infected mice, and the mice were fed normally and their health status after infection was monitored, and their weight changes were recorded. Figure 19 As shown, the survival rate of mice in the control group (Ctrl) was as low as 50%, the survival rates of EcN and EcN-pBbB8K-LT were both 83.3%, and the survival rates of mice in the EcN-pBbB8K-CsgA and EcN-pBbB8K-CLT groups were both 100%.

[0086] 5. Histopathological Examination The duodenum, jejunum, ileum, colorectum and spleen of mice before and after infection were collected and fixed with 4% paraformaldehyde for 24 hours. They were placed in embedding boxes for subsequent dehydration and transparent processing. They were waxed in a 58 ℃ incubator for 30 minutes each for wax 1, wax 2 and wax 3. After waxing, they were immediately paraffinized and embedded in an embedding machine. The slices were 4 μm thick and spread in a 42 ℃ water bath. The slices were placed in an 80 ℃ dry heat box for 1 hour. After drying, the slices were placed in xylene for 8 minutes and different concentrations of alcohol gradient dehydration for 1 minute each, washed with water twice; stained with hematoxylin for 5 minutes, washed with water twice; differentiated with 1% hydrochloric acid alcohol for 5 seconds, washed with water twice; stained with eosin for 2 minutes, washed with water twice; sealed with neutral gum and examined under a microscope. The results are as follows Figure 20-21As shown in the results of histopathological examination, the engineered bacteria reduced the degree of colon crypt destruction and colon area damage in mice, alleviated the inflammatory infiltration caused by Salmonella typhimurium, improved the intestinal mucosal structure, and helped prevent or alleviate S. typhimurium Pathological damage to intestinal tissue was caused. Pathological sections of mouse spleens revealed significant hemorrhage in the control and D-EcN groups, while the EcN group showed intact white and red pulp, with some congestion of small blood vessels and no significant hemorrhage. Compared to the control, EcN, and D-EcN groups, the spleens of the engineered bacteria-immunized and gut-cage groups were healthy and free of lesions.

[0087] Staining of intestinal goblet cells of mice after Salmonella typhimurium infection: 5 μm paraffin sections were cut, dried, and stained using a periodic acid-Schiff (PAS) staining kit. Thirty minutes in advance, the sections were taken out and equilibrated to room temperature, and then the following steps were followed: a. 100 μL of periodic acid solution was added to each slide, oxidized in a dark box for 10 min, soaked in distilled water and placed on a shaker for 5 min; b. 100 μL of Schiff reagent was added, incubated in a dark box at 37°C for 30 min; soaked in distilled water for 5 min; c. 100 μL of hematoxylin reagent was added to each slide, incubated at room temperature for 30 s, and washed with distilled water three times for 3 s each time. The sections were observed under a microscope until the floating color was washed away; d. The sections were soaked in 90% ethanol and anhydrous ethanol for 2 min. min, then soak in xylene twice for 5 min each time; e. After the slides are dry, add anti-fluorescence quenching agent to seal the slides; f. Observe the goblet cell staining under an inverted microscope and count the number of goblet cells on 3 villi to draw a table. Figure 22 As shown, after immunization, the number of goblet cells in the colon and small intestine of mice increased in each group, enabling the intestinal tissue to play an important role in defending the intestinal mucosa against internal and external invasions.

[0088] 7. Effects of engineered probiotics on the intestinal flora of mice After the last immunization, three mice were randomly selected from each group to collect feces for 16S rRNA bacterial diversity testing. Fecal genomic DNA was extracted and PCR amplified for the V3-4 hypervariable region of the bacterial 16S rRNA gene. 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 qualified, paired-end sequencing of the community DNA fragments was performed using the Illumina platform. The results are shown in Figure 2. Figure 23As shown in the results, Bacteroidetes and Firmicutes dominated the intestines of mice in the engineered bacteria group. At the species level, the richness of Allobacterium, Lactobacillus and Bifidobacterium was high, and the abundance of intestinal harmful bacteria (Desulfovibrio) and intestinal conditional pathogens (Pseudomonas aeruginosa) was reduced. This shows that the engineered bacteria EcN-pBbB8K-CLT can effectively increase the abundance of probiotics in the mouse intestine and reduce the proportion of pathogenic bacteria. By improving the richness and diversity of the intestinal flora, it enhances the host animal's anti-infection effect against ETEC K88.

[0089] Analysis of metabolite short-chain fatty acids: Thaw the sample on ice and take an appropriate amount and place it in a 2 mL centrifuge tube. Add 50 μL of 20% phosphoric acid to resuspend it, add 500 μM 4-methylvaleric acid as an internal standard, and shake and mix for 2 minutes. Then centrifuge at 14,000 g for 20 minutes, take the supernatant and place it in an injection bottle for GC-MS detection. The injection volume during detection 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 to a 5977B MSD mass spectrometer (Agilent) for mass spectrometry analysis. The chromatographic peak area and retention time were extracted using MSD ChemStation software. A calibration curve was drawn to calculate the content of short-chain fatty acids in the sample. The results are shown in Figure 2. Figure 24 As shown in the results, we found that engineered bacteria can increase the content of the short-chain fatty acid hexanoic acid metabolite in the intestinal microbiota. It is speculated that the intervention of engineered bacteria regulates the abundance of metabolic bacteria that produce short-chain fatty acids and increases the production of hexanoic acid.

[0090] Although the above describes the specific embodiments of the present invention in conjunction with the accompanying drawings, it 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 on the basis of the technical solution of the present invention without any creative work are still within the scope of protection of the present invention.

Claims

1. A mucosal repairing engineered probiotic, characterized in that: Contains a CsgA-LFCA-TFF3 fusion gene, wherein the CsgA-LFCA-TFF3 fusion gene is Ec The CsgA gene, lactoferrin LFCA gene and intestinal trefoil factor TFF3 gene of the N strain are formed in series. The mucosal repair engineered probiotics are E. coli Nissle 1917 was used as the starting strain.

2. The mucosal repair engineered probiotic according to claim 1, characterized in that 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.

3. The mucosal repair engineered probiotic according to claim 1 or 2, characterized in that: The CsgA-LFCA-TFF3 fusion gene sequence is 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.

4. A fusion gene, characterized in that: The fusion gene is formed by the tandem connection of the CsgA gene, the lactoferrin LFCA gene and the intestinal trefoil factor TFF3 gene, and the sequence of the fusion gene is shown in SEQ ID NO.

4.

5. A fusion protein, characterized in that Encoded by the fusion gene according to claim 3, the amino acid sequence of the fusion protein is shown in SEQ ID NO.

7.

6. An expression vector, characterized in that The expression vector comprises the fusion gene according to claim 3. Preferably, the expression vector is obtained by connecting the fusion gene CsgA-LFCA-TFF3 with the vector pBbB8K.

7. A pharmaceutical composition, pharmaceutical preparation, microbial agent or feed, characterized in that: The invention comprises the mucosal repair probiotics or fermentation products or metabolites thereof according to any one of claims 1 to 3, or the fusion protein according to claim 5.

8. Use of the mucosal-repairing probiotic according to any one of claims 1 to 3 or its fermentation product or metabolite, the fusion gene according to claim 4, the fusion protein according to claim 5, or the expression vector according to claim 6 in the preparation of an anti-bacterial enteritis pharmaceutical composition, pharmaceutical preparation, bacterial agent or feed.

9. The use according to claim 8, characterized in that The bacterial enteritis is caused by Salmonella typhimurium infection.

10. Use of the mucosal-repairing probiotic according to any one of claims 1 to 3, or its fermentation product or metabolite, or the fusion gene according to claim 4, or the fusion protein according to claim 5, or the expression vector according to claim 6, or the pharmaceutical composition, pharmaceutical preparation, bacterial agent, or feed according to claim 7 in any of the following: 1) Preparation of intestinal epithelial cell Caco-2 repair agents and / or preparation of Salmonella typhimurium K88 inhibitors; 2) Activating immune cells and / or preparing products that activate immune cells; 3) Prepare products that maintain the integrity of the intestinal barrier to resist pathogen infection and improve intestinal inflammation.

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