An adhesion-enhanced engineered probiotic and its application

Abstract

The present invention provides an adhesion-enhanced engineered probiotic and its application, wherein the bacterium contains a recombinant plasmid pBbB8K‑LFCA‑FaeG‑FaeC. The engineered probiotic has a strong adhesion to intestinal epithelial cells, and competitively inhibits the adhesion of pathogenic Escherichia coli K88 to intestinal epithelial cells. The engineered probiotic of the present invention is a non-antibiotic Escherichia coli, which is more environmentally friendly and avoids antibiotic tolerance for animals. Immunizing animals with it has a better immune effect, can activate dendritic cells in Peyer's patches and mesenteric lymph nodes, promote T cell and B cell immune responses, maintain the integrity of the intestinal barrier to resist pathogen infection and improve intestinal inflammation, improve the abundance of intestinal probiotics, and reduce the abundance of pathogenic bacteria. The adhesion-enhanced probiotic prepared by the present invention can significantly inhibit bacterial enteritis in mice, providing an effective treatment for inflammatory bowel disease and other gastrointestinal diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to adhesion-enhanced engineered probiotics and applications thereof. Background Art

[0002] The statements in this section merely provide background information related to the present invention and do not necessarily constitute prior art.

[0003] Currently, antibiotics are one of the main treatments for bacterial diseases. However, long-term use of antibiotics can easily lead to bacterial resistance, further disrupting the homeostasis of the intestinal environment. Probiotics can prevent and treat diseases such as ulcerative colitis, Crohn's disease, gastrointestinal tumors, and allergic diseases, and are considered potential alternatives to antibiotics. The introduced probiotics can colonize in the intestines for a short period of time, inhibiting pathogen invasion and infection, and actively regulating intestinal balance to play a probiotic role. Therefore, providing probiotics to the host animal microbiome is one of the most promising strategies for preventing and treating diseases.

[0004] However, oral probiotics are affected by the gastrointestinal environment and their survival and colonization in the intestine are limited, especially in the pathological microenvironment of inflammatory diseases, such as reactive oxygen species (ROS) and depleted mucus layer environments; in addition, probiotics need to maintain sufficient activity and quantity after entering the intestine to play a role in preventing bacterial intestinal diseases. However, gastric acid, bile salts and intestinal peristalsis in the digestive tract environment will also accelerate the clearance and death of probiotics. Different probiotic strains have different preventive effects on bacterial intestinal diseases, so a large number of strains need to be screened and evaluated to find the most suitable strains for subsequent research and development. There is a fierce competitive relationship between probiotics and pathogenic bacteria in the intestine. It is necessary to optimize the functions of probiotics such as their ability to adhere to intestinal mucus or cells and the production of antibacterial substances, so that they can competitively inhibit the colonization of pathogenic microorganisms by occupying the surface of intestinal mucosal tissue. Probiotics cannot effectively prevent or relieve intestinal inflammatory reactions by simply inhibiting the colonization of pathogenic bacteria. The local mucosal immune regulation ability is also one of the key considerations in the development of oral probiotic preparations. However, most of the screened probiotics cannot induce efficient and lasting immune responses after being ingested by animal hosts. At present, the probiotic administration strategy is still limited to continuous and high-dose methods, which leads to high production, transportation and storage costs, and easily causes animal stress reactions. These problems seriously hinder the colonization of highly active probiotics in the intestines and the production of good therapeutic effects and applications.

[0005] Escherichia coli Nissle 1917, referred to as EcN, is the only non-pathogenic Escherichia coli discovered so far and is a probiotic. Clinical results have shown that it can promote the growth of beneficial bacteria in the intestine, inhibit the excessive reproduction of pathogens, enhance the body's antibacterial ability and reduce intestinal mucosal damage; engineered probiotics built with EcN as the chassis are used in the field of intestinal disease prevention and control. However, it cannot colonize in the intestine for a long time to maintain a high concentration of therapeutic molecules, which greatly reduces the immunoregulatory ability of engineered bacteria. Therefore, screening molecules that can improve the adhesion ability of probiotics is crucial to improving the digestive tract colonization ability of engineered bacteria and realizing the clinical application of engineered microbial therapy.

[0006] Patent CN102031262A discloses that by transferring the main pili gene faeG of Escherichia coli into lactobacillus or lactococcus, a safe microorganism that can improve the immune effect is provided; however, a single pili gene often has the problem of weak colonization ability of engineered bacteria and poor anti-enteritis effect. Patent CN1793377A discloses that seven genes, faeC, faeD, faeE, faeF, faeG, faeH, and faeI, are expressed simultaneously in chloroplasts; however, strains expressing 7 genes simultaneously are prone to gene loss and thus lose their original functions. In addition, it is still unknown whether the faeG gene can be correctly expressed in other strains, such as Escherichia coli Nissle1917, whether it does not interfere with other exogenous genes, and whether it can synergize with each other to improve the beneficial effects of Escherichia coli Nissle 1917. Therefore, it is urgent to solve the problem of improving the colonization ability of probiotics in the intestine, improving the inhibitory effect of probiotics on intestinal pathogens, and then preparing drugs such as microbial agents. Summary of the invention

[0007] In order to overcome the deficiencies of the above-mentioned prior art, the present invention provides an adhesion-enhanced engineered probiotic and its application, which contains a recombinant plasmid pBbB8K-LFCA-FaeG-FaeC. The engineered probiotic has a strong adhesion to intestinal epithelial cells and competitively inhibits the adhesion of pathogenic Escherichia coli K88 to intestinal epithelial cells. The engineered probiotic of the present invention is a non-antibiotic Escherichia coli, which is more environmentally friendly and avoids antibiotic tolerance for animals. It has a better immune effect when used to immunize animals, can activate dendritic cells in Peyer's patches and mesenteric lymph nodes, promote T cell and B cell immune responses, maintain the integrity of the intestinal barrier to resist pathogen infection and improve intestinal inflammation, improve the abundance of intestinal probiotics, and reduce the abundance of pathogenic bacteria. The adhesion-enhanced probiotic prepared by the present invention can significantly inhibit bacterial enteritis in mice, providing an effective treatment for inflammatory bowel disease and other gastrointestinal diseases.

[0008] To achieve the above objectives, one or more embodiments of the present invention provide the following technical solutions:

[0009] In a first aspect, the present invention provides an adhesion-enhanced engineered probiotic, which contains a LFCA-FaeG-FaeC fusion gene, wherein the LFCA-FaeG-FaeC fusion gene is formed by a series connection of lactoferrin peptide LFCA, K88 fimbriae subunit FaeG and FaeC genes.

[0010] Literature reports that the gene cluster related to the biosynthesis of K88 pili may include 10 genes (faeA-J), among which the protein FaeG encoded by faeG is the main unit protein and is related to the adhesion characteristics of pili; the polypeptide encoded by faeC is a minor component of the pili structure. Other studies have shown that the interaction between the pili formed by the FaeG subunit on the surface of ETEC bacteria and the receptors on the surface of the host intestinal epithelial cells is a key process in the occurrence of diarrhea; FaeC induces specific immune responses in mice during the pathogenic process of ETEC and can activate the immune cells of mice.

[0011] Lactoferrin (LF) is an iron-chelating multifunctional glycoprotein of the iron chelator transferrin family with a molecular weight of about 80 kDa. It is mainly distributed in colostrum, milk and mucosal secretions. Studies in recent years have shown that lactoferrin can also improve intestinal health by regulating the balance of intestinal flora and reducing intestinal inflammation. It can also promote the development of the immune system and play an important role in regulating immune responses and promoting cell growth. LF is hydrolyzed by pepsin under acidic conditions into two active polypeptides, lactoferricin (Lfcin) and lactoferrampin (Lfampin). Lfcin has been proven to have antimicrobial and immunomodulatory effects. It can block cell apoptosis signal transmission, inhibit programmed cell death of immune cells, regulate T cell differentiation and maturation, regulate the production of cytokines, and enhance the body's immune defense ability. Lfampin has similar antibacterial and immune-regulating functions as Lfcin. However, it is still unknown whether it can be expressed in the same strain as the pili gene without affecting the growth of the host strain; whether it can promote strain colonization.

[0012] In a specific embodiment of the present invention, the genbank numbers of FaeG or FaeC are CP002730.1 and AF450247.2 respectively;

[0013] The nucleotide sequence of the LFCA is shown in SEQ ID NO.4.

[0014] SEQ ID NO.4:

[0015] TTTAAATGCCGTCGTTGGCAATGGCGCATGAAGAAGTTGGGCGCTCCGAGTATTACCTGCGTTCGCCGCGCTTTTGGCGGTGGCTCCAGTGTTGATGGCAAAGAAGATTTGATTTGGAAATTGTTGAGTAAAGCTCAAGAAAAATTTGGCAAAAATAAAAGTCGT.

[0016] In a specific embodiment, the LFCA-FaeG-FaeC fusion gene sequence is as shown in SEQ ID NO.3; or the LFCA-FaeG-FaeC fusion gene is another nucleotide sequence that can express the same amino acid sequence as SEQ ID NO.3 through codon incompatibility;

[0017] Preferably, the LFCA-FaeG-FaeC fusion gene also contains promoter, terminator and restriction site gene sequences;

[0018] Preferably, the restriction enzyme cutting sites are Bgl II and Xho I.

[0019] In a specific embodiment, a gene fragment of pBbB8K is also introduced.

[0020] In a specific embodiment, the probiotics are based on E. coli Nissle 1917 as the starting strain.

[0021] SEQ ID NO.3:

[0022]

[0023] In the research of the present invention, the inventors found that the fusion of LFCA, FaeG and FaeC genes had no effect on the growth of the host bacteria Escherichia coli Nissle 1917 compared with the use of FaeG or FaeG and FaeC genes alone, indicating that the expression of exogenous proteins did not affect the growth performance of the recombinant bacteria. The constructed engineered bacteria EcN / pBbB8K-LFCA-FaeG-FaeC (also known as: EcN(pBbB8K-LFF)) and EcN / pBbB8K-FaeG-FaeC (also known as: EcN(pBbB8K-FF)) increased the adhesion ability of Caco-2 cells by 60.2% and 41.7%, respectively, and the adhesion effect of EcN(pBbB8K-LFF) was significantly higher than that of other strains and EcN(pBbB8K-FF). In the cell adhesion competition test, it was found that both EcN / pBbB8K-LFCA-FaeG-FaeC and EcN / pBbB8K-FaeG-FaeC could effectively inhibit the adhesion of E. coli ETEC K88 to Caco-2 cells, and their cell adhesion rates decreased by 76.5% and 73.7%, respectively, which were significantly better than other strains. In addition, after ETEC K88 infected Caco-2 cells, the two engineered bacteria EcN / pBbB8K-LFCA-FaeG-FaeC and EcN / pBbB8K-FaeG-FaeC significantly increased the expression levels of cell tight junction proteins Occludin and ZO-1, which can inhibit the damage of pathogenic bacteria to epithelial cells by improving tight junctions. At the same time, the study confirmed that the two engineered bacteria can reduce the phosphorylation level of p65 protein caused by ETEC K88 infection, which helps to alleviate the inflammatory response caused by the activation of the NF-κB signaling pathway. Animal test results further confirmed that probiotics expressing LFCA, FaeG and FaeC genes can rapidly activate the proliferation of dendritic cells (DCs) in Peyer's patches (PPs) and mesenteric lymph nodes (MLNs) after oral inoculation of mice, and enhance their uptake and recognition of expressed antigen components. + T lymphocytes and IgA + B lymphocytes differentiate under the action of engineered bacteria, accompanied by an increase in sIgA in intestinal mucus and serum IgG levels. The engineered bacteria inhibit the expression of proinflammatory factors TNF-α and IL-1β through the TLR2-mediated NF-κB signaling pathway, improving intestinal inflammation. In addition, the two strains of engineered bacteria maintain the integrity of the intestinal barrier to resist pathogenic bacteria infection by increasing the expression of tight junction proteins ZO-1 and Occludin. The probiotics can improve the structure of the intestinal flora by increasing the abundance of intestinal probiotics and reducing the level of pathogenic bacteria, thereby maintaining the stability of the intestinal microenvironment and the biological barrier function.

[0024] In a second aspect, the present invention provides a fusion gene LFCA-FaeG-FaeC, which is formed by the lactoferrin peptide LFCA, K88 fimbriae subunit FaeG and FaeC genes connected in series;

[0025] Preferably, the LFCA-FaeG-FaeC fusion gene may also contain promoter, terminator and restriction site gene sequences;

[0026] Preferably, the restriction sites are Bgl II and Xho I;

[0027] Preferably, the fusion gene sequence is shown as SEQ ID NO.3.

[0028] In a third aspect, the present invention provides a fusion protein encoded by the fusion gene LFCA-FaeG-FaeC described in the second aspect, and the amino acid sequence of the fusion protein includes the sequence shown in SEQ ID NO.5.

[0029] SEQ ID NO.5:

[0030] MKKTAIAIAVALAGFATVAQAFKCRRWQWRMKKLGAPSITCVRRAFGGGSSVDGKEDLIWKLLSKAQEKFG

[0031] KNKSRGGGGSGGGGSGGGGSKKTLIALAIAASAASGMAHAWMTGDFNGSVDIGGTITADDYRQKWEWEVG

[0032] TGLNGFGNVLNDLTNGGTKLTITVTGNKPILLGRTKEAFATPVIGGVDGIPQIAFTDYEGASVELRKPDGGTNK

[0033] GLAYFVLPMKNAGGTKVGSVKVNASYAGVLGRGGVTSADGELLSLFADGLSSIFYGGLPRGSELSAGSAAA

[0034] ARTKLFGSLSRDDILGQIQRVNANITSLVDVAGSYRENMEYTDGTVVSAAYALGIANGQTIEATFNQAVTTSTQ

[0035] WSAPLNVAITYYTATATAKKAFLLACVFFLTGGGVSHAAVQKTIFSADVVASVCHVVVDADSTGNSGRLTFGT

[0036] YRKSTGASVPPRDFTVRLYESGATVQGCSAFLAGQVATLDFGNPGQLDAAGVVTRGAGDGIRVDVRAVDAQ

[0037] ADYRGRLTQDNHSVKYPVDFAAKGQFRFRAQPVFPADVKAGEYSGALTFVVTYQ

[0038] In a fourth aspect, the present invention provides a recombinant vector, wherein the expression vector comprises the fusion gene described in the second aspect;

[0039] Preferably, the expression vector is obtained by connecting the fusion gene LFCA-FaeG-FaeC with the vector pBbB8K; the connection method is restriction enzyme connection; and the restriction enzyme sites are Bgl II and Xho I.

[0040] Specifically, the recombinant expression vector is obtained by effectively connecting 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, a 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).

[0041] In a fifth aspect, the present invention provides a pharmaceutical composition or pharmaceutical preparation or bacterial agent or feed, which comprises the adhesion-enhanced engineered probiotic or its fermentation product or metabolite described in the first aspect or the fusion protein described in the third aspect.

[0042] In a sixth aspect, 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 inflammatory bowel disease; the inflammatory bowel disease is enterotoxigenic Escherichia coli infection.

[0043] In a seventh aspect, 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, 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 any of the following:

[0044] 1) enhancing the adhesion to intestinal epithelial cells Caco-2 or competitively inhibiting the adsorption of pathogenic Escherichia coli K88; and / or preparing intestinal epithelial cell Caco-2 adhesives or preparing Escherichia coli K88 inhibitors;

[0045] 2) Activating immune cells and / or preparing products that activate immune cells;

[0046] 3) Activating dendritic cells in Peyer's patches and mesenteric lymph nodes and / or preparing products for activating dendritic cells in Peyer's patches and mesenteric lymph nodes;

[0047] 4) Stimulating the production of specific cytokines, T cells, B cells in the spleen and / or mesenteric lymph nodes and / or preparing products that stimulate specific cytokines in the spleen, mesenteric lymph nodes and / or Peyer's patches; the specific cytokines include CD4 + 、B220 + IgA + , sIgA, IgG, TNF-α;

[0048] 5) Maintaining the integrity of the intestinal barrier to resist infection by pathogens and improve intestinal inflammation and / or preparing products that maintain the integrity of the intestinal barrier to resist infection by pathogens and improve intestinal inflammation.

[0049] One or more of the above technical solutions have the following beneficial effects:

[0050] pBbB8K-LFCA-FaeG-FaeC, which fuses the lactoferrin peptide LFCA, K88 fimbriae subunit FaeG and FaeC genes, increases the adhesion ability of the engineered bacteria to the intestinal epithelial cells Caco-2 by 1.6 times compared to the wild strain, and can competitively inhibit the adhesion of K88 pathogenic bacteria to intestinal epithelial cells, providing an important basis for the engineered bacteria to form sustained colonization and cell interactions in the digestive tract environment.

[0051] 2. By detecting the expression of tight junction proteins in intestinal epithelial cells, the results showed that under the condition of K88 pathogenic bacteria infection, the engineered bacteria EcN / pBbB8K-LFCA-FaeG-FaeC could restore and increase the expression levels of tight junction proteins Occludin and ZO-1 (1.8 times), and reduce the phosphorylation level of p65 protein caused by K88 invasion of cells (1.13 times). Compared with the wild strain, it can inhibit the activation of the NF-κB signaling pathway.

[0052] 3. The engineered bacteria EcN / pBbB8K-LFCA-FaeG-FaeC can be used as a delivery vector and can be orally inoculated into animals. By utilizing its high adhesion to intestinal epithelial cells and competitive inhibition of pathogenic bacteria, it can effectively enhance the interaction between engineered bacteria and immune-related cells, strengthen the activation of antigen-presenting cells (dendritic cells), and promote the proliferation of T and B lymphocytes. It can also enhance specific immune responses and anti-inflammatory responses by increasing sIgA and IgG levels and reducing TNF-α and IL-1β content. The mucosal immune response induced by engineered bacteria can protect mice against ETEC K88 infection and improve intestinal tissue pathological damage by regulating the structure of intestinal flora. Using engineered bacteria as a chassis strain, protective antigens, health or drug genes of animal or human pathogens can be expressed to prepare new functional engineered probiotics. It can be made into a microecological oral preparation, making it a "microbial factory" that produces therapeutic active substances in humans or animals, avoiding the problems of general engineered bacteria colonization cycle and low survival number. As oral preparations, new engineered probiotics have multiple functions such as gene expression, sustained 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.

[0053] Advantages of additional aspects of the present invention will be given in part in the following description, and in part will become obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] The accompanying drawings in the specification, 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.

[0055] Figure 1 Schematic diagram of the synthesis of the target gene of the present invention; (A) BOPA gene model diagram; (B) FaeG-FaeC (FF) fusion gene model diagram; (C) LFCA-FaeG-FaeC (LFF) fusion gene model diagram.

[0056] Figure 2 The plasmid maps of (A) pPG-BOPA, (B) pBbB8K-FF and (C) pBbB8K-LFF in Example 1 of the present invention are shown.

[0057] Figure 3 This is the PCR verification of the recombinant plasmids pBbB8K-FF and pBbB8K-LFF of Example 1 of the present invention; M: DL5,000 DNA Marker; 1: FF; 2: LFF.

[0058] Figure 4This is the plasmid PCR verification of pPG-BOPA, pBbB8K-FF and pBbB8K-LFF in the new Nissle 1917 of Example 1 of the present invention. M: Trans2K Plus and DL5,000DNA Marker; 1: pPG-BOPA; 2: pBbB8K-FF; 3: pBbB8K-LFF.

[0059] Figure 5 This is the growth curve detection result of the EcN / pBbB8K-LFCA-FaeG-FaeC strain in Example 1 of the present invention.

[0060] Figure 6 This is the western blot detection result of the recombinant protein of the EcN / pBbB8K-LFCA-FaeG-FaeC strain in Example 1 of the present invention; M: pre-stained protein Marker; 3: EcN (pBbB8K-LFF); 4: Control.

[0061] Figure 7 This is the immunofluorescence detection result of the EcN / pBbB8K-LFCA-FaeG-FaeC strain in Example 1 of the present invention.

[0062] Figure 8 These are the results of the adhesion and competitive ability test of EcN / pBbB8K-LFCA-FaeG-FaeC on Caco-2 cells in Example 2 of the present invention. The left side shows the adhesion ability test result, and the right side shows the competitive ability test result.

[0063] Fig. 9 This is Example 2 of the present invention, the effect of EcN / pBbB8K-LFCA-FaeG-FaeC on the expression of tight junction proteins in Caco-2 cells infected with K88; (A) The effect of EcN and ETEC K88 strains on the expression level of Occludin in Caco-2 cells; (B) The effect of EcN and ETEC K88 strains on the expression level of ZO-1 in Caco-2 cells; (C) The effect of EcN / pBbB8K-FF and ETEC K88 strains on the expression level of Occludin in Caco-2 cells; (D) The effect of EcN / pBbB8K-FF and ETEC K88 strains on the expression level of ZO-1 in Caco-2 cells; (E) The effect of EcN / pBbB8K-LFF and ETEC K88 strains on the expression level of Occludin in Caco-2 cells; (F) Effect of K88 strain on the expression level of ZO-1 in Caco-2 cells; Note: (left) Western blotting results, (right) gray value of protein bands.

[0064] Fig.10This is the effect of EcN / pBbB8K-LFCA-FaeG-FaeC on the activation of NF-κB pathway in Caco-2 cells stimulated by K88 in Example 2 of the present invention; (A) Effect of EcN on p65 protein expression and its phosphorylation level after ETEC K88 infection of Caco-2 cells; (B) Effect of EcN / pBbB8K-FF on p65 protein expression and its phosphorylation level after ETEC K88 infection of Caco-2 cells; (C) Effect of EcN / pBbB8K-LFF on p65 protein expression and its phosphorylation level after ETEC K88 infection of Caco-2 cells; Note: (left) Western blotting detection results, (right) protein band gray value.

[0065] Fig.11 This is the effect of EcN / pBbB8K-LFCA-FaeG-FaeC on the activation of dendritic cells in PPs and MLN according to Example 3 of the present invention, A: PPs CD11c; B: MLN CD11c.

[0066] Fig.12 This is Example 3 of the present invention, the effect of EcN / pBbB8K-LFCA-FaeG-FaeC on the proliferation of B lymphocytes in PPs.

[0067] Fig.13 This is Example 3 of the present invention, the effect of EcN / pBbB8K-LFCA-FaeG-FaeC on the proliferation of T lymphocytes in PPs.

[0068] Fig.14 This is Example 3 of the present invention, the effect of EcN / pBbB8K-LFCA-FaeG-FaeC on the proliferation of T lymphocytes in the spleen.

[0069] Fig.15 This is the test result of mouse feces sIgA and serum IgG content in Example 3 of the present invention.

[0070] Fig.16 This is the detection result of TNF-α, IL-1β and IL-10 gene transcription levels in mouse small intestinal tissue in Example 3 of the present invention.

[0071] Fig.17 This is the ELISA test result of mouse serum TNF-α, IL-1β and IL-10 in Example 3 of the present invention.

[0072] Fig.18 This is the effect of the engineered bacteria in Example 3 of the present invention on the transcription level of genes related to the intestinal innate immune signaling pathway.

[0073] Fig.19 This is the effect of the engineered bacteria in Example 3 of the present invention on the transcription level of genes related to intestinal tight junction proteins.

[0074] Fig. 20 This is the survival rate of mice after ETEC K88 challenge in Example 3 of the present invention.

[0075] Fig.21 This is the change in body weight of mice after ETEC K88 challenge in Example 3 of the present invention.

[0076] Fig. 22 These are the results of colon tissue pathology testing of each group of mice in Example 3 of the present invention.

[0077] Fig.23 The taxonomic composition of the intestinal microorganisms of each group of mice in Example 3 of the present invention. (A) ASV / OTU table leveling, sequence abundance of ASV or OTU in samples before or after leveling; (B) Taxonomic composition analysis, visualization of composition distribution at six taxonomic levels: phylum, class, order, family, genus, and species; (C) Species composition heat map; (D) Hierarchical clustering analysis, stacked bar chart of the top 10 genera in abundance; (E) Random forest analysis, absolute abundance table of the top 20 species in importance.

[0078] Fig.24 This is the Alpha diversity analysis of intestinal microorganisms of each group of mice in Example 3 of the present invention.

[0079] Fig.25 This is the principal coordinate analysis of the intestinal microbial structure of each group of mice in Example 3 of the present invention.

[0080] Fig.26 It is a taxonomic cladogram (Cladogram) of each group of species in Example 3 of the present invention and a distribution bar graph of LDA values ​​of significantly different species. DETAILED DESCRIPTION

[0081] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0082] The present invention is further described in conjunction with specific examples. The following examples are only for explaining the present invention and do not limit its content. If the specific experimental conditions are not specified in the examples, they are usually carried out according to conventional conditions or the conditions recommended by the reagent company; the reagents and consumables used in the following examples can be obtained from commercial channels unless otherwise specified.

[0083] The main materials and equipment used in the present invention are as follows:

[0084] 1. Strains, plasmids and cell lines

[0085] Table 1 Strains and plasmids

[0086]

[0087] 2. Main Reagents

[0088] Restriction endonucleases Bgl II, Xho I, Nco I and EcoR V, and T4 DNA ligase were purchased from Quanshijin Biotechnology Co., Ltd.; high-purity plasmid mini-extraction kit was purchased from Beijing Kangwei Century Biotechnology Co., Ltd.; DNA gel recovery kit was purchased from Omega Bio-Tek, USA; 10×H buffer and DNA Marker (DL5000, DL10000) were purchased from Takara Biotechnology Co., Ltd.; fetal bovine serum, trypsin (containing EDTA), anti-fluorescence attenuation mounting medium, MEM cell culture, kanamycin and chloramphenicol were purchased from Beijing Solebow Technology Co., Ltd.; developer, DMSO, and BCA protein concentration determination kit were purchased from Biyuntian Biotechnology Co., Ltd.; HRP-labeled rabbit anti-His-Tag IgG was purchased from Bioss; Rat Anti-Mouse CD11c, Rat Anti-MouseCD80, Rat Anti-Mouse CD86, Rat Anti-Mouse CD45R / B220, Rat Anti-Mouse CD3, RatAnti-Mouse IgA, Rat Anti-Mouse CD4 and Rat Anti-Mouse CD8 antibodies were purchased from BD Biosciences.

[0089] 3. Test equipment

[0090] Table 2 Instruments and manufacturers

[0091]

[0092]

[0093] In the present invention, overlap extension PCR technology is used to integrate multiple gene fragments into a large gene fragment, and 6*His and restriction sites are connected to the integrated gene fragment. The schematic diagram of the target gene fragments BOPA, FF, and LFF is shown in FIG. Figure 1 As shown, the nucleotide sequences are shown in SEQ ID NO.1-3 respectively.

[0094] SEQ ID NO.1 BOPA (Bifidobacterium adhesion protein) gene nucleotide sequence: 1782 bp

[0095]

[0096] SEQ ID NO.2FF (FaeG-FaeC fusion gene) gene nucleotide sequence: 1485 bp

[0097]

[0098] Example 1 Construction and verification of adhesion-enhanced engineered probiotic EcN / pBbB8K-LFCA-FaeG-FaeC

[0099] 1. Primer Design and Synthesis

[0100] The FaeG (GenBank: CP002730.1) and FaeC (GenBank: AF450247.2) gene sequences of ETEC K88 strain were obtained from the NCBI database, and the lactoferrin peptide LFCA (nucleotide sequence is shown in SEQ ID NO.4) was genetically synthesized. The three gene amplification primers were designed using Primer 6.0 software. The primer sequences are shown in Table 3.

[0101] Table 3 PCR primer sequences

[0102]

[0103] 2. Construction and identification of MC1061 / pEASY-LFCA-FaeG-FaeC recombinant strain

[0104] 2.1 Preparation of competent E. coli MC1061

[0105] (1) In a clean bench, streak MC1061 on the surface of LB solid medium using an inoculation loop and incubate at 37°C for 16 h;

[0106] (2) In a clean bench, inoculate MC1061, pick a single colony and place it in 5 mL of LB medium, and shake at 37°C overnight (about 16 hours);

[0107] (3) The next day, in a clean bench, add 1.5% of the culture medium to 100 mL of LB and culture at 37°C in a shaking incubator until the OD 600 = about 0.5 (2.5-3h);

[0108] (4) In a clean bench, transfer the cultured bacterial solution into a sterile 50 mL centrifuge tube, place on ice for 10 min, and centrifuge at 5000 rpm at 4°C for 10 min. Discard the supernatant in a clean bench.

[0109] (5) In a clean bench, gently suspend the bacteria with 10 mL of cold washing buffer, place on ice for 10 min, centrifuge at 5000 rpm at 4°C for 10 min, and discard the supernatant in a clean bench;

[0110] (6) In a clean bench, gently suspend the bacteria with 10 mL of cold washing buffer, place on ice for 20 min, centrifuge at 5000 rpm for 10 min at 4°C, and discard the supernatant in a clean bench;

[0111] (7) In a clean bench, gently resuspend the bacteria in pre-cooled storage buffer, place on ice, and cool for 30 min. In a clean bench, dispense the competent cells into pre-cooled 1.5 mL EP tubes at 50 μL / tube and store at -80°C.

[0112] Table 4 Washing buffer recipe

[0113]

[0114] The pH was adjusted to 6.4, and the solution was filtered through a disposable Nalgene filter (0.45 μm pore size) and stored at 4°C after aliquoting.

[0115] Preparation of storage buffer: Add glycerol to the washing buffer to make the final concentration 25% (V / V), use a disposable Nalgene filter (0.45 μm pore size), and store at 4°C after aliquoting.

[0116] 2.2 Construction of MC1061 / pEASY-LFCA-FaeG-FaeC recombinant strain

[0117] Lactoferrin peptide LFCA, K88 fimbriae subunits FaeG and FaeC were amplified by PCR, and the LFCA (165 bp), FaeG (873 bp) and FaeC (546 bp) genes were fused by SOE-PCR. The fusion fragment was cloned into the pEASY-Blunt vector, which was transformed into the competent Escherichia coli MC1061 to screen positive clones. The recombinant strain MC1061 / pEASY-LFCA-FaeG-FaeC was obtained by PCR, double enzyme digestion reaction and DNA sequencing. The schematic diagram of the fusion gene fragment LFCA-FaeG-FaeC is shown in Figure 1 shown.

[0118] 3. Construction and identification of the engineered strain EcN / pBbB8K-LFCA-FaeG-FaeC

[0119] 3.1 Preparation of competent E. coli Nissle 1917

[0120] (1) Streak E. coli Nissle 1917 on the surface of LB agar using an inoculating loop and incubate at 37°C for 16 h;

[0121] (2) Pick a single colony and place it in 5 mL of LB broth at 37°C, shaking at 200 rpm overnight (about 16 h);

[0122] (3) Inoculate 100 mL of LB broth at a ratio of 1.5% and culture at 37°C in a shaking incubator until OD 600 = about 0.5 (2.5-3h);

[0123] (4) When OD 600 = 0.5. Ice bath for at least 15 minutes (if necessary, the culture solution can be stored at 4°C for several hours);

[0124] (5) Centrifuge at 5000 rpm for 10 min at 4°C to collect the cells;

[0125] (6) Add 50 mL of sterile deionized water (precooled to 4°C), resuspend the cells, centrifuge at 5000 rpm for 10 min at 4°C, discard the supernatant, and repeat once;

[0126] (7) Resuspend the cells in 50 mL of sterile 10% glycerol (precooled to 4°C), centrifuge at 5000 rpm for 10 min at 4°C, discard the supernatant, and repeat once;

[0127] (8) Resuspend the cells in 10% glycerol (precooled to 4°C) to a final volume of 2 mL.

[0128] (9) Aliquot the competent cells into pre-cooled 1.5 mL EP tubes at 100 μL / tube and store at -80°C.

[0129] 3.2 Construction of pBbB8K-LFCA-FaeG-FaeC recombinant plasmid

[0130] The LFCA-FaeG-FaeC fusion gene was amplified by PCR and introduced into the Bgl II and Xho I sites. The pBbB8K plasmid was digested with Bgl II and Xho I and purified and recovered. The LFCA-FaeG-FaeC fusion gene was cloned into the pBbB8K plasmid under the action of T4 ligase. The resulting plasmid was named: pBbB8K-LFF or pBbB8K-LFCA-FaeG-FaeC. The plasmid map Figure 2 As shown on the right, the PCR identification results are as follows Figure 3 .

[0131] In the present invention, the above method is also used to fuse FaeG (873 bp) and FaeC (546 bp) genes. The schematic diagram of the fusion gene FF is as follows: Figure 1 The sequence is shown in SEQ ID NO. 2, and the vector is constructed and constructed into the pBbB8K plasmid to obtain the vector pBbB8K-FF. The plasmid map of pBbB8K-FF is shown in Figure 2 As shown in the figure, the PCR identification results are as follows Figure 3 .

[0132] In the present invention, the BOPA gene (Bifidobacterium bifidum S17-BOPA, GenBank: CP002220.1) is fused to the gene Figure 1The sequence is shown in SEQ ID NO.1, and is constructed into plasmid pPG612 through restriction sites Nco I and EcoR V to obtain vector pPG-BOPA. The plasmid map is shown in Figure 2 Shown on the left.

[0133] pPG612 plasmid sequence (4233 bp):

[0134]

[0135] 3.3 Electroporation of recombinant plasmid into competent E. coli Nissle 1917

[0136] (1) Thaw competent cells on ice, add 10 μL of plasmid, mix gently, and incubate on ice for 5 min;

[0137] (2) Precool the cuvette to 4°C, transfer the plasmid / competent cell mixture to the cuvette (try to avoid creating bubbles), and place on ice for 10 min.

[0138] (3) Electric shock conditions: 2 kV, 200 Ω, 25 μF;

[0139] (4) After the electroporation, quickly add 900 μL of preheated antibiotic-free LB broth and culture at 37°C, 200 rpm for 60 min.

[0140] (5) Take 100 μL of bacterial solution and spread it evenly on LB resistance agar. Single colonies will be visible after 12-16 hours.

[0141] (6) Pick a single colony and place it in 10 mL of LB broth. Culture it at 37°C to preserve the bacteria and extract the plasmid for subsequent experiments.

[0142] The recombinant plasmids pPG-BOPA, pBbB8K-FF, and pBbB8K-LFF were transformed into E. coli Nissle 1917 competent cells to obtain recombinant strains. The target gene of the recombinant plasmid in E. coli Nissle 1917 was amplified by PCR to prove that the recombinant expression plasmid was successfully transferred into E. coli Nissle 1917. The plasmid PCR results are as follows: Figure 4 As shown, target bands were visible at 1824 bp, 1485 bp, and 1695 bp, indicating that the plasmid transformation was successful.

[0143] 3.4 Determination of growth curve of engineered bacteria

[0144] Inoculate 100 μL of engineered bacteria into 10 mL of LB broth and incubate at 37°C, 200 rpm overnight. The next day, inoculate 1% of the inoculum into 100 mL of LB liquid culture medium and incubate at 37°C, 200 rpm. Take an appropriate amount of bacterial solution every hour to measure the OD 600 The absorbance at nm was continuously monitored for 12 h and the growth curve of the strain was plotted. Figure 5 As shown, there is no significant difference in the growth rate of the engineered bacteria and the wild strain, indicating that the expression of the target protein has no effect on the growth of the host bacteria.

[0145] 3.5 Expression and identification of recombinant proteins in engineered bacteria

[0146] The overnight cultured engineered bacteria precipitate was collected by centrifugation, washed twice with sterile PBS, 400 μL of resuspended bacteria solution was taken, mixed evenly with 100 μL 5× SDS PAGE Buffer, and boiled for 10 min. Centrifuged at 12000 rpm for 1 min, the supernatant was collected, and the expression of recombinant protein was detected by Western blot. After observation by the imaging instrument, a 59 kDa recombinant protein was successfully obtained, which was consistent with the expected size, such as Figure 6 shown.

[0147] The bacterial suspension was evenly spread on a glass slide and dried naturally. Mouse His Taq primary antibody was used for incubation at 37°C for 30 min. After washing with PBS buffer, FITC-labeled secondary antibody was used for incubation at 37°C for 30 min. The fluorescence signal on the bacterial surface was observed under a fluorescence microscope. The recombinant protein was anchored and expressed on the bacterial surface. Figure 7 As shown. The immunofluorescence results show that the fusion protein can be secreted and expressed in the form of mature peptide chains with the help of signal peptide sequences. Example 2 Detection of the regulatory effect of adhesion-enhanced engineered probiotics on intestinal epithelial cell function

[0148] 1. Cell Adhesion Ability Detection

[0149] The wild strain and the engineered strain were inoculated into LB medium, and the bacterial solution at the stationary stage was centrifuged at 5000 rpm for 10 min at 4°C to collect the bacteria, which were washed three times with pre-cooled PBS and resuspended in MEM complete medium. The bacterial solution was inoculated into a monolayer of Caco-2 cells (2.5×10 7 bacteria and 2.5×10 5 The cells were cultured in a 37°C, 5% CO2 incubator. After 1 hour, the complete medium was discarded and the cells were washed three times with pre-warmed PBS. After washing, 200 μL of 1% Triton X-100 was added to each well and the cells were cultured in a 37°C incubator for 30 minutes. The lysate was diluted with PBS at a ratio of 1:10 and counted on a plate. The cells were cultured in a 37°C incubator overnight. The results are shown in Figure 2. Figure 8As shown on the left, compared with the wild-type EcN strain, the adhesion ability of EcN (pPG-BOPA) decreased by 32.6%, with a significant difference (P<0.01); the adhesion ability of EcN (pPG-BOPA, pBbB8K-FF) decreased by 48.7%, with a significant difference (P<0.001); the adhesion ability of EcN (pPG-BOPA, pBbB8K-LFF) decreased by 25.6%, with a significant difference (P<0.001); while the adhesion ability of EcN (pBbB8K-FF) increased by 41.7%, with a significant difference (P<0.01); the adhesion ability of EcN (pBbB8K-LFF) increased by 60.2%, with a significant difference (P<0.0001). The above results show that pBbB8K-FF and pBbB8K-LFF can enhance the adhesion ability of EcN to cells. The engineered bacteria EcN / pBbB8K-LFCA-FaeG-FaeC (EcN(pBbB8K-LFF)) has strong adhesion to intestinal epithelial cells, providing an important basis for the sustained colonization of the engineered bacteria in the digestive tract environment.

[0150] It was also found that in E. coli Nissle 1917, the expression of the exogenous gene fragment LFCA-FaeG-FaeC can promote the adhesion ability of the engineered bacteria better than the fragment FaeG-FaeC, indicating that LFCA and FaeG-FaeC can synergistically promote the colonization ability of E. coli Nissle1917, which provides an important basis for the engineering bacteria to form colonization in the digestive tract environment.

[0151] 2. Cell competition ability detection

[0152] The wild strain and engineered strain were inoculated into LB medium, and K88 was inoculated into Amp-resistant LB broth (for subsequent screening, Amp-resistant plasmid was introduced into K88). The bacterial solution at the stationary phase was centrifuged at 5000 rpm for 10 min at 4°C, the cells were collected, washed three times with pre-cooled PBS, and resuspended with MEM complete medium. The wild strain, engineered strain and K88 strain were mixed in a ratio of 1:1 (100 μL of bacterial solution, 2.5×10 7 CFU bacterial count) was inoculated into a monolayer of Caco-2 cells in a 24-well plate. After 2 hours of action, the complete medium was discarded and the cells were washed three times with pre-warmed PBS to remove non-adherent bacteria. After washing, 200 μL of 1% Triton X-100 was added to each well and incubated at 37°C for 30 minutes. The lysate was diluted with PBS at a ratio of 1:10 and counted on an Amp-resistant LB plate. The cells were incubated at 37°C overnight. The results are shown in Figure 8As shown on the right, compared with the control group, EcN, EcN(pPG-BOPA), EcN(pPG-BOPA, pBbB8K-FF), EcN(pPG-BOPA, pBbB8K-LFF), EcN(pBbB8K-FF) and EcN(pBbB8K-LFF) strains can inhibit the adhesion of K88 to epithelial cells, and their cell adhesion rates decreased by 84.1%, 85.5%, 44.4%, 60.9%, 73.7% and 76.5%, respectively, which were significantly lower than those of the control group (P<0.0001). The results showed that EcN, EcN(pPG-BOPA), EcN(pPG-BOPA, pBbB8K-FF), EcN(pPG-BOPA, pBbB8K-LFF), EcN(pBbB8K-FF) and EcN(pBbB8K-LFF) could effectively inhibit the adhesion of pathogenic bacteria to intestinal epithelial cells. In addition, through the above results, this study found that the two engineered probiotics EcN(pBbB8K-FF) and EcN(pBbB8K-LFF) had strong adhesion to cells and could effectively inhibit the adhesion of K88 to cells; indicating that LFCA and FaeG-FaeC could synergistically inhibit the adhesion of K88 to cells.

[0153] 3. Detection of protein expression of infected cells by adhesion-enhanced engineered bacteria

[0154] Caco-2 cells were cultured at 2 × 10 5 The cells were inoculated into a 6-well plate at a concentration of 100 / mL, and each well was suspended with 2 mL of complete medium. The cells were cultured in a 37°C, 5% CO2 incubator. Caco-2 cells were treated with adhesion-enhanced engineered bacteria at an infection multiplicity (MOI) of 100:1, and cultured in a 37°C, 5% CO2 incubator for 6 hours. The complete medium was discarded, and the cells were washed three times with pre-warmed PBS. The cells were challenged with K88 at an infection multiplicity (MOI) of 50:1. 2 mL of complete medium (without double antibody) was added to each well, and the cells were mixed and cultured in a 37°C, 5% CO2 incubator 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 Fig. 9 , Fig.10 As shown in the figure, the engineered bacteria can enhance the expression levels of Occludin and ZO-1 proteins after K88 infection of cells, indicating that it can improve the damage of pathogenic bacteria to the barrier function of intestinal epithelial cells ( Fig. 9 ), and the engineered bacteria can reduce the phosphorylation level of p65 protein caused by K88 invasion of cells, indicating that it can inhibit the activation of NF-κB signaling pathway ( Fig.10 ).

[0155] Example 3 Immunoprotective effect of adhesion-enhanced engineered probiotics against ETEC K88

[0156] 1. Engineered probiotics vaccination program

[0157] Six-week-old C57BL / 6J female mice were acclimated to the laboratory environment for 1 week, fasted for 2 hours before immunization, and resumed normal feeding 1 hour after immunization. Immunization was performed once every week, and each time was orally administered for 3 consecutive days, for a total of 3 immunizations. The immunization dose for each mouse was 1.0×10 9 CFU / ml (0.2 mL), and mice in the control group were inoculated with an equal amount of sterile PBS buffer, and flow cytometry was performed on the 29th day.

[0158] 2. Detection of Proliferation and Differentiation of Immune-related Cells

[0159] Mouse spleen (SP), mesenteric lymph nodes (MLNs), and Peyer's lymph nodes (PPs) were sterilely collected to prepare single cell suspensions, and 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, and CD8-PE antibodies were added and incubated at 4°C in the dark for 30 minutes to detect T lymphocyte proliferation. The results are as follows: Fig.11 , Fig.12 , Fig.13 , Fig.14 As shown, CD11c in PPs and MLN of mice in the engineered bacteria EcN / pBbB8K-LFCA-FaeG-FaeC group + The number of DCs cells was significantly higher than that of the control group, indicating that the adhesion-enhanced engineered bacteria constructed in this study can quickly stimulate the activation of dendritic cells in the intestinal lymph nodes of mice through oral immunization, which is beneficial to the recognition, uptake and immune-related signal transduction of pathogenic bacteria and their antigens by antigen-presenting cells. In addition, the engineered bacteria promoted the expression of CD45R + IgA + B lymphocyte proliferation and promoted CD3 + CD4 + The proliferation of T lymphocytes was significantly higher than that of the control group.

[0160] 3. Detection of immunoglobulin and cytokine levels

[0161] 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. The results showed that the engineered bacteria EcN / pBbB8K-LFCA-FaeG-FaeC could significantly increase the levels of sIgA in feces and IgG in serum of mice ( Fig.15 ), reduced serum TNF-α, IL-10 and IL-1β levels ( Fig.17 ).

[0162] Total RNA was extracted from the small intestine of mice and reverse transcribed into cDNA. RT-PCR was used to detect the expression levels of TNF-α, IL-1β, IL-10, ZO-1, Occludin, TLR2, TLR3 and p65 genes. Fig.15 , Fig.16 , Fig.17 , Fig.18 , Fig.19 As shown, the engineered bacteria significantly downregulated the expression levels of TNF-α, IL-1β, p65 and TLR2 genes, and upregulated the expression levels of TLR3, ZO-1 and Occludin genes.

[0163] 4. Detection of survival rate and weight change of mice challenged with ETEC K88

[0164] One week after the last immunization, 10×LD 50 K88 strain (5.34×10 7 CFU / ml) were injected intraperitoneally once, and the animals were fed and observed for 3 days. The number of deaths and body weight were counted every 12 hours, pathological changes were detected, and mortality and body weight changes were calculated. Fig. 20 , Fig.21 As shown, the survival rate of mice in the engineered bacteria group after challenge was 66.67%, the weight loss trend of mice in the engineered bacteria group after challenge was significantly lower than that in the control group, and 60 hours after challenge, the weight of mice in the engineered bacteria group returned to the level before challenge.

[0165] 5. Histopathological examination

[0166] The duodenum, jejunum, ileum, and colorectum of mice before and after the infection were obtained, fixed with 4% paraformaldehyde for 24 hours, and placed in an embedding box for subsequent dehydration and transparent treatment. Wax was immersed in a 58°C incubator for 30 minutes each for wax 1, wax 2, and wax 3. After the wax immersion was completed, the slices were immediately paraffined and embedded in an embedding machine. The slice thickness was 4μm, and the slices were spread in a 42°C water bath. The slices were placed in an 80°C dry heat box for 1 hour. The dried slices were placed in xylene for 8 minutes and different concentrations of alcohol gradient dehydration for 1 minute each, and washed twice with water; hematoxylin staining for 5 minutes, washed twice with water; 1% hydrochloric acid alcohol differentiation for 5s, washed twice with water; eosin staining for 2 minutes, washed twice with water; neutral gum sealing and microscopic examination. The results are as follows Fig. 22 The results of pathological histological examination showed that the engineered bacteria reduced the degree of colon crypt destruction and colon area damage in mice, alleviated the inflammatory infiltration caused by ETEC K88, improved the intestinal mucosal structure, and helped prevent or alleviate the pathological damage of intestinal tissue caused by ETEC K88.

[0167] 6. Effects of engineered probiotics on intestinal flora of mice

[0168] After the last immunization, 5 mice were randomly selected from each group to collect feces for 16S rRNA bacterial diversity detection. Fecal genomic DNA was extracted and PCR amplification of the V3-4 hypervariable region of the bacterial 16S rRNA gene was performed. The library was constructed using the DNA PCR-Free Sample Preparation Kit. The constructed library was quantified by Qubit and Q-PCR. After the library was qualified, the Illumina platform was used to perform paired-end sequencing on the community DNA fragments. Fig.23 , Fig.24 , Fig.25 , Fig.26 As shown in the data, in the intestine of the mice in the engineered bacteria group, the Bacteroidetes_Muribaculaceae genus dominated (33.38%), the relative abundance of Lactobacillus was 18.46%, the relative abundance of Firmicutes_Lachnospiraceae genus NK4A136 was 10.47%, and the relative abundance of opportunistic pathogens such as Streptococcus was 0%, indicating that the engineered bacteria EcN / pBbB8K-LFCA-FaeG-FaeC can effectively increase the abundance of probiotics in the intestine of mice and reduce the proportion of pathogenic bacteria, and enhance the anti-infection effect of host animals against ETEC K88 by improving the richness and diversity of intestinal flora.

[0169] Although the above describes the specific implementation mode 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 creative work are still within the scope of protection of the present invention.

Claims

1. An adhesion-enhanced engineered probiotic, characterized in that: It contains a fusion gene, and the fusion gene sequence is shown in SEQ ID NO.3; The probiotics E. coli Nissle 1917 is the chassis strain.

2. The adhesive engineered probiotic according to claim 1, wherein the engineered probiotic further introduces a gene fragment of pBbB8K.

3. A fusion gene, the sequence of which is shown in SEQ ID NO.

3. A fusion protein encoded by the fusion gene according to claim 3.

5. An expression vector, characterized in that: The expression vector comprises the fusion gene according to claim 3.

6. An expression vector according to claim 5, characterized in that The expression vector is obtained by connecting the fusion gene and the vector pBbB8K; the connection method is restriction enzyme connection; the restriction enzyme site is Bgl II and XOt I.

7. A pharmaceutical composition or pharmaceutical preparation or bacterial agent, comprising the adhesion-enhanced engineered probiotic according to any one of claims 1 to 2 or the fusion protein according to claim 4.

8. Use of the adhesion-enhanced engineered probiotic according to any one of claims 1-2, or the fusion gene according to claim 3, or the fusion protein according to claim 4 in the preparation of drugs, vaccines or bacterial agents for preventing and / or treating inflammatory bowel disease; the inflammatory bowel disease is enterotoxigenic Escherichia coli infection.

9. The use according to claim 8, characterized in that: 1) The drug, vaccine or bacterial agent is used to enhance the adhesion to intestinal epithelial cells Caco-2 or competitively inhibit the adsorption of pathogenic Escherichia coli K88; 2) The drug, vaccine or bacterial agent is used to activate the body's immune cells; 3) The drug, vaccine or bacterial agent is used to activate dendritic cells in Peyer's patches and mesenteric lymph nodes; 4) The drug, vaccine or bacterial agent is used to stimulate specific cytokines in the spleen, mesenteric lymph nodes and / or Peyer's patches to resist K88 infection; the specific cytokines include CD4 + 、B220 + IgA + , sIgA, IgG, TNF-α; 5) The drug, vaccine or bacterial agent is used to maintain the integrity of the intestinal barrier to resist K88 pathogen infection and improve intestinal inflammation.

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