Expression cassette, recombinant expression vector, genetically engineered bacterium, construction method, culture method and application

Abstract

The invention provides an expression cassette, a recombinant expression vector, a genetically engineered bacterium, a construction method, a culture method and an application, and particularly belongs to the technical field of genetic engineering. The invention provides an expression cassette. The expression cassette comprises a SpaC truncated protein gene, an antioxidant enzyme three-enzyme coupling system fusion protein gene and a cytokine fusion protein gene, the expression cassette further comprises a surface display system for presenting the SpaC truncated protein on the surface of the cell; the antioxidant enzyme three-enzyme coupling system fusion protein is fused with catalase, superoxide dismutase and glutathione peroxidase. The cytokine fusion protein is fused with a YebF protein and a trefoil factor 3 protein. The expression cassette provided by the invention has the function of improving the adhesion colonization ability of the EcN strain, can improve the active oxygen scavenging ability of the EcN strain and repair the intestinal epithelial barrier function, is high in safety, and can realize more effective treatment of inflammatory bowel diseases.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering, and particularly relates to an expression cassette, a recombinant expression vector, a genetically engineered bacterium and a construction and culture method and application. BACKGROUND

[0002] Inflammatory Bowel Disease (IBD) is a chronic and recurrent inflammatory disease of the gastrointestinal tract, mainly including Crohn's Disease (CD) and Ulcerative Colitis (UC). Its pathological features are abnormal activation of the intestinal mucosal immune system, leading to persistent inflammatory response, tissue damage and intestinal barrier dysfunction. The pathogenesis of IBD has not been fully elucidated, and it is currently believed to be closely related to genetic susceptibility, intestinal flora imbalance, environmental factors and immune dysregulation. IBD patients often show clinical symptoms such as abdominal pain, diarrhea and weight loss, and may cause extraintestinal complications, seriously affecting the quality of life. Existing treatment methods such as aminosalicylates, glucocorticoids and immunomodulators can alleviate symptoms, but have limitations such as low response rate, obvious side effects or high cost. Therefore, developing new targeted therapies or optimizing existing treatment regimens is still a key issue to be addressed in the current IBD field.

[0003] Escherichia coli Nissle 1917 (EcN) as a non-pathogenic strain has been shown to have potential efficacy in the treatment of IBD. Its mechanisms of action include: (1) regulating intestinal flora homeostasis and reducing the proliferation of harmful bacteria; (2) regulating immune response, such as promoting the secretion of anti-inflammatory cytokines (IL-10) and inhibiting the secretion of pro-inflammatory cytokines (TNF-α, IL-6); (3) enhancing intestinal barrier function by up-regulating the expression of tight junction proteins (such as Occludin, ZO-1) to maintain mucosal integrity. In addition, EcN has become an ideal chassis microorganism for developing engineered strains targeting IBD treatment due to its clear genetic background, convenient genetic manipulation and low culture cost. The therapeutic efficacy of engineered strains is closely related to their adhesion and colonization ability in the intestine. However, the binding capacity of EcN strains to cell and protein targets present in the inflammatory intestinal environment is limited, which limits their adhesion and colonization, and there is currently a lack of products or methods that can improve the adhesion and colonization ability of EcN strains. SUMMARY

[0004] The present application aims to provide an expression cassette, a recombinant expression vector, a genetically engineered bacterium and a construction and culture method and application. The expression cassette described in the present application has the function of improving the adhesion and colonization ability of EcN strains, and can improve the activity oxygen clearance capacity of EcN strains and repair the intestinal epithelial barrier function, has high safety and can achieve more effective treatment of inflammatory bowel disease.

[0005] This invention provides an expression cassette comprising a SpaC truncated protein gene, an antioxidant enzyme three-enzyme coupling system fusion protein gene, and a cytokine fusion protein gene; the expression cassette also includes a surface display system for presenting the SpaC truncated protein on the cell surface; The nucleotide sequence of the SpaC truncated protein gene is as shown in SEQ ID NO.2 or is a nucleotide sequence that has greater than or equal to 80% homology with the sequence shown in SEQ ID NO.2 and has the adhesion ability of the SpaC truncated protein. The nucleotide sequence of the antioxidant enzyme three-enzyme coupling system fusion protein gene is as shown in SEQ ID NO.8 or is a nucleotide sequence that has greater than or equal to 80% homology with the sequence shown in SEQ ID NO.8 and has catalase, superoxide dismutase and glutathione peroxidase activities. The nucleotide sequence of the cytokine fusion protein gene is as shown in SEQ ID NO.10 or is a nucleotide sequence that has greater than or equal to 80% homology with the sequence shown in SEQ ID NO.10 and has the activity of YebF protein and trefoil factor 3 protein.

[0006] Preferably, the surface display system includes an ice nucleoprotein surface display system; the ice nucleoprotein display system includes *Pseudomonas syringae*. Pseudomonas syringae The ice nucleoprotein is derived from [a specific source], and the nucleotide sequence of the gene encoding the ice nucleoprotein is shown in SEQ ID NO.3; the expression cassette further includes a SpaC truncated protein promoter, which includes an L-arabinose inducible promoter; the L-arabinose inducible promoter includes an AraC regulatory protein gene, an araBAD promoter gene, and a ribosome binding site gene, and the nucleotide sequence of the L-arabinose inducible promoter is shown in SEQ ID NO.4.

[0007] Preferably, the expression cassette further includes a green fluorescent protein gene and a promoter for the green fluorescent protein gene; the promoter for the green fluorescent protein gene includes the constitutive promoter J23119.

[0008] Preferably, the promoter of the antioxidant enzyme three-enzyme coupling system fusion protein and the cytokine fusion protein includes a ykgMO promoter that responds to calprotectin; the nucleotide sequence of the ykgMO promoter that responds to calprotectin is shown in SEQ ID NO. 6.

[0009] The present invention also provides a recombinant expression vector for the expression cassette described in the above technical solution.

[0010] The present invention also provides genetically engineered bacteria comprising the recombinant expression vector described in the above technical solution.

[0011] Preferably, the host bacteria of the genetically engineered bacteria comprises Escherichia coli Nissle 1917.

[0012] The application further provides a construction method of the genetically engineered bacteria according to the above technical solution, comprising the following steps: transforming the recombinant expression vector according to the above technical solution into a host bacteria competent cell to obtain the genetically engineered bacteria.

[0013] The application further provides a culture method of the genetically engineered bacteria according to the above technical solution, comprising the following steps: inoculating the genetically engineered bacteria into a seed culture medium for culture to obtain a genetically engineered bacteria seed liquid; fermenting and culturing the genetically engineered bacteria seed liquid in a fermentation culture medium to induce expression of the SpaC truncated protein, and obtaining a genetically engineered bacteria fermentation product.

[0014] The application further provides application of the expression cassette according to the above technical solution, the recombinant expression vector according to the above technical solution, the genetically engineered bacteria according to the above technical solution, the genetically engineered bacteria constructed by the construction method according to the above technical solution, or the genetically engineered bacteria obtained by the culture method according to the above technical solution in preparation of a product for improving adhesion and colonization ability, active oxygen scavenging ability and / or repairing intestinal epithelial barrier function of host bacteria, or in preparation of a drug for improving target treatment of inflammatory bowel disease of host bacteria.

[0015] The application provides an expression cassette. The expression cassette has the functions of improving adhesion and colonization ability of EcN strains, improving active oxygen scavenging ability and repairing intestinal epithelial barrier function of EcN strains, is high in safety, and can achieve more effective treatment of inflammatory bowel disease. The genetically engineered bacteria constructed based on the expression cassette are expected to be probiotic drugs for target treatment of inflammatory bowel disease, and have application potential in the field of biological medicine.

[0016] Specifically, based on the expression cassette, the application constructs a recombinant expression plasmid containing the expression cassette; based on the recombinant expression plasmid, the application constructs genetically engineered bacteria. The genetically engineered bacteria have enhanced adhesion and colonization ability, and have the functions of scavenging active oxygen and repairing intestinal epithelial barrier function by secreting cytokines. The genetically engineered bacteria having enhanced adhesion and colonization ability and the function of target treatment of IBD are expected to be used as biological medicine preparations for development of IBD treatment preparations. BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the accompanying drawings needed in the embodiments will be briefly introduced as follows. Obviously, the accompanying drawings in the following description only constitute some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained from these drawings without any creative effort.

[0018] Figure 1 A plasmid map of pET22b-SpaC-mGFP-TE-TFF3 provided by the present application; Figure 2 SDS-PAGE and Western blot results of genetically engineered bacteria displaying and expressing SpaC truncated protein provided by the present application; Figure 3 Immunofluorescence staining results of SpaC truncated protein on the surface of genetically engineered bacteria provided by the present application; Figure 4 Adhesion experiment results of genetically engineered bacteria and control strains on a protein model in vitro provided by the present application; wherein, A is a mucin model result graph; B is a type IV collagen protein model result graph; Figure 5 Adhesion rate results of genetically engineered bacteria and control strains in an intestinal epithelial cell model formed by Caco-2 cells provided by the present application; Figure 6 ABTS rapid method detection results of total antioxidant activity of the antioxidant enzyme tri-enzyme coupling system expressed by genetically engineered bacteria under the induction of different concentrations of calprotectin provided by the present application; Figure 7 FRAP method detection results of total antioxidant activity of the antioxidant enzyme tri-enzyme coupling system expressed by genetically engineered bacteria under the induction of different concentrations of calprotectin provided by the present application; Figure 8 SDS-PAGE and Western blot results of TFF3 cytokine fusion protein secreted and expressed by genetically engineered bacteria under the induction of different concentrations of calprotectin provided by the present application; Figure 9 Results of the influence of genetically engineered bacteria on the body weight of healthy mice provided by the present application; Figure 10 Live imaging results of genetically engineered bacteria and control strains adhering and colonizing in the intestinal tract of healthy mice provided by the present application; wherein, A is a control strain result graph; B is a genetically engineered bacteria result graph; Figure 11 Fluorescence intensity analysis results of live imaging of genetically engineered bacteria and control strains adhering and colonizing in the intestinal tract of healthy mice provided by the present application; Figure 12A graph of the weight loss protection results of the genetically engineered bacteria and the control strain provided by the present application in a DSS-induced mouse colitis model; Figure 13 A graph of the disease index scoring results of the genetically engineered bacteria and the control strain provided by the present application in a DSS-induced mouse colitis model; Figure 14 A graph of the colon length measurement results of the genetically engineered bacteria and the control strain provided by the present application in a DSS-induced mouse colitis model; Figure 15 A graph of the spleen weight measurement results of the genetically engineered bacteria and the control strain provided by the present application in a DSS-induced mouse colitis model. DETAILED DESCRIPTION

[0019] The present application provides an expression cassette, which comprises a SpaC truncated protein gene, an antioxidant enzyme trimer fusion system fusion protein gene and a cytokine fusion protein gene; the expression cassette further comprises a surface display system for presenting the SpaC truncated protein on the cell surface; The nucleotide sequence of the SpaC truncated protein gene is shown in SEQ ID NO. 2 or a nucleotide sequence with a homology of greater than or equal to 80% with the sequence shown in SEQ ID NO. 2 and having the adhesion ability of the SpaC truncated protein; The nucleotide sequence of the antioxidant enzyme trimer fusion system fusion protein gene is shown in SEQ ID NO. 8 or a nucleotide sequence with a homology of greater than or equal to 80% with the sequence shown in SEQ ID NO. 8 and having the activities of catalase, superoxide dismutase and glutathione peroxidase; The nucleotide sequence of the cytokine fusion protein gene is shown in SEQ ID NO. 10 or a nucleotide sequence with a homology of greater than or equal to 80% with the sequence shown in SEQ ID NO. 10 and having the activities of YebF protein and trefoil factor 3 protein.

[0020] The expression cassette comprises a SpaC truncated protein gene, and the nucleotide sequence of the SpaC truncated protein gene is shown in SEQ ID NO. 2 or a nucleotide sequence with a homology of greater than or equal to 80% with the sequence shown in SEQ ID NO. 2 and having the adhesion ability of the SpaC truncated protein. In a specific embodiment, the SpaC truncated protein gene is Lactobacillus rhamnosus Lactobacillus rhamnosusA truncated protein of an adhesion protein SpaC from GG, a truncated protein of an adhesion domain of an adhesion protein SpaC at the top of a pilus of Lactobacillus rhamnosus GG is designed based on the adhesion protein SpaC, and the SpaC truncated protein has an effect of enhancing the adhesion ability of a host bacterium. In specific embodiments, the SpaC truncated protein gene is codon-optimized, and the nucleotide sequence is shown in SEQ ID NO. 2. In specific embodiments, the amino acid sequence of the SpaC truncated protein is shown in SEQ ID NO. 1. The expression cassette described in the present application further comprises a surface display system for presenting the SpaC truncated protein on the cell surface. In specific embodiments, the surface display system comprises an ice nucleation protein surface display system; the ice nucleation protein display system comprises a Pseudomonas syringae Pseudomonas syringae ice nucleation protein from Pseudomonas syringae, and the nucleotide sequence of the gene encoding the ice nucleation protein is shown in SEQ ID NO. 3. In specific embodiments, the expression cassette further comprises a SpaC truncated protein promoter, and the SpaC truncated protein promoter comprises an L-arabinose inducible promoter; the L-arabinose inducible promoter comprises an AraC regulatory protein gene, an araBAD promoter gene, and a ribosome binding site gene, and the nucleotide sequence of the L-arabinose inducible promoter is shown in SEQ ID NO. 4. In specific embodiments, the L-arabinose inducible promoter, the ice nucleation protein surface display system, and the adhesion protein SpaC truncated protein constitute an adhesion module. Specifically, the present application places the fusion protein of the ice nucleation protein surface display system and the SpaC truncated protein downstream of the L-arabinose promoter, so that the engineered strain can present the SpaC truncated protein on the cell surface, thereby improving the adhesion ability of the engineered strain to intestinal protein targets and epithelial cells.

[0021] In specific embodiments, the expression cassette further comprises a green fluorescent protein gene (mNeonGreen, mGFP) and a promoter of the green fluorescent protein gene; the promoter of the green fluorescent protein gene comprises a constitutive promoter J23119. In specific embodiments, the promoter of the green fluorescent protein gene and the green fluorescent protein constitute a chromogenic marker module, and the nucleotide sequence of the chromogenic marker module is shown in SEQ ID NO. 5. The constitutively expressed green fluorescent protein mGFP can facilitate detection and tracking of the genetically engineered bacteria constructed based on the expression cassette.

[0022] The expression cassette comprises an antioxidant enzyme tri-enzyme cascade system fusion protein gene and a cytokine fusion protein gene, the nucleotide sequence of the antioxidant enzyme tri-enzyme cascade system fusion protein gene is shown in SEQ ID NO. 8 or a nucleotide sequence with homology greater than or equal to 80% with the sequence shown in SEQ ID NO. 8 and having the activities of catalase, superoxide dismutase and glutathione peroxidase; the nucleotide sequence of the cytokine fusion protein gene is shown in SEQ ID NO. 10 or a nucleotide sequence with homology greater than or equal to 80% with the sequence shown in SEQ ID NO. 10 and having the activities of YebF protein and trefoil factor 3 protein. In specific embodiments, the promoters of the antioxidant enzyme tri-enzyme cascade system fusion protein and the cytokine fusion protein comprise a calprotectin-responsive ykgMO promoter; the nucleotide sequence of the calprotectin-responsive ykgMO promoter is shown in SEQ ID NO. 6. In specific embodiments, the antioxidant enzyme tri-enzyme cascade system fusion protein is composed of a 3×Flag tag, a Lactobacillus plantarum Lactobacillus plantarum CAT from ATCC14431, superoxide dismutase SOD from Bacillus subtilis Bacillus subtilis 168 and Lactobacillus casei Lactobacillus casei GPX from ATCC393. In specific embodiments, the amino acid sequence of the antioxidant enzyme tri-enzyme cascade system fusion protein is shown in SEQ ID NO. 7. In specific embodiments, the cytokine fusion protein is composed of E. coli autosecretory protein YebF, human-derived trefoil factor 3 (TFF3) and 6×His tag, and the amino acid sequence of the cytokine fusion protein is shown in SEQ ID NO. 9. Based on the antioxidant enzyme tri-enzyme cascade system fusion protein gene and the cytokine fusion protein gene, the present application realizes the treatment of IBD by inhibiting the abnormal increase of reactive oxygen species and repairing the intestinal barrier, and in combination with the calprotectin-responsive ykgMO promoter, the present application has the functions of reactive oxygen species scavenging and mucosal repair in response to the inflammatory environment and has the function of targeted treatment of IBD.

[0023] This invention also provides a recombinant expression vector for the expression cassette described in the above-mentioned technical solution. The recombinant expression plasmid of this invention can confer enhanced adhesion and colonization ability, especially on EcN strains, and targeted therapy function for IBD. This invention does not particularly limit the type of plasmid vector; those skilled in the art can conventionally select vectors suitable for E. coli replication and gene expression for construction. In a specific embodiment, the base plasmid of the recombinant expression vector includes the pET-22b(+) plasmid. When the expression cassette of this invention includes the SpaC truncated protein gene, the green fluorescent protein gene, the antioxidant enzyme three-enzyme coupling system fusion protein gene, and the cytokine fusion protein gene, and the base plasmid is the pET-22b(+) plasmid, this invention can prepare the pET22b-SpaC-mGFP-TE-TFF3 plasmid based on the expression cassette and the base plasmid. In a specific embodiment, the map of the pET22b-SpaC-mGFP-TE-TFF3 plasmid is shown below. Figure 1 As shown. Specifically, in the pET22b-SpaC-mGFP-TE-TFF3 plasmid, the L-arabinose inducible promoter is inserted into the pET-22b(+) vector. Bgl The T7 promoter is replaced between the II and Nde I restriction sites. Further, *Pseudomonas syringae* Pseudomonas syringae The ice nucleoprotein surface display system, after codon optimization, was inserted downstream of the pelB signal peptide in the pET-22b(+) vector. More specifically, the ice nucleoprotein surface display system was inserted into... Nco I and Not Between the I restriction sites. Furthermore, the SpaC truncated protein, an adhesion protein derived from *Lactobacillus rhamnosus* GG, was codon-optimized and inserted into... Not I and Xho Between the I restriction sites, the SpaC protein is fused to the C-terminus of the ice nucleoprotein surface display system, and the 6×His tag carried on the pET-22b(+) vector is fused to the C-terminus of the SpaC truncated protein. Furthermore, a fluorescent protein expression module composed of the constitutive promoter J23119 and green fluorescent protein mGFP is inserted downstream of the surface-displaying SpaC truncated protein module. Further, to endow the engineered bacteria with the functions of sensing and responding to inflammatory environments, scavenging reactive oxygen species, and repairing mucosal damage, the calprotectin-responsive promoter ykgMO is designed upstream of both the antioxidant enzyme three-enzyme coupling system fusion protein and the cytokine fusion protein to regulate the expression of the two fusion proteins; these two gene expression modules are then inserted downstream of the fluorescent protein expression module.

[0024] The application further provides a genetically engineered bacterium comprising the recombinant expression vector described in the above technical solution. In specific embodiments, the host bacterium of the genetically engineered bacterium comprises Escherichia coli Nissle 1917. When the genetically engineered bacterium described in the application uses Escherichia coli probiotic Nissle 1917 as a chassis strain, and the expression cassette described in the application comprises a surface display system (ice nucleation protein surface display system) for presenting the SpaC truncated protein on the cell surface, and a ykgMO promoter responsive to calprotectin, the genetically engineered bacterium described in the application can present the adhesion domain of the adhesion protein SpaC of Lactobacillus rhamnosus GG on the cell surface through the ice nucleation protein surface display system, enhance the adhesion and colonization ability of the strain, and regulate the expression of the antioxidant enzyme trimer coupling system and the cytokine secretion module through the induction of the inflammation marker calprotectin, so that the genetically engineered bacterium can perceive and respond to intestinal inflammation to produce a therapeutic effect. Experimental results show that the genetically engineered bacterium of the application not only shows enhanced adhesion and binding ability in protein models and cell models, but also shows active oxygen scavenging ability and cytokine secretion function under the induction of the inflammation marker calprotectin. In addition, the genetically engineered bacterium of the application has good biological safety and shows good relief effect on symptoms related to colitis. The genetically engineered bacterium of the application is expected to be used as a probiotic drug for targeted treatment of inflammatory bowel disease, and has application potential in the field of biological medicine. Further, in specific embodiments, when the promoter of the SpaC truncated protein is an L-arabinose inducible promoter, the genetically engineered bacterium presents the adhesion protein SpaC truncated protein on the cell surface after induction by L-arabinose. In vitro protein models, the genetically engineered bacterium has enhanced adhesion to mucin and type IV collagen. Cell experiment results show that, in an intestinal epithelial model formed by Caco-2 monolayer cells, the genetically engineered bacterium has significantly enhanced adhesion compared with a control strain that does not display and express SpaC protein. Under different concentrations of calprotectin induction conditions, the genetically engineered bacterium shows concentration-dependent enhanced ROS decomposition ability and increased cytokine secretion level, and shows good inflammation relief effect in an intestinal inflammation model. The genetically engineered bacterium of the application with enhanced adhesion and colonization ability and targeted IBD treatment function is expected to be used as a biological medicine preparation for the development of IBD treatment preparations.

[0025] The application further provides a construction method of the genetically engineered bacterium, comprising the following steps: transforming the recombinant expression vector into a host bacterium competent cell to obtain the genetically engineered bacterium. In specific embodiments, the host bacterium comprises Escherichia coli. In specific embodiments, the Escherichia coli comprises Escherichia coli Nissle1917. The application does not have special limitations on the preparation method of the competent cell, and the calcium chloride method can be used to prepare the competent cell. The application does not have special limitations on the transformation method, and the conventional plasmid transformation method into the competent cell can be used, such as the heat shock method. After transformation, the genetically engineered bacterium can be obtained through resistance plate screening and sequencing.

[0026] The application further provides a culture method of the genetically engineered bacterium, comprising the following steps: Inoculating the genetically engineered bacterium into a seed culture medium for culture to obtain a genetically engineered bacterium seed liquid; Fermenting and culturing the genetically engineered bacterium seed liquid in a fermentation culture medium to induce SpaC truncated protein expression, and obtaining a genetically engineered bacterium fermentation product.

[0027] After the genetically engineered bacterium is activated, the genetically engineered bacterium is inoculated into a seed culture medium for culture to obtain a genetically engineered bacterium seed liquid. In specific embodiments, the seed culture medium can be an LB culture medium. In specific embodiments, the culture time can be 8-12h, and specifically can be overnight culture.

[0028] After the genetically engineered bacterium seed liquid is obtained, the genetically engineered bacterium seed liquid is inoculated into a fermentation culture medium for fermentation culture to induce SpaC truncated protein expression, and a genetically engineered bacterium fermentation product is obtained. In specific embodiments, the inoculation amount can be 2-4%. In specific embodiments, when the cell concentration OD 600 is 0.4-0.6, the induction is performed. When the promoter of the SpaC truncated protein gene is an L-arabinose inducible promoter, the induction is performed using L-arabinose. When L-arabinose is used for induction, the final concentration of L-arabinose can be 0.2%. In specific embodiments, the fermentation culture temperature is 37℃. In specific embodiments, the fermentation culture time is 16-24h.

[0029] The application further provides application of the genetically engineered bacterium prepared by the expression cassette, the recombinant expression vector, the genetic engineering bacterium or the construction method or the culture method in preparation of a product for improving adhesion and colonization ability of a host bacterium, active oxygen scavenging ability and / or repair of intestinal epithelial barrier function or in preparation of a drug for improving targeted treatment of inflammatory bowel disease of the host bacterium. In specific embodiments, the host bacterium includes Escherichia coli Nissle 1917. The truncated protein of the adhesion protein SpaC can enhance the adhesion ability of the host bacterium, the catalase, the superoxide dismutase and the glutathione peroxidase can improve the active oxygen scavenging ability, and the secreted expression of the cytokine fusion protein can enable the host bacterium to obtain the function of repairing the intestinal epithelial barrier. The expression cassette or the recombinant expression vector can achieve treatment of IBD. In specific embodiments, the inflammatory bowel disease includes colitis. The genetically engineered bacterium or the fermentation product of the genetically engineered bacterium exhibits adhesion enhancement to the protein and cell target points present in the intestinal tract and active oxygen scavenging and cytokine secretion function induced by calprotectin, and can achieve more efficient treatment of inflammatory bowel disease. In specific embodiments, the drug further includes a pharmaceutically acceptable excipient. In specific embodiments, the excipient can be a biologically safe carrier such as gelatin, chitosan, sodium alginate, starch and the like. The dosage form of the drug is not particularly limited in the application and can be a microcapsule, a granule, a hydrogel and the like. The results of the embodiments show that the genetically engineered bacterium of the application exhibits good biological safety and no toxic side effects in a healthy mouse model, and exhibits inhibition effects on body weight loss, diarrhea and hematochezia, colon shortening and splenomegaly in a colitis mouse model.

[0030] In order to further illustrate the application, the expression cassette, the recombinant expression vector, the genetically engineered bacterium and the construction and culture method and application provided by the application are described in detail below in combination with embodiments, but they should not be understood as limiting the protection scope of the application.

[0031] In the following embodiments, the experimental methods used are conventional methods, and the materials, reagents and the like used are commercially available unless otherwise specified.

[0032] Embodiment 1 Preparation, culture and adhesion protein characterization of the genetically engineered bacterium.

[0033] The embodiment provides an experimental scheme for preparing a genetically engineered bacterium with enhanced adhesion and colonization ability and targeted treatment function of IBD, including the following steps: (1) Synthesis and transformation of the recombinant plasmid: The recombinant plasmid pET22b-SpaC-mGFP-TE-TFF3 was synthesized by Beijing Qingke Biotechnology Co., Ltd. Specifically, the plasmid synthesis required pET-22b(+) as the expression vector, with the L-arabinose inducible promoter (SEQ ID NO. 4) inserted between the Bgl II and Nde I restriction sites of the pET-22b(+) vector to replace the T7 promoter. The ice nucleoprotein surface display system (SEQ ID NO. 3) was inserted into... Nco I and Not Between the I restriction sites, the codon-optimized nucleotide sequence (SEQ ID NO.2) of the adhesion protein SpaC truncated protein gene is inserted into... Not I and Xho The I restriction sites form a surface-displaying SpaC protein module; further, the J23119 promoter and the nucleotide sequence of green fluorescent protein mNeonGreen (mGFP) (SEQ ID NO.5) are synthesized 6 bp downstream of the terminator of the SpaC protein module to form a fluorescent labeling module; further, two ykgMO promoters (SEQ ID NO.6) that regulate the coding genes of the antioxidant enzyme three-enzyme coupling system (SEQ ID NO.8) and the coding genes of the cytokine fusion protein secretion expression system (SEQ ID NO.10) are synthesized immediately downstream of the T7te terminator of the fluorescent labeling module to obtain the complete recombinant plasmid pET22b-SpaC-mGFP-TE-TFF3, as shown in the plasmid map. Figure 1 As shown. Since the multiple functional modules of the recombinant plasmid are all independent expression cassette structures, inserting each module into different positions of the plasmid vector without destroying key sequences such as terminators, replicons, and resistance genes on the plasmid does not affect the normal transcription and translation of genes, and can achieve the same effect as the plasmid described in this invention patent.

[0034] Competent cells of strain EcN (purchased from Beina Biotechnology, product number BNCC361741) were prepared using the calcium chloride method. The recombinant plasmid pET22b-SpaC-mGFP-TE-TFF3 was transformed into the competent cells using the heat shock method. Recombinants were screened on ampicillin-resistant plates and sequenced for verification. The resulting recombinant genetically engineered bacteria (EcN-EB) were stored in 15% glycerol solution at -80°C for later use.

[0035] (2) Preparation of seed culture of genetically engineered bacteria: The recombinant genetically engineered bacteria described in step (1) were streaked onto an ampicillin-resistant plate for activation. Single colonies were picked and inoculated into LB medium containing 120 μg / mL ampicillin. The culture was shaken at 37°C for 12 h to obtain recombinant Escherichia coli seed culture.

[0036] (3) Fermentation of genetically engineered bacteria: The seed liquid was inoculated into LB medium at a ratio of 2%, and when the bacterial concentration OD 600 0.4 was reached, 0.2% L-arabinose was added to induce the expression of recombinant protein, and the genetically engineered bacteria were cultured at 37°C for 18 h.

[0037] (4) Characterization of surface-displayed SpaC truncated protein The genetically engineered bacteria culture solution obtained in step (3) was centrifuged and resuspended in PBS buffer containing 1% PMSF. Using wild-type EcN (EcN-WT) culture solution as a control group, the ultrasonic disrupter was used for low-temperature ultrasonic disruption at a power of 150 W for 25-35 min. At 4°C, centrifugation was performed at 8000 rpm for 10 min, and the supernatant (S) and precipitate (P) of the cell disruption solution were separated. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting are shown in Figure 2 , and the target band (74.4 kDa) of the expected size was detected in the precipitate component of the cell disruption solution of EcN-EB, and the corresponding band was in the wild-type EcN sample. The experimental results show that the genetically engineered bacteria EcN-SpaC can effectively display the expressed SpaC truncated protein.

[0038] (5) Immunofluorescence staining of genetically engineered bacteria: In order to further prove that the SpaC truncated protein is presented on the surface of the genetically engineered bacteria, immunofluorescence staining was used to detect the His tag carried by the surface-presented protein of the genetically engineered bacteria. The control strain EcN-Con was obtained by transforming the wild-type EcN with the pET22b-mGFP plasmid (J23119 promoter and mGFP coding gene inserted between the BglII and XhoI The fermentation broth of the genetically engineered bacteria and the control strain prepared according to the method described in step (3) was centrifuged to collect the bacterial precipitate, which was resuspended in PBS buffer and then incubated with mouse anti-His tag primary antibody, followed by incubation of the strain with Alexa Fluor 488-conjugated goat anti-mouse fluorescent secondary antibody. The imaging parameters of the laser confocal microscope were adjusted, and the mGFP fluorescence interference was filtered out, and the results are shown in Figure 3 . The EcN-EB sample showed green fluorescence produced by the Alexa Fluor 488 group, proving the presence of His tag on the surface of the strain. The experimental results show that the SpaC truncated protein fused with His tag is successfully presented on the surface of the genetically engineered bacteria.

[0039] Example 2 Protein model adhesion of genetically engineered bacteria.

[0040] The genetically engineered bacteria were cultured as described in Example 1, and the in vitro protein model adhesion experiment was performed according to the following scheme: (1) Coating of protein plates: Two proteins present in the intestinal tract were selected as models in this example: mucin is a protein secreted by goblet cells of the intestinal epithelium and is the main component of the intestinal mucus layer; collagen type IV is a matrix protein exposed at sites of intestinal inflammatory damage. The mucin used in this example was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (S12066), and the collagen type IV was purchased from Hangzhou Yunyilian Biotechnology Co., Ltd. (GD68630). The mucin and collagen type IV were dissolved in a sodium carbonate-sodium bicarbonate buffer at pH 9.4, and 200 μg / mL of the mucin or collagen type IV was used to incubate the Nunc MaxiSorp 96-well plates at low temperature overnight to allow the proteins to bind to the surface of the plates. The unbound proteins were washed away with sodium bicarbonate buffer.

[0041] (2) Adhesion experiment of genetically engineered bacteria: 1 x 10 6 CFU of genetically engineered bacteria (the concentration of the genetically engineered bacteria culture solution obtained in Example 1 was adjusted, and the adjusted bacterial solution contained 1 x 10 6 CFU of viable bacteria per 200 μL) and a control strain EcN-Con expressing only a fluorescent protein were added to the protein-coated plates described in step (1), and incubated at 37°C for 3 h. The unadhered strains were removed by washing with PBS buffer 3 times. The strains adhered to the plates were scraped off, and colony counting was performed using the dilution plating method. The adhesion results in the mucin and collagen type IV models are shown in Figure 4 The adhesion of the genetically engineered bacteria was increased by 4.79 times (238:49.67, Figure 4 A in the mucin model) and by 6.86 times (305.33:44.5, Figure 4 B in the collagen type IV model) compared to the control strain. This result demonstrates that the adhesion and binding of the genetically engineered bacteria to protein targets in the intestinal tract are enhanced.

[0042] Example 3 Cell model adhesion of genetically engineered bacteria.

[0043] In this example, a Caco-2 cell monolayer was used to simulate the structure of intestinal epithelial cells.

[0044] The genetically engineered bacteria were cultured as described in Example 1, and the in vitro cell model adhesion experiment was performed according to the following scheme: (1) Caco-2 cell culture: Caco-2 cells were cultured in DMEM medium containing 10% fetal bovine serum, 1% non-essential amino acids, and 1% penicillin-streptomycin mixture. 1 x 10 6 CFU Caco-2 cells, overnight adherent cell monolayer.

[0045] (2) The genetically engineered bacteria and the control strain EcN-Con culture solution were cultured according to the culture method described in Example 1. After centrifugation to collect the bacterial cells, the bacterial cells were resuspended and adjusted to a concentration of 1 x 10 7 CFU / mL. 1 mL of resuspended bacterial solution was added to each well of a six-well plate, and incubated at 37°C in an environment containing 5% CO2 for 3 h. Unattached strains were removed by washing 3 times with PBS buffer. The adherent bacterial cells were released by Triton X-100 lysis, and colony counting was performed using the dilution plating method. As shown in Figure 5 , the adhesion rate of the genetically engineered bacteria was 11.91 times that of the control strain without surface-displayed SpaC truncated protein (14.77:1.24), which proved that the genetically engineered bacteria provided by the present application had enhanced adhesion to the epithelial cell model.

[0046] Example 4 Expression of the antioxidant enzyme trimer coupling system of the genetically engineered bacteria.

[0047] This example provides an experimental scheme for characterizing the ROS scavenging ability of the genetically engineered bacteria in response to the expression of the antioxidant enzyme trimer coupling system of calprotectin, including the following steps: (1) Transformation of recombinant plasmid: Calcium chloride method was used to prepare competent cells of EcN strain, and the recombinant plasmid pET22b-SpaC-mGFP-TE-TFF3 described above was transformed into the competent cells by heat shock method. The recombinants were screened on ampicillin-resistant plates and verified by sequencing. The obtained genetically engineered bacteria were stored in 15% glycerol solution and frozen at -80°C for standby use; (2) Preparation of seed solution of genetically engineered bacteria: The recombinant genetically engineered bacteria described in step (1) were streaked onto ampicillin-resistant plates for activation, and a single colony was inoculated into LB medium containing 120 μg / mL ampicillin and incubated at 37°C for 12 h with shaking to obtain a seed solution of recombinant Escherichia coli.

[0048] (3) Fermentation culture of genetically engineered bacteria: The seed solution was inoculated into LB medium at a 2% inoculation amount, and the bacterial concentration OD 600When the OD600 reached 0.4, the final concentrations of calprotectin were added at 0 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL, and 800 μg / mL, respectively, to induce the expression of the antioxidant enzyme triose system, and the culture was shaken at 37°C for 18 h.

[0049] (4) Measurement of total antioxidant level: The genetically engineered bacteria obtained in step (3) were collected by centrifugation, resuspended with PBS buffer, and then broken by ultrasonic wave at a power of 150 W for 25-35 min at low temperature. The supernatant was separated by centrifugation at 8000 rpm for 10 min at 4°C. The total antioxidant activity of the supernatant was detected by ABTS rapid method and FRAP method, respectively. The results of the total antioxidant activity detected by ABTS rapid method are shown in Figure 6 , and the results of the total antioxidant activity detected by FRAP method are shown in Figure 7 In both independent total antioxidant activity detection methods, the antioxidant enzyme triose system showed a concentration-dependent increase in antioxidant activity.

[0050] Example 5 Secretion of cytokines by genetically engineered bacteria.

[0051] The genetically engineered bacteria were cultured according to the scheme described in Example 4, and the secretion of cytokines was characterized according to the following scheme: (1) After fermentation, the supernatant was collected, concentrated, and the secreted protein was collected from the supernatant of the culture medium using an ultrafiltration tube with a 10 kD cutoff. The concentrated protein was collected after washing with PBS buffer for 3 times.

[0052] (2) The secreted proteins induced by different concentrations of calprotectin were detected by SDS-PAGE and Western blotting. Since the cytokine fusion protein carries a 6×His tag, a mouse anti-His tag primary antibody and a goat anti-mouse fluorescent secondary antibody were used for Western blotting detection. The results are shown in Figure 8 , under the induction of 400 μg / mL and 800 μg / mL calprotectin, the genetically engineered bacteria successfully expressed and secreted the TFF3 cytokine fusion protein.

[0053] Example 6 Safety evaluation of genetically engineered bacteria The genetically engineered bacteria were cultured according to the method described in Example 1, and the safety evaluation scheme was as follows: (1) Mouse feeding: 6-8 week old C57BL / 6 male mice were used as experimental animals, and the mice were fed in a SPF level feeding standard animal house, with free diet and water, and the environmental temperature was maintained at 25±1°C. After 7 days of adaptive feeding, the mice were treated by gavage.

[0054] (2) Monitoring of body weight change and blood biochemical test: the control group (Control) mice were given 200 μL of PBS buffer by gavage every day, and the genetically engineered bacteria group (EcN-EB) mice were given 200 μL of 1 x 10 9 CFU genetically engineered bacteria liquid sample. The gavage period lasted for 5 days, during which the body weight change of the mice was monitored every day, and the mice were still monitored for body weight for 1 week after the end of the gavage period.

[0055] Clinical observation showed that all mice were in good mental condition, could normally eat and drink water, and there was no abnormality in feces, and no mice died during the experiment. The body weight change of the mice is shown in Figure 9 The genetically engineered bacteria group did not show weight loss compared with the control group, which proved that the genetically engineered bacteria did not have toxic side effects on the mice.

[0056] Some mice were euthanized 24 h after the end of the gavage period, and blood samples were collected for blood biochemical analysis. The analysis results are shown in Table 1. The total protein (TP), albumin (ALB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), phosphatase (ALP), urea nitrogen (BUN) and creatinine (CREA) indicators of the mice in the control group and the genetically engineered bacteria group all met the reference value indicators, which proved that the genetically engineered bacteria had no safety hidden dangers in the clinic.

[0057] Table 1 Blood biochemical indicators of genetically engineered bacteria and its control group

[0058] Example 7 Intestinal colonization of genetically engineered bacteria In this embodiment, the genetically engineered bacteria were cultured by the method described in Example 1, and 6-8 week old C57BL / 6 male mice were used as experimental animal models to evaluate the intestinal colonization of genetically engineered bacteria, and the implementation scheme is as follows: (1) Mouse pretreatment: After the mice adapted to the feeding were anesthetized with isoflurane, the abdominal hair was removed. After the depilated mice recovered for 3 days, they were given gavage treatment.

[0059] (2) The control group mice were given gavage with EcN-Con liquid sample expressing only green fluorescent protein, and the genetically engineered bacteria group mice were given gavage with EcN-EB liquid sample, and the gavage period lasted for 5 days, 1 x 10 9 CFU liquid sample, and the mice were imaged in vivo 24 h after the last gavage. The imaging results are shown in Figure 10 , and the fluorescence intensity analysis results are shown in Figure 11 , and the experimental results showed that the average adhesion colonization ability of the genetically engineered bacteria in the intestine was increased by 1.52 times compared with the control strain without expressing adhesion protein.

[0060] Example 8 Remission effect of genetically engineered bacteria on DSS-induced colitis in mice.

[0061] In this example, genetically engineered bacteria were cultured in the same way as described in Example 1, and 6-8 week old C57BL / 6 male mice were used as experimental animal models. The implementation is as follows: (1) Mice colitis modeling and genetically engineered bacteria treatment: The healthy group of mice kept normal drinking water, and the drinking water of other groups of mice was added with 2.5% dextran sodium sulfate (DSS) to induce the occurrence of colitis. After 7 days of colitis modeling, the genetically engineered bacteria were administered by gavage. The healthy group (PBS) and the inflammation group (DSS) of mice were treated with PBS buffer by gavage; the control group (Con+DSS) of mice was treated with EcN-Con bacterial liquid sample expressing only green fluorescent protein by gavage, and the genetically engineered bacteria group (EB+DSS) of mice was treated with EcN-EB bacterial liquid sample by gavage. The gavage period lasted for 5 days, 1x10 9 CFU bacterial liquid sample, and the drinking water of the inflammation group, the control group and the genetically engineered bacteria group of mice was replaced with a solution containing 2.5% DSS and 10 g / L arabinose during the gavage period.

[0062] (2) Body weight monitoring and fecal sample disease index (DAI) scoring of mice: The mice were weighed and fecal samples were collected every day during the colitis modeling and gavage period to check the diarrhea and hematochezia index, and the colitis disease index of the mice was scored in combination with the body weight loss. The body weight changes and DAI scoring results of the mice are shown in Figure 12 and Figure 13 respectively. The mice's body weight began to decrease significantly and the disease index increased on the 3rd day of DSS modeling, indicating that the colitis modeling was successful. The genetically engineered bacteria produced stronger body weight protection and disease index inhibition than the control strain. At the end of the gavage period, the body weight of the genetically engineered bacteria group of mice had recovered to a level close to that of the healthy mice. The experimental results proved that the genetically engineered bacteria had good remission efficacy on DSS-induced colitis.

[0063] (3) Intestinal and spleen evaluation of mice: Since colitis usually shows shortening of the colon length and splenomegaly, the mice were euthanized 24 hours after the end of the gavage period, and intestinal tissue was collected to measure the colon length. The results are shown in Figure 14 . The intestinal tract of the genetically engineered bacteria group of mice showed significant colon length protection. The results of spleen weighing are shown in Figure 15 . The spleen of the genetically engineered bacteria group of mice recovered to a level close to that of the healthy group of mice. The above results all indicate the remission function of the genetically engineered bacteria on colitis.

[0064] Although the above embodiments have been described in detail, it should be understood that these are only some embodiments of the present application, but not all embodiments. Other embodiments can be obtained based on the above embodiments without creativity, and these embodiments all belong to the protection scope of the present application.

Claims

1. An expression cassette comprising, The expression cassette comprises a SpaC truncated protein gene, an antioxidant enzyme three-enzyme cascade system fusion protein gene, and a cytokine fusion protein gene; the expression cassette further comprises a surface display system for presenting the SpaC truncated protein on the surface of cells; The nucleotide sequence of the SpaC truncated protein gene is shown in SEQ ID NO. 2 or is a nucleotide sequence with homology of greater than or equal to 80% to the sequence shown in SEQ ID NO. 2 and having the adhesion ability of the SpaC truncated protein; The nucleotide sequence of the antioxidant enzyme three-enzyme cascade system fusion protein gene is shown in SEQ ID NO. 8 or is a nucleotide sequence with homology of greater than or equal to 80% to the sequence shown in SEQ ID NO. 8 and having the catalytic activity of catalase, superoxide dismutase, and glutathione peroxidase; The nucleotide sequence of the cytokine fusion protein gene is shown in SEQ ID NO. 10 or is a nucleotide sequence with homology of greater than or equal to 80% to the sequence shown in SEQ ID NO. 10 and having the catalytic activity of YebF protein and trefoil factor 3 protein.

2. The expression cassette of claim 1, wherein, The surface display system comprises an ice nuclein protein surface display system; the ice nuclein protein display system comprises an ice nuclein protein of Pseudomonas syringae Pseudomonas syringae The nucleotide sequence of the gene encoding the ice nuclein protein is shown as SEQ ID NO. 3; the expression cassette further comprises a SpaC truncated protein promoter, the SpaC truncated protein promoter comprises an L-arabinose inducible promoter; the L-arabinose inducible promoter comprises an AraC regulatory protein gene, an araBAD promoter gene and a ribosome binding site gene, and the nucleotide sequence of the L-arabinose inducible promoter is shown as SEQ ID NO.

4.

3. The expression cassette of claim 1, wherein, The expression cassette further comprises a green fluorescent protein gene and a promoter of the green fluorescent protein gene; the promoter of the green fluorescent protein gene comprises a constitutive promoter J23119.

4. The expression cassette of claim 1, wherein, The promoters of the antioxidant enzyme three-enzyme cascade system fusion protein and the cytokine fusion protein comprise a ykgMO promoter responsive to calprotectin; the nucleotide sequence of the ykgMO promoter responsive to calprotectin is shown in SEQ ID NO.

6.

5. A recombinant expression vector comprising the expression cassette of any one of claims 1-4.

6. A genetically engineered bacterium comprising the recombinant expression vector of claim 5.

7. The genetically engineered bacteria according to claim 6, characterized in that, The host bacterium of the genetically engineered bacterium comprises Escherichia coli Nissle 1917.

8. The method for constructing the genetically engineered bacteria according to claim 6 or 7, characterized in that, The method comprises the following steps: transforming the recombinant expression vector of claim 5 into a host bacterium competent cell to obtain the genetically engineered bacterium.

9. The method for culturing the genetically engineered bacteria according to claim 6 or 7, characterized by, The method comprises the following steps: Inoculating the genetically engineered bacterium into a seed culture medium for culture to obtain a genetically engineered bacterium seed liquid; Inoculating the genetically engineered bacterium seed liquid into a fermentation culture medium for fermentation culture to induce the expression of the SpaC truncated protein and obtain a genetically engineered bacterium fermentation product.

10. Use of the genetically engineered bacterium obtained by the method of claim 8 or the culture method of claim 9 in the preparation of a product for improving the adhesion and colonization ability of the host bacterium, the active oxygen scavenging ability, and / or the repair of the intestinal epithelial barrier function or in the preparation of a drug for targeted treatment of inflammatory bowel disease.

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