A probiotic ecn△lpp-a5 strain and a construction method and application thereof

Abstract

The application discloses a probiotic EcN△lpp-A5 strain and a construction method and application thereof, the probiotic EcN△lpp-A5 strain takes the probiotic Escherichia coli Nissle 1917 as a chassis, knocks out the outer membrane lipoprotein gene lpp, introduces a fusion protein formed by annexin V (Annexin V) and an Lpp signal peptide sequence and an OmpA anchoring domain sequence, and expresses the annexin V fusion protein on the surface of bacterial cells. The probiotic EcN△lpp-A5 constructed in the application has the characteristics of genetic stability and no exogenous resistance; the strain retains the original probiotic characteristics of EcN, realizes intestinal target colonization and long-time retention, and has the performance of continuously expressing and secreting exogenous recombinant proteins. The strain has strong intestinal targeting and high colonization ability, is safe, is suitable for oral administration, has a mature preparation process, and is convenient for large-scale production.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a probiotic strain EcN△lpp-A5, its construction method, and its application. Background Technology

[0002] The probiotic *E. coli* Nissle 1917 (EcN) has been approved in multiple countries for early intervention in ulcerative colitis (UC). Compared to traditional drugs, EcN offers advantages such as low production cost, in vivo proliferation, and ease of genetic modification. However, its poor intestinal colonization ability limits its clinical efficacy. Previous studies have often enhanced EcN's colonization ability through chemical modification or nanoparticle encapsulation to increase efficacy. However, as the bacteria proliferate, these modified compounds or nanomaterials are gradually diluted and cannot maintain their effects long-term. We believe that by leveraging EcN's ease of genetic modification and selecting promising and rational targets based on the biological mechanisms of inflammatory bowel disease, we can perform multifunctional targeted modification to enhance its therapeutic effects on colitis.

[0003] Intestinal epithelial cell death and apoptosis-mediated damage to the intestinal mucosal barrier plays a crucial role in the development and progression of ulcerative colitis (UC). Phosphatidylserine eversion is a recognized marker of early apoptosis, while Annexin V, due to its specific binding to phosphatidylserine, is widely used for in vitro apoptosis detection.

[0004] This invention presents a bold and original concept that can express Annexin V on the surface of the probiotic EcN through genetic modification, targeting phosphatidylserine on the surface of capillary endothelial cells in the intestine (especially the colon) and early apoptotic intestinal epithelial cells, thereby increasing the colonization of EcN at the enteritis site.

[0005] Currently, surface display of *E. coli* mainly relies on systems such as OmpA, Lpp-OmpA, and Autotransporter. The Lpp-OmpA chimeric system anchors target proteins to the outer membrane via the signal peptide and pre-nonapeptide of Lpp, while the transmembrane domain of OmpA provides surface exposure. It has been used for the presentation of antigens, enzymes, and peptides. The outer membrane lipoprotein Lpp, encoded by the lpp gene, is one of the most abundant outer membrane proteins in Gram-negative bacteria, and its peptidoglycan anchoring structure makes the outer membrane highly dense. Previous studies have shown that complete knockout of lpp can significantly increase outer membrane permeability and enhance the secretion level of exogenous proteins, but it also weakens the mechanical strength of the strain. A balance between stability and functional output needs to be achieved through surface display systems. This invention proposes to further modify *EcN* by displaying Annexin V on its surface to increase EcN intestinal colonization, knocking out the lipoprotein lpp gene that hinders protein secretion, thereby achieving a dual-functional design of "surface-targeted ligand + secreted therapeutic protein."

[0006] In summary, there is an urgent need in this field for an integrated technology solution based on EcN that combines enhanced secretion with surface targeting to address bottlenecks such as low secretion efficiency of exogenous proteins, lack of targeting in intestinal colonization, and insufficient production of functional proteins.

[0007] Currently, there is a lack of a probiotic strain EcN△lpp-A5-aTN, its construction method, and its application. Summary of the Invention

[0008] The purpose of this invention is to overcome the deficiencies in the prior art and provide a probiotic strain EcN△lpp-A5-aTN, its construction method, and its application.

[0009] The technical solutions adopted in this invention are as follows: Firstly, this application provides a probiotic strain EcN△lpp-A5-aTN. Secondly, this application provides a method for constructing the probiotic strain EcN△lpp-A5-aTN. Thirdly, this application provides an intermediate strain EcN△lpp-A5. Fourthly, this application provides a method for constructing the intermediate strain EcN△lpp-A5.

[0010] The first aspect of this application provides a probiotic strain EcN△lpp-A5, the preservation name of which is TPEC-LA034, and its classification name is Escherichia coli. Escherichia coli It is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, on September 26, 2025, with accession number CGMCC No. 36084.

[0011] Furthermore, using *Escherichia coli* Nissle 1917 as the substrate bacteria, the outer membrane lipoprotein coding gene *lpp* was completely knocked out using the CRISPR-Cas9 system, and an exogenous DNA fragment was integrated at the deletion site. The exogenous DNA fragment contains the coding sequence of the Annexin V fusion protein. This nucleotide sequence is composed of the coding sequence of the Lpp signal peptide, the coding sequence of the OmpA anchoring domain, and the coding sequence of Annexin V, which are linked sequentially from 5′ to 3′. Under the guidance of the Lpp signal peptide, the Annexin V fusion protein is secreted into the bacterial periplasmic space and then anchored to the outer membrane through the OmpA domain, realizing the outer membrane-anchored display of Annexin V on the bacterial surface.

[0012] Furthermore, the coding sequence of the Annexin V fusion protein is shown in SEQ ID NO:1, the coding sequence of the Lpp signal peptide is shown in SEQ ID NO:3, and the coding sequence of the OmpA anchoring domain is shown in SEQ ID NO:4.

[0013] The second aspect of this application provides a method for constructing the probiotic strain EcNΔlpp-A5, characterized by the following steps: (1) knocking out the lpp gene in the EcN genome using a CRISPR-Cas9 system, wherein the CRISPR-Cas9 system contains an sgRNA targeting lpp and a donor template; (2) inserting the coding sequence of Annexin V fusion protein into the lpp gene deletion site using a CRISPR-Cas9 system to obtain the EcNΔlpp-A5 strain.

[0014] Further, in step (1), the nucleotide sequence of sgRNA is shown in SEQ ID NO:5, and the donor template of sgRNA is from the genome of E. coli Nissle 1917, and the genome sequence number of E. coli Nissle 1917 is NCBI ReferenceSequence: NZ_CP007799.1; in step (2), the coding sequence of the Annexin V fusion protein is formed by sequentially linking the Lpp signal peptide coding sequence, the OmpA anchoring domain coding sequence and the Annexin V (ANXA5) coding sequence from 5′ to 3′.

[0015] The third aspect of this application provides the use of the probiotic strain EcN△lpp-A5 of claim 1 in the preparation of a probiotic carrier for enhancing targeted colonization of intestinal inflammatory sites and for expressing and secreting recombinant proteins.

[0016] Further, the probiotic carrier contains a nucleotide sequence encoding an anti-TNF-α nanobody αTN, as shown in SEQ ID NO:2; or the probiotic carrier contains a nucleotide sequence encoding a red fluorescent protein RFP, as shown in SEQ ID NO:6, and a promoter nucleotide sequence as shown in SEQ ID NO:7; or the probiotic carrier contains a nucleotide sequence encoding a tracer protein luxCDABE, as shown in SEQ ID NO:8.

[0017] Furthermore, targeted colonization is achieved through the specific binding of Annexin V to phosphatidylserine on the surface of capillary endothelial cells in the intestine (especially the colon) and to phosphatidylserine everted at sites of enteritis.

[0018] Furthermore, probiotic carriers are used to load and / or express therapeutic or tracer protein or nucleic acid drugs.

[0019] Furthermore, the therapeutic protein includes any one of anti-TNF-α nanobody, IL-10, IL-22, or antimicrobial peptide; the tracer protein is any one of red fluorescent protein RFP, EGFP, or luxCDABE.

[0020] Furthermore, the probiotic carrier is an oral preparation.

[0021] Beneficial effects: This invention exhibits strong targeting, high colonization ability, and efficient secretion of recombinant proteins, including anti-TNF-α nanobodies, red fluorescent protein RFP, and luxCDABE. It boasts high safety, is suitable for oral administration, and its preparation process is mature, facilitating large-scale production.

[0022] Compared with the prior art, the present invention has the following advantages: (1) Strong targeting and high colonization ability: By knocking out the lpp gene to enhance the permeability of the outer membrane, and combining Annexin V with the specific binding of phosphatidylserine on the everted enteritis site, the colonization efficiency of engineered bacteria EcN△lpp-A5-αTN in the intestine is significantly higher than that of wild-type EcN, thus achieving precise delivery.

[0023] (2) Efficient expression and secretion of recombinant proteins: Using the Lpp-OmpA display system, stable expression and secretion of recombinant proteins such as anti-TNF-α nanobody αTN, red fluorescent protein RFP, and tracer protein luxCDABE were achieved.

[0024] (3) High safety and suitable for oral administration: The engineered bacteria are constructed based on the probiotic EcN. No obvious toxic side effects were observed after continuous gavage, and no abnormal liver or kidney function or organ pathological changes were caused. It has good biocompatibility and clinical translation potential.

[0025] (4) The preparation process is mature and easy to scale up production: The construction method is based on CRISPR-Cas9 gene editing and conventional plasmid transformation technology. The operation process is clear, the genetic stability is good, and it is suitable for industrial fermentation preparation and has industrialization prospects.

[0026] (5) It has the characteristics of genetic stability and no exogenous resistance; the strain retains the original beneficial characteristics of EcN, while achieving intestinal targeted colonization and long-term retention, and has the ability to continuously express and secrete exogenous recombinant proteins. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 This diagram illustrates the binding of Annxin V of the present invention to intestinal epithelial cells. Figure 1 Figure A shows the effect of CCK-8 assay on the viability of CT26 cells as described in this invention; Figure 1 B is a graph showing the proportion of apoptosis induced by CHX and TNFα treatment detected by flow cytometry according to the present invention; Figure 1 Figure C shows the purification of Annexin V-RFP protein using the affinity chromatography method of this invention; Figure 1 D is a diagram showing the binding of Annexin V-RFP protein to normal cells and apoptotic cells as detected by immunofluorescence according to the present invention. Figure 1 E is a diagram showing the distribution of Annexin V in the colon tissue of normal and enteritis mice according to the present invention.

[0029] Figure 2 This is a diagram illustrating the construction and secretion efficiency verification of the EcN△lpp-A5 engineered strain of the present invention. Figure 2 A is a growth curve diagram of the EcN△lpp-A5 engineered strain and the wild strain of the present invention; Figure 2 B is a diagram showing the colony morphology observed in plate culture according to the present invention; Figure 2 C is a scanning electron microscope image of the size and morphology of bacteria observed according to the present invention; Figure 2 D is a graph showing the detection efficiency of red fluorescent protein (RFP) secretion in the culture medium supernatant of this invention.

[0030] Figure 3 This is a comparison of the therapeutic effects of the EcN△lpp-A5 engineered strain of the present invention with wild-type EcN. Figure 3 A represents the effect of the engineered EcN△lpp-A5 strain and wild-type EcN on colonic inflammation in mice with enteritis. Figure 3 B represents the effect of the engineered EcN△lpp-A5 strain and wild-type EcN on the feces of mice with enteritis. Figure 3 C represents the effect of the engineered EcN△lpp-A5 strain and wild-type EcN on the colon morphology of mice with enteritis.

[0031] Figure 4 This demonstrates the colonization ability of the EcN△lpp-A5 engineered bacteria of the present invention in the colon of healthy mice. Figure 4A represents the colonization level of wild-type EcN carrying RFP red fluorescent protein in the colon of healthy mice at different time points after administration of the present invention. Figure 4 B represents the colonization level of EcN△lpp-A5 engineered bacteria carrying RFP red fluorescent protein in the colon of healthy mice at different time points after administration of the present invention, observed by fluorescence microscopy.

[0032] Figure 5 This invention demonstrates the colonization ability of the EcN△lpp-A5 engineered bacteria in the colon of DSS enteritis mice. Figure 5 A represents the colonization level of wild-type EcN carrying RFP red fluorescent protein in the colon of mice with enteritis, observed by fluorescence microscopy at different time points after administration of the present invention. Figure 5 B represents the colonization level of EcN△lpp-A5 engineered bacteria carrying RFP red fluorescent protein in the colon of mice with enteritis, observed under a fluorescence microscope at different time points after administration of the present invention.

[0033] Figure 6 In vivo imaging of the EcN△lpp-A5 engineered strain of the present invention in the mouse colon. Figure 6 A represents the in vivo observation of the distribution of wild-type EcN and EcNΔlpp-A5 engineered bacteria carrying the luxCDABE fluorescent protein in healthy mice and mice with enteritis, respectively. Figure 6 B is a quantitative fluorescence distribution diagram of the strain of the present invention in healthy mice; Figure 6 C is a quantitative fluorescence distribution diagram of the strain of the present invention in mice with enteritis; Figure 6 DE is the method used in this invention to observe the distribution of EcN and EcNΔlpp-A5 engineered bacteria in various organs of healthy and enteritis mice after dissection. Figure 7 This serves as a verification of the construction of the EcN△lpp-A5-αTN engineered bacteria of this invention. Figure 7 A is a comparison of the growth curves of EcN△lpp-A5-αTN and EcN△lpp-A5-αTN in this invention; Figure 7 B is a comparison of the colony morphology of EcN△lpp-A5-αTN and EcN△lpp-A5-αTN in this invention; Figure 7 C is the verification of the αTN secretion expression effect of the EcN△lpp-A5-αTN engineered bacteria of this invention.

[0034] Figure 8 This is a schematic diagram showing the ANXA5 on the surface of the EcN△lpp-A5 of the present invention. The outer membrane lipoprotein encoding gene lpp was completely knocked out using the CRISPR-Cas9 system, and the exogenous DNA fragment Annexin V was integrated into the deletion site. Detailed Implementation

[0035] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as described in detail in the claims. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply.

[0036] The first aspect of this application provides a probiotic strain EcN△lpp-A5, whose preservation name is TPEC-LA034 and whose classification name is Escherichia coli. Escherichia coli It is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, on September 26, 2025, with accession number CGMCC No. 36084.

[0037] In some embodiments, using *Escherichia coli* Nissle 1917 as the substrate bacteria, the outer membrane lipoprotein coding gene *lpp* is completely knocked out using the CRISPR-Cas9 system, and an exogenous DNA fragment is integrated at the deletion site. The exogenous DNA fragment contains the coding sequence of the Annexin V fusion protein. This nucleotide sequence is composed of the coding sequence of the Lpp signal peptide, the coding sequence of the OmpA anchoring domain, and the coding sequence of Annexin V, which are linked sequentially from 5′ to 3′. Under the guidance of the Lpp signal peptide, the Annexin V fusion protein is secreted into the bacterial periplasmic space and then anchored to the outer membrane through the OmpA domain, realizing the outer membrane-anchored display of Annexin V on the bacterial surface.

[0038] In some embodiments, the coding sequence of the Annexin V fusion protein is shown in SEQ ID NO:1, the coding sequence of the Lpp signal peptide is shown in SEQ ID NO:3, and the coding sequence of the OmpA anchoring domain is shown in SEQ ID NO:4.

[0039] The second aspect of this application provides a method for constructing the probiotic strain EcN△lpp-A5, comprising the following steps: (1) knocking out the lpp gene in the EcN genome using a CRISPR-Cas9 system, wherein the CRISPR-Cas9 system contains an sgRNA targeting lpp and a donor template; sgRNA sequence: 5'-TAACCGTCGCTGGACAA-3', and the nucleotide sequence of the sgRNA is shown in SEQ ID NO:5. The donor template for sgRNA was derived from the genome of E. coli Nissle 1917, and the NCBI Reference Sequence number for the E. coli Nissle 1917 genome is NZ_CP007799.1. (2) The coding sequence for Annexin V fusion protein was inserted into the lpp gene deletion site using the CRISPR-Cas9 system to obtain the EcNΔlpp-A5 strain.

[0040] In some embodiments, in step (2), the coding sequence of the Annexin V fusion protein is formed by linking the Lpp signal peptide coding sequence, the OmpA anchoring domain coding sequence and the Annexin V coding sequence from 5′→3′.

[0041] The third aspect of this application provides the use of the probiotic strain EcN△lpp-A5 in the preparation of a probiotic vector for enhancing targeted colonization of intestinal inflammatory sites and in the expression and secretion of recombinant proteins.

[0042] In some embodiments, the probiotic carrier comprises a nucleotide sequence encoding an anti-TNF-α nanobody αTN, as shown in SEQ ID NO:2; or the probiotic carrier comprises a nucleotide sequence encoding a red fluorescent protein RFP, as shown in SEQ ID NO:6, and a promoter nucleotide sequence as shown in SEQ ID NO:7; or the probiotic carrier comprises a nucleotide sequence encoding a tracer protein luxCDABE, as shown in SEQ ID NO:8.

[0043] In some embodiments, targeted colonization is achieved by Annexin V specifically binding to phosphatidylserine on the surface of capillary endothelial cells in the intestine (especially the colon) and to phosphatidylserine everted at sites of enteritis.

[0044] In some embodiments, the probiotic carrier is used to load and / or express therapeutic or tracer protein or nucleic acid drugs.

[0045] In some embodiments, the therapeutic protein is an anti-TNF-α nanobody.

[0046] In some embodiments, the therapeutic protein is IL-10; In some embodiments, the therapeutic protein is IL-22; In some embodiments, the therapeutic protein is an antimicrobial peptide; In some embodiments, the tracer protein is the red fluorescent protein RFP.

[0047] In some embodiments, the tracer protein EGFP.

[0048] In some embodiments, the tracer protein is luxCDABE.

[0049] In some embodiments, the probiotic carrier is an oral formulation. Example 1

[0050] This invention relates to a probiotic strain EcN△lpp-A5, whose preservation name is TPEC-LA034 and its classification name is Escherichia coli. Escherichia coli It is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, on September 26, 2025, with accession number CGMCC No. 36084.

[0051] Using Escherichia coli Nissle 1917 as the substrate bacteria, the outer membrane lipoprotein coding gene lpp was completely knocked out using the CRISPR-Cas9 system, and an exogenous DNA fragment was integrated at the deletion site. The exogenous DNA fragment contained the coding sequence of the Annexin V fusion protein. The nucleotide sequence was composed of the coding sequence of the Lpp signal peptide, the coding sequence of the OmpA anchoring domain, and the coding sequence of Annexin V, which were linked from 5′ to 3′. Under the guidance of the Lpp signal peptide, the Annexin V fusion protein was secreted into the bacterial periplasmic space and then anchored to the outer membrane through the OmpA domain, realizing the outer membrane-anchored display of Annexin V on the bacterial surface.

[0052] The coding sequence of the Annexin V fusion protein is shown in SEQ ID NO:1, the coding sequence of the Lpp signal peptide is shown in SEQ ID NO:3, and the coding sequence of the OmpA anchoring domain is shown in SEQ ID NO:4. Example 2

[0053] The present invention discloses a method for constructing the probiotic strain EcN△lpp-A5, comprising the following steps: (1) knocking out the lpp gene in the EcN genome using a CRISPR-Cas9 system, wherein the CRISPR-Cas9 system contains an sgRNA targeting lpp and a donor template; sgRNA sequence: 5'-TAACCGTCGCTGGACAA-3', the nucleotide sequence of the sgRNA is shown in SEQ ID NO:5, and the donor template of the sgRNA is derived from the genome of E. coli Nissle 1917, the NCBI Reference Sequence number of the E. coli Nissle 1917 genome is NZ_CP007799.1. Method for constructing EcN△lpp-A5 using the CRISPR-Cas9 system: the EcN-pCas9 strain is cultured in LB medium until the OD600 value is 0.2, and 10 mM L-arabinose is added to induce Cas9 protein expression. After culturing until the OD600 reached 0.7, bacterial cells were collected to prepare EcN-pCas9 competent cells. Subsequently, 100 ng of pTargetT plasmid (containing an sgRNA sequence targeting the lpp gene (5'-TAACCGTCGCTGGACAA-3') and an LPP-OmpA-ANXA5 fusion fragment) was introduced into the competent cells via electroporation. Transformed cells were incubated at 30°C for 1 hour and plated on LB agar plates containing 30 μg / mL kanamycin and 50 μg / mL ampicillin. After overnight incubation at 30°C, transformants were screened.

[0054] (2) The coding sequence of Annexin V fusion protein was inserted into the lpp gene deletion site using the CRISPR-Cas9 system to obtain the EcNΔlpp-A5 strain. The coding sequence of Annexin V fusion protein is composed of the Lpp signal peptide coding sequence, the OmpA anchoring domain coding sequence and the Annexin V coding sequence linked together from 5′→3′. Example 3

[0055] The present invention relates to the application of a probiotic strain EcN△lpp-A5 in the preparation of a probiotic vector for enhancing intestinal targeted colonization and expressing and secreting recombinant proteins. Example 4

[0056] The difference between Example 4 and Example 3 is that the probiotic carrier contains a nucleotide sequence encoding the anti-TNF-α nanobody αTN, as shown in SEQ ID NO:2. Targeted colonization is achieved through the specific binding of Annexin V to phosphatidylserine residues on the everted enteritis site. The probiotic carrier is an oral formulation. Example 5

[0057] The difference between Example 5 and Example 3 lies in the application of the probiotic strain EcN△lpp-A5 of the present invention in the preparation of a probiotic carrier for enhancing intestinal targeted colonization and expressing and secreting recombinant proteins. Targeted colonization is achieved through the specific binding of Annexin V to phosphatidylserine on the surface of intestinal (especially colonic) capillary endothelial cells and to phosphatidylserine everted at sites of enteritis. The probiotic carrier is used to load and / or express therapeutic or tracer proteins or nucleic acid drugs. Example 6

[0058] The difference between Example 6 and Example 5 is that the probiotic carrier is used to load and / or express a therapeutic protein, which is IL-10. Example 7

[0059] The difference between Example 7 and Example 5 is that the probiotic carrier is used to load and express a therapeutic protein, which is IL-22. Example 8

[0060] The difference between Example 8 and Example 5 is that the probiotic carrier is used to load and express a therapeutic protein, which is an antimicrobial peptide. Example 9

[0061] The difference between Example 9 and Example 5 is that the probiotic carrier contains a nucleotide sequence encoding the red fluorescent protein RFP, as shown in SEQ ID NO:6, and a promoter nucleotide sequence as shown in SEQ ID NO:7. Example 10

[0062] The difference between Example 10 and Example 5 is that the tracer protein EGFP is used. Example 11

[0063] The difference between Example 11 and Example 5 is that the probiotic carrier contains a nucleotide sequence encoding the tracer protein luxCDABE, as shown in SEQ ID NO:8. Example 12

[0064] This invention relates to the application of a probiotic strain EcN△lpp-A5 in the preparation of a probiotic carrier for enhancing intestinal targeted colonization and in the expression and secretion of recombinant proteins. The probiotic carrier contains a nucleotide sequence encoding a red fluorescent protein (RFP), as shown in SEQ ID NO:5. The probiotic carrier is an oral formulation. The probiotic strain EcN△lpp-A5 expressing the RFP can be used to trace the metabolism and distribution of the probiotic EcN△lpp-A5 in vivo. Example 13

[0065] The difference between Example 13 and Example 12 is that Example 13 involves the application of a probiotic strain EcN△lpp-A5 of the present invention in the preparation of a probiotic vector for enhancing intestinal targeted colonization and in the expression and secretion of recombinant proteins. The probiotic vector is used to load and / or express a tracer protein, which is the fluorescent protein EGFP. The probiotic strain EcN△lpp-A5 expressing the fluorescent protein EGFP can be used to trace the metabolism and distribution of the probiotic EcN△lpp-A5 in vivo. Example 14

[0066] The difference between Example 14 and Example 12 is that Example 14 involves the application of a probiotic strain EcN△lpp-A5 of the present invention in the preparation of a probiotic carrier for enhancing intestinal targeted colonization and in the expression and secretion of recombinant proteins. The probiotic carrier is used to load and / or express a tracer protein, luxCDABE. The probiotic strain EcN△lpp-A5 expressing the fluorescent protein luxCDABE can be used to trace the metabolism and distribution of the probiotic EcN△lpp-A5 in vivo. Example 15

[0067] This invention aims to modify EcN in two aspects: increasing secretory function and intestinal colonization ability, resulting in EcN△lpp-A5. Specifically, knocking out the major lipoprotein lpp in the outer membrane of *E. coli* helps to increase its protein secretion function. Furthermore, utilizing the increased phosphatidylserine eversion characteristic of enteritis tissue, this invention constructed an engineered bacterium, EcN△lpp-A5, expressing Annexin V protein that specifically binds to phosphatidylserine, to further enhance its colonization ability in enteritis tissue. Subsequently, using EcN△lpp-A5 as the substrate bacterium, this invention constructed an engineered bacterium, EcN△lpp-A5-αTN, capable of successfully secreting and expressing the anti-TNF-α nanobody αTN, as well as an engineered bacterium expressing RFP and luxCDABE.

[0068] 1. Experimental Materials 1.1 Laboratory animals, bacterial strains, and plasmids The experimental animals and strains are the same as in 2.1.1.

[0069] 1.2 The experimental apparatus is shown in Table 1: Table 1

[0070] Other instruments are the same as in 2.1.2.

[0071] 1.3 Experimental materials and reagents are shown in Table 2: Table 2

[0072] 1.4 Preparation of experimental solutions 100 mg / mL Ampicillin Sodium Stock Solution: Accurately weigh 1 g of ampicillin sodium powder, add it to a centrifuge tube containing 10 mL of ddH2O, vortex to dissolve it completely, filter it through a 0.22 μm filter membrane for sterilization, and then aliquot it into 1.5 mL centrifuge tubes (1 mL / tube) and store it at -20℃ for long-term storage.

[0073] 40 mg / mL kanamycin sulfate stock solution: Accurately weigh 400 mg kanamycin sulfate powder, add it to a centrifuge tube containing 10 mL ddH2O, vortex to dissolve it completely, filter it through a 0.22 μm filter membrane for sterilization, and then aliquot it into 1.5 mL centrifuge tubes (1 mL / tube) and store it at -20℃ for long-term storage.

[0074] 50×TAE buffer: Weigh 242 g Tris and 37.2 g Na2EDTA.2H2O, add 800 mL ddH2O to dissolve them, then add 57.1 mL glacial acetic acid, and finally bring the volume to 1 L with ddH2O. Dilute to 1×TAE buffer before use.

[0075] 1% Agarose Gel: Taking the preparation of 30 mL agarose solution as an example, weigh 0.3 g of agarose and add it to a conical flask containing 30 mL of 1×TAE. Place the flask in a microwave oven and heat on high until boiling. After each boiling, remove the conical flask, gently shake, and repeat 3 times until the solution is clear and transparent. After slightly cooling, add 3 μL of Gel-red nucleic acid dye, shake to mix, and pour the agarose solution into a gel casting mold with combs inserted. After about 40 minutes of complete solidification, nucleic acid electrophoresis can be performed.

[0076] Coomassie Brilliant Blue staining solution: Measure 500 mL of anhydrous ethanol and 100 mL of glacial acetic acid, add them to a beaker, then add 2.5 g of Coomassie Brilliant Blue R250 and ddH2O to make up to 1 L. Transfer the solution to a special bottle (with a rotor) and stir overnight.

[0077] Decolorizing solution: Measure 300 mL of anhydrous ethanol, 100 mL of glacial acetic acid and 600 mL of ddH2O, and mix vigorously.

[0078] Intestinal tissue digestion solution: Add 150 mg collagenase I (1 mg / mL) to 150 mL RPMI-1640 culture medium, vortex to mix, and store at -20℃ for later use.

[0079] 100% Percoll: Mix Percoll stock solution and 10xPBS buffer at a ratio of 9:1 and shake vigorously to mix. Example 16

[0080] 2 Experimental Methods 2.1 A5-RFP protein purification Equilibrate the column: Equilibrate the nickel column with 5-10 column volumes of binding buffer.

[0081] Sample loading: The cell lysate containing the protein is slowly loaded onto the nickel column, so that the target protein A5-RFP in the solution binds to the nickel ions on the nickel column.

[0082] Washing: Wash the column with 10-25 column volumes of washing buffer to remove non-specifically bound proteins other than A5-RFP.

[0083] Elution: Elute proteins using imidazole elution buffer at different concentration gradients of 50-500 mM. A5-RFP is red, and the eluted protein will be visible as red eluent.

[0084] Dialysis: The collected eluent containing A5-RFP was placed in a dialysis bag for dialysis. A stir bar was added, and the mixture was dialyzed overnight at 4°C to purify the protein and remove small molecule impurities such as imidazole.

[0085] 2.2 Polymerase chain reaction (PCR) The reaction system was prepared in 0.2 mL PCR tubes. The specific PCR amplification reaction system is shown in Table 3 below.

[0086] Table 3

[0087] After gently mixing the above reaction solution with a pipette, briefly insulate it and perform PCR amplification. The specific PCR reaction program settings are shown in Table 4 below.

[0088] Table 4 After the PCR reaction is complete, take an appropriate amount of PCR product and perform electrophoresis on a 1% agarose gel at 140 V for 30-40 min, and then detect the size of the PCR product bands.

[0089] 2.3 Agarose gel electrophoresis and gel recovery Separating target DNA fragments by agarose gel electrophoresis: Prepare an agarose gel of appropriate concentration (0.8%-2%) according to the size of the target DNA fragment. When the agarose solution cools to about 60°C, add the corresponding volume of nucleic acid dye SYBR Green at a ratio of 1:10000, mix well, pour into a gel mold, insert a comb, and let stand at room temperature for 30 min until the agarose solution is completely solidified. Then, mix the plasmid digestion product or PCR product with 6× DNA loading buffer and add it to the sample wells. Electrophoresis is performed at 110 V for 30-50 min. Stop electrophoresis when bromophenol blue migrates to 2 / 3 of the gel.

[0090] Gel cutting: Use a UV gel imaging system to locate the target DNA band. Carefully cut the gel block containing the target DNA fragment with a clean scalpel, removing as much excess gel as possible. Place the gel block in a 2 mL EP tube and weigh it. Perform subsequent gel recovery operations according to the instructions of the Cisco Gel Recovery / PCR Product Purification Kit (AE0101).

[0091] Gel dissolution: Add 3 times the volume of sol / binding solution DB, place in a 56℃ water bath for about 10 minutes to dissolve the gel. During this period, invert the EP tube every 3 minutes to help the gel dissolve quickly.

[0092] DNA adsorption: After dissolving the gel in hot solution, let it stand at room temperature for 2-5 min, then centrifuge briefly to collect the droplets on the tube wall. Add the solution to the EC adsorption column, let it stand at room temperature for 1 min to allow it to fully bind to the adsorption column, then centrifuge at 12000 rpm for 1 min and discard the waste liquid in the collection tube.

[0093] DNA washing: Add 600 μL of wash buffer (with anhydrous ethanol added) to the adsorption column, centrifuge at 12000 rpm for 30 s, discard the waste liquid, and repeat the washing once. Centrifuge the empty column at 12000 rpm for 2 min to completely remove residual wash buffer.

[0094] DNA elution: Place the EC adsorption column into a new 1.5 mL EP tube, add 30-120 μL LEPC water to the center of the adsorption membrane, and incubate at room temperature for 3 min to allow the DNA to fully dissolve in the water. Centrifuge at 12000 rpm for 1 min to collect the eluent containing DNA. Measure the concentration and purity of DNA at 260 nm and 280 nm wavelengths using a NanoDrop One spectrophotometer. The A260 / A280 ratio of pure DNA should be between 1.8 and 2.0. Store at -20℃ for later use.

[0095] 2.4 Seamless DNA Cloning Vector linearization: The vector was linearized by restriction endonuclease digestion or reverse PCR amplification, and the digested or PCR products were purified using a gel extraction kit to determine the concentration of the linearized vector.

[0096] Insert fragment amplification: Homologous sequences (homologous arms) from both ends of the linearized vector were introduced into the 5' end of the forward and reverse amplification primers for the target gene. The target fragment was then amplified by PCR using high-fidelity polymerase 2× Phanta Flash Master Mix, resulting in the amplified insert fragment carrying sequences at both the 5' and 3' ends that are completely identical to those at the ends of the linearized vector. The PCR product was purified using a gel extraction kit, and the insert fragment concentration was determined.

[0097] Recombination reaction: The recombination cloning reaction was carried out according to the instructions of the CloneUFO One Step Cloning kit. The specific recombination cloning reaction system is shown in Table 5 below.

[0098] Table 5

[0099] After gently mixing the above reaction solution by pipetting, centrifuge briefly and incubate at 37°C for 30 min to complete the recombination reaction. The recombinant product can be used immediately for conversion or temporarily stored at -20°C and thawed for conversion when needed.

[0100] 2.5 Plasmid Extraction Bacterial culture: Select a single colony of *E. coli* containing the target plasmid, inoculate it into LB liquid medium containing the corresponding antibiotic, and incubate it on a shaker at 37°C and 200 rpm for 16-18 h until the bacterial culture reaches the plateau growth phase. Perform plasmid extraction according to the instructions of Kangwei Century EndoFree Plasmid Midi Kit (CW2105).

[0101] Bacterial cell collection: Take 10-15 mL of overnight cultured Escherichia coli, centrifuge at 13000 rpm for 1 min, discard as much supernatant as possible, and collect the bacterial cell precipitate.

[0102] Bacterial resuspension: Add 500 μL of Buffer P1 to the centrifuge tube containing the bacterial pellet, mix thoroughly with a vortex mixer and pipette to fully suspend the bacterial pellet.

[0103] Cell lysis: Add 500 μL of Buffer P2 to the centrifuge tube, gently invert the tube 8-11 times, and incubate at room temperature for 5 minutes to lyse the bacterial culture until it becomes a homogeneous, clear, and viscous solution. Avoid vigorous shaking, as this can lead to genomic DNA contamination.

[0104] Column equilibration: Add 200 μL of Buffer PS to the adsorption column (Spin Columns DL) that has been placed in the collection tube, centrifuge at 13000 rpm for 2 min, discard the waste liquid in the collection tube, and keep the adsorption column for subsequent DNA adsorption.

[0105] Neutralization and renaturation: Add 500 μL of Buffer E3 to the cell lysis buffer, and immediately invert the container 8-11 times until a white, bean curd-like precipitate appears. The number of inversions can be adjusted as needed. Let stand at room temperature for 5 min. Centrifuge at 13000 rpm for 5 min, and transfer the supernatant to an Endo-Remover FM filter column. Centrifuge at 13000 rpm for 1 min, and filter twice. Collect the filtrates from both filtrations into the same centrifuge tube.

[0106] Plasmid extraction: Add 450 μL of isopropanol to the centrifuge tube containing the filtrate, invert and mix well, add this mixture to the adsorption column that has been equilibrated in step 5, centrifuge at 13000 rpm for 1 min, and load the mixture onto the column in multiple portions.

[0107] Plasmid washing: Add 750 μL of washing buffer PW to the adsorption column, wash at 13000 rpm for 1 min, discard the waste liquid in the collection tube, put the adsorption column back into the collection tube, and air-displace at 13000 rpm for 1 min to remove residual ethanol in the adsorption column.

[0108] Plasmid elution: Place the adsorption column containing the plasmid in a new centrifuge tube, add approximately 100 μL of preheated deionized water (70°C) to the center of the adsorption membrane, let stand at room temperature for 2-6 min to fully dissolve the plasmid, and centrifuge at 13000 rpm for 2 min to collect the plasmid. Determine the plasmid concentration and store at -20°C for later use.

[0109] 2.6 Preparation of competent Escherichia coli cells Strain activation: Take a small amount of the strain stored at -80℃ and streak it on an LB agar plate. Incubate it upside down in a 37℃ incubator for 14-16 h. Pick a single colony and inoculate it into 2 mL of LB liquid medium. Incubate at 37℃ and 200 rpm for about 13 h.

[0110] Large-scale culture: Transfer 500 μL of the activated bacterial culture to 50 mL of LB medium (1% inoculum). Incubate at 37°C and 200 rpm for 2-3 h, monitoring carefully until E. coli reaches the logarithmic growth phase, at which point the OD of the bacterial culture will be measured. 600 It should reach 0.4-0.6.

[0111] Preparation of competent cells: Transfer the bacterial culture to a pre-chilled 50 mL sterile centrifuge tube, cool on ice for 10 min, centrifuge at 4°C and 5000 rpm for 7 min, and discard the supernatant. Add 25 mL of pre-chilled 0.1 M CaCl2 solution to the centrifuge tube, gently pipette to mix, and suspend the bacterial precipitate. Incubate on ice for 20 min.

[0112] Competent cell preservation: Centrifuge at 5000 rpm for 5 min at 4℃, discard the supernatant, add 5 mL of pre-chilled 0.1 M CaCl2 solution (containing 15% glycerol), and gently pipette to mix the cells. This yields competent cells. Immediately aliquot 100 μL into 1.5 mL sterile centrifuge tubes on ice, flash-freeze in liquid nitrogen, and store at -80℃ for later use. If used directly for transformation experiments, glycerol can be omitted when resuspending the cells.

[0113] 2.7 Chemical transformation method of Escherichia coli Take 100 μL of DH5α competent cells thawed on ice, add an appropriate amount of ligation product (not exceeding 1 / 10 of the competent cell volume), gently rotate and mix with a pipette tip, and let stand on ice for 30 min.

[0114] Place in a 42℃ water bath for 45-90 seconds for heat shock, then immediately place on ice for 2-5 minutes.

[0115] Add 700 μL of antibiotic-free LB liquid medium and incubate at 37°C and 200 rpm for 1 h to restore bacterial resistance.

[0116] Centrifuge at 5000 rpm for 5 min, discard 700 μL of culture medium supernatant, gently mix the bacterial cells with the remaining 100-150 μL of culture medium, spread all the bacterial suspension evenly on LB solid culture plates containing the corresponding antibiotics, and incubate upside down at 37 ℃ for 14-16 h.

[0117] 2.8 EcN Growth Curve Determination Bacterial inoculation and culture preparation: Pick a single colony of *E. coli* from a fresh LB solid culture plate and inoculate it into 2 mL of LB liquid medium. Incubate at 37°C and 200 rpm until it reaches the logarithmic growth phase. Dilute with fresh LB liquid medium to the initial OD value. 600 The value is 0.05-0.1. This step is performed entirely in a clean bench to avoid contamination by other microorganisms.

[0118] Microplate loading: In a clean bench, add 200 μL of diluted bacterial culture to each well of a 96-well microplate. Set up 3-5 replicate wells to reduce error. Simultaneously, add 200 μL of LB broth without bacterial culture to a few wells as blank controls. Seal the microplate with sealing film to minimize liquid evaporation during incubation. Take care to avoid generating air bubbles during loading, as this may affect subsequent detection results.

[0119] Microplate reader setup and culture detection: Place the sealed microplate into the microplate reader and set the parameters. The culture temperature is set to 37℃. Automatic plate shaking for 1 minute every 15 minutes is enabled to prevent bacterial sedimentation and ensure uniform mixing of the bacterial solution. Then, measure the absorbance of each well at 600 nm wavelength. The microplate reader automatically performs continuous monitoring and records the OD value of each well at each time point. 600 The detection period lasts 20-24 hours. Throughout the detection process, try to keep the environment around the microplate reader stable and avoid vibration and temperature fluctuations.

[0120] Data processing and growth curve plotting: After the assay, the data is exported from the microplate reader. First, the OD of all replicate wells at each time point is calculated. 600 Average value, minus the OD of the blank control well at the corresponding time point. 600 The OD value was adjusted to eliminate the influence of the light absorption of the culture medium itself. The corrected OD value was calculated with incubation time as the x-axis. 600 The average value was used as the ordinate to plot the growth curve of E. coli. Example 17

[0121] Detection of exogenous proteins secreted by EcN Escherichia coli expansion culture: Escherichia coli was inoculated into LB medium and cultured at 37°C and 200 rpm until the Escherichia coli reached the logarithmic growth phase.

[0122] Supernatant collection: Transfer the bacterial culture to a centrifuge tube, centrifuge at 4℃ and 6000-8000 rpm for 10-15 min, and collect the supernatant. The rotation speed should not be too high to avoid bacterial cell rupture and affecting the accuracy of the experiment.

[0123] Protein precipitation: Take an appropriate volume of supernatant, add 1 / 4 volume of methanol, mix vigorously for about 15 seconds, add 1 / 4 volume of chloroform, mix again, add 3 / 4 volume of deionized water, shake vigorously, and centrifuge at 4℃ and 12000 rpm for 5 minutes. At this time, the solution is divided into three layers, and the protein precipitate is located at the interface between the middle layer and the lower layer.

[0124] Precipitation washing: Carefully discard the supernatant and subnatant, retaining the protein precipitate. Add 1 mL of methanol to resuspend the precipitate, centrifuge at 11,000 rpm for 5 min at 4 °C, and discard the supernatant. Repeat the washing process once, and then incubate the precipitate at 50 °C for 5 min to allow it to dry.

[0125] Protein dissolution: Add an appropriate amount of protein dissolution buffer, gently pipette to dissolve the precipitate, add 5× loading buffer, denature at 100℃ for 10 min, and then store the protein sample at -80℃ for later use. Example 18

[0126] EcN electroporation conversion Preparation of EcN electrocompetent cells: The initial culture method for EcN is the same as that for E. coli. After centrifuging to obtain EcN cells, add an equal volume of pre-chilled 10% glycerol to gently suspend the cells. Centrifuge at 5000 rpm for 6 minutes at 4°C, and discard the supernatant. Wash once more with 10% glycerol. Finally, add 2 mL of pre-chilled 10% glycerol to suspend the cells. This yields EcN electrocompetent cells, which should be prepared and used immediately.

[0127] Electroporation: Take 100 μL of EcN for electroporation of competent cells, add 1-10 μL of plasmid (total plasmid content: 500-1000 ng). Gently mix and transfer to a pre-chilled 0.2 cm electroporation cuvette. Incubate on ice for 5-20 min. Wipe the cuvette dry and place it in an electroporator. Electroporate at 2.5 kV and 200 ohms. Immediately after electroporation, add 700 μL of antibiotic-free LB medium. Transfer the bacterial culture to a centrifuge tube using a pipette and incubate at 37°C and 150 rpm for 1 hour.

[0128] Plate screening: Centrifuge the revived bacterial cells at 5000 rpm for 6 min, discard the supernatant, and take 100-150 μL of bacterial solution to spread on LB solid plates containing the corresponding antibiotics. Incubate upside down at 37°C overnight. Example 19

[0129] Live imaging of small animals Mice were anesthetized in a gas anesthesia device containing isoflurane, and became comatose within 1-3 minutes. The anesthetized animals were carefully placed on the sample stage of the imaging system, and their positions were adjusted so that their heads and noses were aligned with the internal anesthesia device to prevent awakening during imaging. Images were then taken to obtain images of LUX bioluminescent EcN and EcN-engineered bacteria within the mice.

[0130] Colonization of EcN engineered bacteria in the colon After mice were administered EcN-modified bacteria containing red fluorescent protein via gavage, colon tissue samples were collected from the mice at five time points: 6 h, 12 h, 24 h, 48 h, and 72 h.

[0131] Tissue fixation: The colon tissue was quickly placed in 4% paraformaldehyde fixative and fixed at 4°C for 2-4 hours to stabilize the morphology and structure of the colon tissue.

[0132] Tissue dehydration: Discard paraformaldehyde, wash the fixed tissue three times in PBS to remove excess fixative, then transfer the tissue to 30% sucrose solution and soak overnight at 4°C to allow the tissue to settle.

[0133] Embedding: The dehydrated colon tissue is taken out and placed in a disposable embedding mold. An appropriate amount of OCT embedding agent is added so that the colon tissue is located in the center and completely embedded. The colon tissue forms a ring shape. Then it is quickly placed in liquid nitrogen for flash freezing, so that the OCT embedding agent can be quickly solidified and the colon can be fixed.

[0134] Sectioning: The embedded colon tissue block was mounted on a cryostat, the microtome parameters were adjusted, and the section thickness was set to 10 μm. The excised colon tissue was arc-shaped and adhered to a glass slide.

[0135] Section pretreatment: First, allow the frozen sections to thaw at room temperature for 5-10 minutes. Then, slowly rinse the sections with PBS buffer to remove the OCT embedding agent from the surface of the sections. Wash the sections three times, five minutes each time, being gentle to prevent tissue detachment.

[0136] Nuclear staining and mounting: Mount the slides using anti-fluorescence quenching mounting medium containing DAPI, cover with a coverslip, and fix with nail polish to prevent slippage.

[0137] Example 20

[0138] EcN scanning electron microscope image The EcN strain, stored at -80°C, was removed from the refrigerator and inoculated into 1 mL of LB liquid medium using a sterile pipette tip under laminar flow hood conditions. It was then placed in a 37°C shaker for overnight growth.

[0139] Take 200 μL of EcN cultured overnight and transfer it to 20 mL of fresh LB liquid medium. Continue to culture at 37℃ and 250 rpm for 4-7 h with shaking to expand the culture.

[0140] Immobilize the bacteria: Collect 20 mL of the cultured EcN bacterial solution into a 50 mL centrifuge tube, centrifuge at 5000 rpm for 8 minutes at 4℃, discard the supernatant, and collect the bacterial pellet.

[0141] Gently resuspend the EcN cell pellet in 10 mL of PBS, and repeat the washing steps twice to remove culture medium impurities from the surface of the EcN.

[0142] Add 3 mL of 2.5% (v / v) glutaraldehyde fixative to the washed EcN cell precipitate, gently pipette the EcN cells to disperse them evenly, and fix them in a 4°C refrigerator for 2 hours.

[0143] After fixation, centrifuge at 5000 rpm for 10 minutes, discard the fixative, and then wash the EcN cell precipitate three times with 2 mL PBS for 10 minutes each time to remove unreacted glutaraldehyde.

[0144] Add 8 mL of 30% (v / v) ethanol solution to the EcN cell precipitate to suspend the cells, and treat at room temperature for 12 min for preliminary dehydration.

[0145] After the initial dehydration, the mixture was centrifuged at 5000 rpm for 10 min, and then EcN was dehydrated sequentially with 50%-100% ethanol solutions, with each concentration treated for 12 min.

[0146] Finally, dehydrate with 100% ethanol 2-3 times, 12 minutes each time, to ensure that the water in the EcN bacteria is fully replaced.

[0147] The dehydrated EcN cell suspension was transferred to a sample basket and then placed in the sample chamber of a critical point dryer.

[0148] Liquid carbon dioxide is slowly and continuously introduced into the critical point dryer to replace the ethanol in the sample until the ethanol in the sample is completely replaced. The temperature of the sample chamber is slowly increased until the liquid carbon dioxide reaches above its critical temperature, at which point it transitions to a supercritical state. The pressure is then slowly released, allowing the supercritical carbon dioxide to escape from the sample chamber, thus completing the sample drying process.

[0149] Carefully remove the dried sample from the sample basket with tweezers and place it on the sample stage coated with conductive adhesive, ensuring the sample adheres firmly to the conductive adhesive. Then, gently disperse the EcN bacteria using a toothpick.

[0150] The sample stage with EcN bacteria attached was placed in an ion sputtering coating instrument, then a vacuum was drawn, and finally argon gas was introduced into the coating instrument.

[0151] Then the ion sputtering power supply is turned on. Under the action of the electric field, argon ions are accelerated to bombard the gold target. Gold atoms are sputtered from the target surface and deposited on the sample surface to form a uniform gold film. Example 21

[0152] Annxin V binding to intestinal epithelial cells During cell death, phosphatidylserine undergoes significant eversion. In this invention, the binding of Annexin V to normal colon cells and apoptotic colon cells was investigated. First, CT26 cells were treated with 25 μg / ml CHX and 20 ng / ml TNFα (tumor necrosis factor α) for 24 h to simulate cell death under conditions of in vivo enteritis. CCK8 assay results showed a significant decrease in cell viability (…). Figure 1 A), flow cytometry analysis showed an increased proportion of apoptotic cells. Figure 1 B), indicating that the present invention successfully induced apoptosis. Next, the present invention purified Annexin V-RFP (Annexin V fusion red fluorescent protein) protein. High-purity Annexin V-RFP protein was successfully obtained by affinity chromatography under 50 mM imidazole conditions. Figure 1 C). Annexin V-RFP protein was incubated with normal cells and apoptotic cells, respectively. Immunofluorescence assays showed that during apoptosis, Annexin V binding on the cell membrane increased, and red fluorescence was significantly enhanced. Figure 1 D). Animal-level results showed that Annexin V signaling in the colon of mice with enteritis was significantly enhanced (D). Figure 1 These results indicate that enhancing the colonization ability of EcN at enteritis sites by expressing Annxin V on the surface of EcN is highly feasible.

[0153] Figure 1 This diagram illustrates the binding of Annxin V of the present invention to intestinal epithelial cells. Figure 1 Figure A shows the effect of CCK-8 assay on the viability of CT26 cells as described in this invention; Figure 1 B is a graph showing the proportion of apoptosis induced by CHX and TNFα treatment detected by flow cytometry according to the present invention; Figure 1 Figure C shows the purification of Annexin V-RFP protein using the affinity chromatography method of this invention; Figure 1 D is a diagram showing the binding of Annexin V-RFP protein to normal cells and apoptotic cells as detected by immunofluorescence according to the present invention. Figure 1 E is a diagram showing the distribution of Annexin V in the colon tissue of normal and enteritis mice according to the present invention.

[0154] 3.2 Construction of the EcN△lpp-A5 engineered strain Genetically engineering bacteria to enhance the secretion levels of exogenous therapeutic proteins and their colonization ability in the gut is an important direction in EcN modification. LPP is a major lipoprotein component of the E. coli outer membrane. Multiple studies have shown that knocking out the lpp gene can enhance bacterial outer membrane permeability, thereby facilitating the secretion of exogenous proteins carried by engineered bacteria. Therefore, this invention first successfully knocked out the lpp gene in EcN, constructing engineered bacteria EcNΔlpp. Previous studies in our research group have shown that phosphatidylserine eversion is increased in the enteritis site, and Annexin V has been widely reported to bind to phosphatidylserine in the presence of calcium ions. The LPP-OmpA fusion display system is a technique for displaying recombinant proteins on the surface of E. coli. In this system, the signal sequence and the first nine amino acids of Lpp can target and anchor the fusion protein to the outer membrane, while OmpA, as the most abundant outer membrane protein in E. coli, has a C-terminal domain that can promote the expression of the fusion protein on the cell surface. Using genetic engineering techniques, the target protein gene Annexin V was fused with the Lpp signal sequence and the C-terminal domain of OmpA to construct a fusion protein. The Annexin V protein was successfully displayed on the cell surface. Based on EcN△lpp, the EcN△lpp-A5 engineered bacteria were successfully constructed to further enhance the colonization ability of the engineered bacteria at the enteritis site.

[0155] Using CRISPR-Cas9 technology, this invention successfully knocked out the lpp lipoprotein gene in the EcN genome and inserted lpp-ompa-Annexin V into that region, successfully obtaining the engineered bacterium EcN△lpp-A5. The growth curve of the engineered bacterium was almost identical to that of the wild type, indicating that the knockout of the lpp gene and the insertion of A5 had no significant effect on bacterial growth. Figure 2 A). After incubation on plates, the modified and wild-type bacteria showed identical colony size, smooth surfaces, and no significant changes in morphology. Figure 2 B). Scanning electron microscopy revealed no significant changes in the size and morphology of the modified and wild-type bacteria, indicating that the cell structure of the modified bacteria was not significantly affected. Figure 2 C). The red fluorescent protein gene RFP was transferred into engineered and wild-type bacteria. Protein secretion efficiency was assessed by detecting the red fluorescence intensity in the culture supernatant. It was found that the red fluorescence intensity of the engineered bacteria was significantly higher than that of the wild-type bacteria. Figure 2 (D) indicates that the modified bacteria have a significant advantage in secreting heterologous proteins.

[0156] Figure 2 This is a diagram illustrating the construction and secretion efficiency verification of the EcN△lpp-A5 engineered strain of the present invention. Figure 2 A is a growth curve diagram of the engineered bacteria and wild bacteria of the present invention; Figure 2 B is a diagram showing the colony morphology observed in plate culture according to the present invention; Figure 2 C is a scanning electron microscope image of the size and morphology of bacteria observed according to the present invention; Figure 2 D is a graph showing the detection efficiency of red fluorescent protein (RFP) secretion in the culture medium supernatant of this invention.

[0157] 3.4 Comparison of therapeutic effects of engineered EcN△lpp-A5 strain and wild-type EcN on enteritis. To evaluate the impact of engineered EcN on therapeutic efficacy, we compared the therapeutic effects of engineered EcN△lpp-A5 strain and wild-type EcN in a DSS-induced colitis mouse model. No significant differences were observed between the two strains across any of the indicators. Specifically, Figure 3 A comparison of colon length and Figure 3 Fecal analysis of B showed that the engineered strain was comparable to the wild-type EcN in alleviating colitis and reducing disease severity. Figure 3 Morphological observation of the colonic tissue of C further confirmed this result. Both the engineered strain treatment group and the wild-type EcN treatment group were able to effectively repair colonic mucosal damage, reduce inflammatory cell infiltration and edema, and the degree of improvement in histopathology was similar.

[0158] In summary, the EcN△lpp-A5 engineered strain constructed in this study maintained the effectiveness of wild-type EcN in the treatment of colitis.

[0159] Figure 3 This study compares the therapeutic effects of the EcN△lpp-A5-aTN engineered strain of the present invention with those of wild-type EcN. Figure 3 A represents the effect of the engineered EcN△lpp-A5-aTN strain and wild-type EcN on colonic inflammation in mice with enteritis. Figure 3 B represents the effect of the engineered EcN△lpp-A5-aTN strain of this invention and wild-type EcN on the feces of mice with enteritis. Figure 3 C represents the effect of the engineered EcN△lpp-A5-aTN strain and wild-type EcN on the colon morphology of mice with enteritis.

[0160] Example 22 The modified EcN△lpp-A5 strain exhibited superior gut colonization ability compared to the wild-type EcN. This invention describes the electrochemical transformation of wild-type EcN bacteria and EcNΔlpp-A5 modified bacteria into the red fluorescent protein RFP. The probiotic vector contains a nucleotide sequence encoding the red fluorescent protein RFP, as shown in SEQ ID NO:6, and a promoter nucleotide sequence, as shown in SEQ ID NO:7. The vector is used for fluorescence microscopy imaging. This invention systematically compared the colonization effects of two strains in enteritis tissues of healthy controls and DSS enteritis model mice at different time points (6 h, 12 h, 24 h, 48 h, and 72 h) after oral administration. In healthy control mice, both the wild-type and modified strains showed the highest colonic retention at 6 h. With increasing time, bacterial retention decreased, with the modified strain exhibiting a stronger red fluorescent signal in the colon, indicating a stronger colonization ability in the modified strain. Figure 4 AB). In DSS enteritis mice, the colonization rates of both modified and wild-type bacteria were higher than in normal mice. Figure 5 (AB) This may be due to impaired intestinal barrier function caused by intestinal inflammation, which makes it easier for bacteria to colonize. The number of colonized bacteria at each time point was significantly higher than that of wild bacteria, which may be related to the higher level of phosphatidylserine eversion in enteritis tissue.

[0161] Figure 4 This demonstrates the colonization ability of the EcN△lpp-A5 engineered bacteria of the present invention in the colon of healthy mice. Figure 4 A represents the colonization level of wild-type EcN carrying RFP red fluorescent protein in the colon of healthy mice at different time points after administration of the present invention. Figure 4 B represents the colonization level of EcN△lpp-A5 engineered bacteria carrying RFP red fluorescent protein in the colon of healthy mice at different time points after administration of the present invention, observed by fluorescence microscopy.

[0162] Figure 5 This invention demonstrates the colonization ability of the EcN△lpp-A5 engineered bacteria in the colon of DSS enteritis mice. Figure 5 A represents the colonization level of wild-type EcN carrying RFP red fluorescent protein in the colon of mice with enteritis, observed by fluorescence microscopy at different time points after administration of the present invention. Figure 5 B represents the colonization level of EcN△lpp-A5 engineered bacteria carrying RFP red fluorescent protein in the colon of mice with enteritis, observed under a fluorescence microscope at different time points after administration of the present invention.

[0163] This invention describes the electrochemical conversion of luxCDABE fluorescent protein into EcN wild-type bacteria and EcNΔlpp-A5 modified bacteria. The nucleotide sequence encoding luxCDABE fluorescent protein is shown in SEQ ID NO:8, and the promoter nucleotide sequence is shown in SEQ ID NO:7 (the pEcN-αTN promoter nucleotide sequence is shown in SEQ ID NO:7). This is used for in vivo imaging of small animals. The results are consistent with those observed by fluorescence microscopy. Observation using the IVIS small animal in vivo imaging system revealed that the fluorescence intensity of the EcNΔlpp-A5 modified bacteria in the intestine was higher than that of the control bacteria. Furthermore, at each observation time point, the fluorescence intensity of the modified bacteria in the intestine of enteritis mice was significantly higher than that in the control mice treated with the modified bacteria. Figure 6 AC). Anatomical results of various organs in each group of mice showed that no bacterial colonization was found in the heart, liver, spleen, lungs, and kidneys. Figure 6 DE) Figure 6 In vivo imaging of the EcN△lpp-A5 engineered strain of the present invention in the mouse colon. Figure 6 A represents the in vivo observation of the distribution of wild-type EcN and EcNΔlpp-A5 engineered bacteria carrying the luxCDABE fluorescent protein in healthy mice and mice with enteritis, respectively. Figure 6 B is a quantitative fluorescence distribution diagram of the strain of the present invention in healthy mice; Figure 6 C is a quantitative fluorescence distribution diagram of the strain of the present invention in mice with enteritis; Figure 6 DE is the result of this invention, which involves dissecting and observing the distribution of EcN and EcNΔlpp-A5 engineered bacteria in various organs of healthy and enteritis mice. Example 23

[0164] Construction of EcN△lpp-A5-aTN engineered bacteria This invention first transforms a plasmid expressing an anti-TNF-α nanobody into a modified chassis bacterium EcN△lpp-A5, constructing an engineered bacterium expressing aTN, EcN△lpp-A5-aTN. The growth curve of EcN△lpp-A5-aTN showed no significant difference from that of the EcN△lpp-A5 control bacterium. Figure 7 A), the edges of the colonies are all relatively smooth ( Figure 7 B). Western blot results showed that aTN was successfully expressed and secreted into the culture medium ( Figure 7 C).

[0165] Figure 7 This serves as a verification of the construction of the EcN△lpp-A5-αTN engineered bacteria of this invention. Figure 7 A is a comparison of the growth curves of EcN△lpp-A5-αTN and EcN△lpp-A5 in this invention; Figure 7B is a comparison of the colony morphology of EcN△lpp-A5-αTN and EcN△lpp-A5 in this invention; Figure 7 C is the verification of the αTN secretion expression effect of the EcN△lpp-A5-αTN engineered bacteria of this invention.

[0166] This invention successfully constructed the probiotic strain EcN△lpp-A5, which is genetically stable and free from exogenous resistance. This strain retains the original growth and probiotic characteristics of EcN, while achieving targeted colonization and long-term retention in the intestine, and has the ability to continuously express and secrete exogenous recombinant proteins. Figure 8 This is a schematic diagram illustrating the working principle of the EcN△lpp-A5 strain of the present invention. The main conclusions are as follows: Knocking out the outer membrane lipoprotein gene lpp significantly improved the secretion efficiency of exogenous proteins in engineered bacteria. The introduction of Annexin V, by binding to phosphatidylserine residues everted at enteritis sites, enhanced the targeted colonization of engineered bacteria in the intestine and inflamed tissues. The engineered bacterium EcN△lpp-A5 can continuously express and secrete various recombinant proteins, such as the anti-TNF-α nanobody αTN, enhancing the therapeutic efficacy of the strain in treating enteritis; it can also secrete and express tracer proteins RFP and luxCDABE, enabling the tracking of the strain's metabolism and distribution in vivo, laying a solid foundation for drug development.

[0167] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above-described experimental examples. The experimental examples and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope. The scope of protection of the present invention is defined by the appended claims, specification, and their equivalents.

Claims

1. A probiotic strain EcN△lpp-A5, characterized in that: The probiotic strain EcN△lpp-A5 is deposited under the name TPEC-LA034 at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The deposit date is September 26, 2025, and the accession number is CGMCC No. 36084.

2. The probiotic strain EcN△lpp-A5 according to claim 1, characterized in that: The probiotic strain EcN△lpp-A5 uses Escherichia coli Nissle 1917 as the substrate bacteria. The outer membrane lipoprotein coding gene lpp is completely knocked out using the CRISPR-Cas9 system, and a foreign DNA fragment is integrated at the deletion site. The foreign DNA fragment contains the coding sequence of Annexin V fusion protein. The nucleotide sequence is composed of the coding sequence of Lpp signal peptide, the coding sequence of OmpA anchoring domain and the coding sequence of Annexin V from 5′ to 3′. Under the guidance of Lpp signal peptide, Annexin V fusion protein is secreted into the bacterial periplasmic space and then anchored to the outer membrane through the OmpA domain, realizing the outer membrane-anchored display of Annexin V on the bacterial surface.

3. The probiotic strain EcN△lpp-A5 according to claim 1, characterized in that: The coding sequence of the Annexin V fusion protein is shown in SEQ ID NO:1, the coding sequence of the Lpp signal peptide is shown in SEQ ID NO:3, and the coding sequence of the OmpA anchoring domain is shown in SEQ ID NO:

4.

4. A method for constructing the probiotic strain EcN△lpp-A5 as described in any one of claims 1 to 3, characterized in that... The steps include: (1) knocking out the lpp gene in the EcN genome using the CRISPR-Cas9 system, wherein the CRISPR-Cas9 system contains an sgRNA targeting lpp and a donor template; (2) inserting the coding sequence of Annexin V fusion protein into the lpp gene deletion site using the CRISPR-Cas9 system to obtain the EcNΔlpp-A5 strain.

5. The construction method according to claim 4, characterized in that: In step (1), the nucleotide sequence of the sgRNA is shown in SEQ ID NO:

5. The donor template of the sgRNA is derived from the genome of E. coli Nissle 1917, and the E. coli Nissle 1917 genome sequence number is NCBI Reference Sequence: NZ_CP007799.1; In step (2), the coding sequence of the Annexin V fusion protein is formed by sequentially linking the Lpp signal peptide coding sequence, the OmpA anchoring domain coding sequence and the Annexin V (ANXA5) coding sequence from 5′ to 3′.

6. The use of the probiotic strain EcN△lpp-A5 according to claim 1 in the preparation of a probiotic vector for enhancing targeted colonization of intestinal inflammatory sites and for expressing and secreting recombinant proteins.

7. The application according to claim 6, characterized in that: The probiotic carrier contains a nucleotide sequence encoding an anti-TNF-α nanobody αTN, as shown in SEQ ID NO:2; or the probiotic carrier contains a nucleotide sequence encoding a red fluorescent protein RFP, as shown in SEQ ID NO:6, and a promoter nucleotide sequence as shown in SEQ ID NO:7; or the probiotic carrier contains a nucleotide sequence encoding a tracer protein luxCDABE, as shown in SEQ ID NO:

8.

8. The application according to claim 6, characterized in that: The targeted colonization is achieved through the specific binding of Annexin V to phosphatidylserine on the surface of capillary endothelial cells in the intestine (especially the colon) and to phosphatidylserine everted at sites of enteritis.

9. The application according to claim 6, characterized in that: The probiotic carrier is used to load and / or express therapeutic or tracer protein or nucleic acid drugs.

10. The application according to claim 6, characterized in that: The therapeutic protein includes any one of anti-TNF-α nanobody, IL-10, IL-22, or antimicrobial peptide; the tracer protein is any one of red fluorescent protein RFP, EGFP, or luxCDABE.

11. The use of the probiotic strain EcN△lpp-A5 according to claim 1 in the preparation of a probiotic vector for enhancing targeted colonization of intestinal inflammatory sites and for expressing and secreting recombinant proteins.

12. The application according to claim 6, characterized in that: The probiotic carrier contains a nucleotide sequence encoding an anti-TNF-α nanobody αTN, as shown in SEQ ID NO:2; or the probiotic carrier contains a nucleotide sequence encoding a red fluorescent protein RFP, as shown in SEQ ID NO:6, and a promoter nucleotide sequence as shown in SEQ ID NO:7; or the probiotic carrier contains a nucleotide sequence encoding a tracer protein luxCDABE, as shown in SEQ ID NO:

8.

13. The application according to claim 6, characterized in that: The targeted colonization is achieved through the specific binding of Annexin V to phosphatidylserine on the surface of intestinal capillary endothelial cells and to phosphatidylserine everted at the site of enteritis.

14. The application according to claim 6, characterized in that: The probiotic carrier is used to load and / or express therapeutic or tracer protein or nucleic acid drugs.

15. The application according to claim 6, characterized in that: The therapeutic protein includes any one of anti-TNF-α nanobody, IL-10, IL-22, or antimicrobial peptide; the tracer protein is any one of red fluorescent protein RFP, EGFP, or luxCDABE.

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