A fusion protein, gene, expression vector, genetically engineered bacteria and its applications

Abstract

This invention belongs to the field of biomedical technology and relates to a fusion protein, gene, expression vector, genetically engineered bacteria, and their applications. The fusion protein is derived from the fusion of bacterial outer membrane vesicle protein and human PD-L1, or from the fusion of bacterial outer membrane vesicle protein, human PD-L1, and human Fc protein. Genetically engineered bacteria constructed using the fusion gene provided by this invention can release PD-L1-rich outer membrane vesicles, which can effectively penetrate the lamina propria of the mucosa and regulate inflammation inside and outside the intestine. This genetically engineered bacterium exhibits good biosafety, effectively regulating intestinal flora and metabolism, and improving intestinal barrier function; moreover, it shows good oral administration compliance, which is beneficial for clinical trials.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a fusion protein, gene, expression vector, genetically engineered bacteria and its applications. Background Technology

[0002] Inflammatory bowel disease (IBD) is a chronic, progressive, relapsing / remissionable intestinal disease that severely impacts patients' quality of life. Due to its refractory, relapsing, disabling, and carcinogenic characteristics, IBD causes immense suffering for patients and imposes a significant economic burden on their families and society. Furthermore, its incidence rate in my country has been increasing annually, drawing considerable attention in recent years. Current research considers IBD an autoimmune disease. Traditional anti-inflammatory treatments, including hormones, have been used for forty years, but the treatment outcomes are unsatisfactory. Therefore, increasing research focuses on biological therapies for IBD. However, most immunosuppressive therapies suppress the systemic immune system, such as infliximab (IFX), adalimumab (ADA), and ustekinumab (UST) in first-line treatment regimens; and small molecule drugs like the JAK inhibitor tofacitinib, all of which have significant adverse reactions and safety concerns. Therefore, the treatment of IBD requires a new type of drug with higher safety and lower toxicity.

[0003] Regulating gut microbiota and nutrient metabolism is one of the new strategies for treating IBD, and current research mainly focuses on the use of probiotics. Probiotics have the effects of anti-oxidative stress, repairing the intestinal barrier, immune regulation, gut microbiota regulation, and improving nutrition and metabolism, and can be used as an adjunct therapy for IBD. However, some clinical trials have suggested that the efficacy of probiotics is not significant. To improve the clinical efficacy of probiotics in treating IBD, some scholars believe that multi-strain compound preparations or combined use of prebiotics should be used, while others have proposed fecal microbiota transplantation, but its actual efficacy still needs further improvement. Therefore, simply regulating gut microbiota and nutrient metabolism through probiotics is insufficient to treat / allergic to IBD.

[0004] In recent years, some scholars have attempted to treat IBD by modifying probiotics. These include engineered yeast strains with self-regulating functions that can sense and neutralize pro-inflammatory molecules; engineered Saccharomyces cerevisiae that can synthesize butyrate; engineered Escherichia coli (EcN) 1917 that overexpresses catalase and superoxide dismutase; EcN coupled with nanoparticles that can scavenge reactive oxygen species; and engineered EcN that secretes trefoil factors (TFFs). Studies have confirmed that these engineered bacteria can effectively deliver corresponding treatments to the intestines and have a certain alleviating or inhibitory effect on IBD, but their overall efficacy still needs further verification. However, engineered probiotics may be an effective means of treating IBD.

[0005] Inflammatory responses can ideally be completely resolved through regulation by the body's immune system. However, in IBD patients, impaired intestinal immune regulation leads to chronic relapsing immune activation and gastrointestinal lesions. Relief of intestinal inflammation and mucosal healing are key steps in IBD treatment; addressing local chronic inflammation and maintaining intestinal homeostasis are crucial. Intestinal immune balance is closely related to the programmed cell death protein-1 (PD-1) / programmed cell death-ligand (PD-L1) pathway, and studies have shown abnormalities in the PD-1 / PD-L1 signaling pathway in IBD. Blocking the PD-1 / PD-L1 signaling pathway can lead to varying degrees of colonic inflammation, while PD-L1 can alleviate enteritis symptoms in mice with dextran sulfate sodium (DSS)-induced colitis. Therefore, PD-L1 may be a novel approach for treating IBD. However, systemic application of PD-L1 can broadly and comprehensively suppress the immune system, increasing the risk of infection and tumors.

[0006] Therefore, by combining the advantages of engineered probiotics and PD-L1, constructing engineered probiotics that highly express PD-L1-Fc can achieve intestinal-specific immunosuppression, reduce the impact on the systemic immune system, and, with the added benefits of probiotics in anti-oxidative stress, repairing the intestinal barrier, immune regulation, intestinal flora regulation, and improving nutrition and metabolism, a new strategy can be provided for the treatment of IBD.

[0007] IBD-related treatments mainly fall into three categories: traditional drugs, biologics, and novel small molecule drugs. Traditional drugs include aminosalicylic acid preparations, glucocorticoids, and immunosuppressants. Aminosalicylic acid preparations have poor efficacy against small intestinal Crohn's disease (CD) and have numerous side effects, while glucocorticoids and immunosuppressants have high systemic side effects. Biologics include IFX, ADA, VDZ, and UST, but they have drawbacks such as moderate efficacy, immunogenicity, parenteral administration, relatively poor tolerability, high cost, and risks of infection and tumor formation. Novel small molecule drugs include JAK inhibitors and S1P receptor modulators, but their clinical efficacy and risks still require further observation and evaluation. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides a fusion protein, a gene, an expression vector, a genetically engineered bacterium, and its applications. The genetically engineered bacterium constructed using the fusion gene provided by this invention can release PD-L1-rich outer membrane vesicles, which can effectively penetrate the lamina propria of the mucosa and regulate intestinal inflammation.

[0009] The technical solution provided by this invention is as follows:

[0010] The present invention provides a fusion protein, which is derived from the fusion of bacterial outer membrane vesicle protein and human PD-L1 or from the fusion of bacterial outer membrane vesicle protein, human PD-L1 and human Fc protein.

[0011] Furthermore, the bacterial outer membrane vesicle protein is bacterial ClyA, and the amino acid sequence of the fusion protein is any one of the following 1)-2): 1) An amino acid sequence as shown in SEQ ID NO.1 or SEQ ID NO.2; 2) An amino acid sequence that has the same function as the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2, obtained by replacing, deleting or inserting one, several or dozens of amino acids in the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2.

[0012] The present invention also provides a fusion gene, wherein the fusion gene is any one of the following 1)-3): 1) It has a nucleotide sequence as shown in SEQ ID NO.3 or SEQ ID NO.4; 2) Possesses a polynucleotide that can encode a protein sequence as shown in SEQ ID NO.1 or SEQ ID NO.2; 3) Has 75% or more identity with the DNA fragment defined in 1) or 2), and encodes a protein that is functionally equivalent to the protein shown in SEQ ID NO.1 or SEQ ID NO.2.

[0013] The present invention also provides an expression vector comprising a gene expressing the fusion protein described above and a base vector.

[0014] The present invention also provides a genetically engineered bacterium, which includes probiotics and the expression vector described above.

[0015] Furthermore, the probiotics include any one or more of the following: *Escherichia coli*, *Aktermannii*, *Bifidobacterium adolescentis*, *Bifidobacterium lactis*, *Bifidobacterium bifidum*, *Bifidobacterium breve*, *Bifidobacterium longum*, *Bifidobacterium infantis*, *Lactobacillus acidophilus*, *Lactobacillus curvatureii*, *Lactobacillus bulgaricus*, *Lactobacillus delbrueckii* subsp. *lactobacter*, *Lactobacillus gasseri*, *Lactobacillus helveticus*, *Lactobacillus casei*, *Lactobacillus paracasei*, *Lactobacillus rhamnosus*, *Lactobacillus fermentum*, *Lactobacillus reuteri*, *Lactobacillus plantarum*, *Lactobacillus salivarius*, *Lactobacillus curvatureii*, *Lactobacillus sakei*, *Streptococcus thermophilus*, *Lactococcus lactis* subsp. *lactobacter*, *Lactococcus lactis*, *Lactococcus lactis* subsp. *lactobacter*, *Lactococcus lactis* subsp. *lactobacter*, *Propionibacterium freundii* subsp. *she*, *Propionibacterium propionate*, *Leuconostoc mesenteroides* subsp. *enteroides*, *Pediococcus lactis*, *Pediococcus pentosaceus*, *Bacillus coagulans*, *Staphylococcus calf*, *Staphylococcus xylose*, *Staphylococcus carminatus*, and

[0016] The present invention also provides the application of the above-described fusion protein, the above-described fusion gene, the above-described expression vector, or the above-described genetically engineered bacteria in the preparation of drugs for digestive system diseases, systemic autoimmune diseases, intestinal aGVHD, or liver aGVHD.

[0017] Furthermore, the digestive system diseases include inflammatory bowel disease.

[0018] Beneficial effects

[0019] 1. This invention utilizes probiotics to express the PD-L1 protein, which is secreted outside the probiotics via bacterial outer membrane vesicles (OMVs) and enters the human digestive tract. OMVs containing PD-L1 protein can enter the digestive tract mucosa and submucosal lymphatic system, inhibiting the activation and proliferation of T cells within the digestive tract, thereby alleviating tissue damage. Secondly, when the OMV contains Fc, dendritic cells (DCs) can capture PD-L1 and present it to T cells through cell-cell interactions, inhibiting T cell activation. These genetically engineered bacteria can regulate the intestinal immune system through their expressed target protein, thus directly applicable to various digestive system diseases and transplant-related aGVHD (acute graft versus host disease). Furthermore, the target protein secreted or expressed by genetically engineered bacteria in the intestine can act on the liver through the enterohepatic circulation and on the central nervous system through the gut-brain axis, regulating the hepatic and central immune systems.

[0020] 2. The engineered bacteria constructed using the fusion gene of this invention can highly express PD-L1 protein on outer membrane vesicles (OMVs), resulting in high PD-L1 expression on the surface of OMVs. OMVs are delivered orally to the intestines via engineered probiotics. OMVs penetrate into various layers of the intestinal mucosa and directly inhibit the activation and proliferation of infiltrating T cells through the ClyA-PD-L1-Fc fusion protein. On the other hand, OMVs are recognized and taken up by dendritic cells (DCs) and macrophages through the interaction of Fc and FcR. These dendritic cells and macrophages then present PD-L1 to effector T cells, thereby suppressing the local immune system. Furthermore, OMVs can be channeled to submucosal lymph nodes and other sites, binding to PD-1 on cytotoxic T lymphocytes and memory T cells, activating the PD-1 / PD-L1 signaling pathway, and inhibiting T cell activity and function. This exerts a strong local intestinal immune effect and reduces systemic immunosuppression. Meanwhile, probiotics can improve intestinal barrier function and regulate IBD-related gut microbiota dysbiosis, providing a new strategy with high clinical feasibility for IBD treatment.

[0021] 3. PD-1 is expressed during activation of T cells, B cells, macrophages, and dendritic cells, and has been shown to play a crucial role in regulating peripheral tolerance and autoimmunity. PD-1 binds to its ligands PD-L1 and PD-L2, transmitting inhibitory signals to T cells in chronic infections and tumors. Furthermore, PD-1 and its ligand, the negative regulator Th17, play a pathogenic role in the development of autoimmune diseases. Soluble PD-L1-Ig protein can alleviate the severity of collagen-induced arthritis in mice and inhibit cell proliferation and the production of IL-17 and IL-23 by splenocytes. In a chronic colitis model, both PD-L1 and PD-1 are significantly overexpressed. PD-L1 expressed in the colon inhibits the proliferation of activated CD4+ and CD8+ effector T cells and plays an important role in mucosal tolerance to symbiotic bacteria and dietary antigens. Blocking the PD-1 / PD-L1 pathway can lead to varying degrees of inflammatory lesions in the colon, while administration of PD-L1 can alleviate enteritis symptoms in mice with dextran sulfate sodium (DSS)-induced colitis. PD-1 inhibitors (e.g., nivolumab, pembrolizumab, and avelumab) and PD-L1 inhibitors (e.g., atezolizumab) have been reported to induce immune-related colitis. Therefore, PD-L1 administration may be an option for treating IBD. However, PD-L1 has a strong immunosuppressive effect, and systemic administration of PD-L1 increases the risk of infection and carcinogenesis. Therefore, the technical challenge this invention aims to solve is to accurately target and deliver PD-L1 to the inflamed intestinal mucosa to achieve local intestinal immunity and avoid the side effects of systemic use.

[0022] 4. Genetically engineered bacteria have good biosafety, regulate intestinal flora and metabolism, and improve intestinal barrier function; and have good oral administration compliance, which is beneficial for clinical trials.

[0023] 5. The engineered bacteria synthesis method of the present invention is simple, the resulting product has a uniform morphology, high safety, and low production cost, making it suitable for industrial-scale production. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the plasmid structure of a mouse-derived fusion protein.

[0025] Figure 2 It involves the construction of engineered probiotics and the identification of their phenotype and function.

[0026] Figure 3 It is an in vitro functional analysis of engineered probiotics.

[0027] Figure 4 It is engineered probiotics + Ara that improve colitis in mice.

[0028] Figure 5 This is the mechanism by which engineered probiotics treat IBD.

[0029] Figure 6 It is engineered probiotics + Ara that improve the gut microbiota and metabolism in mice.

[0030] Figure 7 This is an in vivo safety assessment of engineered probiotics.

[0031] Figure 8 It is the expression and identification of engineered probiotics. Detailed Implementation

[0032] Example 1

[0033] I. Design of Fusion Proteins and Their Encoding Genes

[0034] (1) Human origin

[0035] This invention provides a fusion protein (denoted as fusion protein ClyA-Flag-PD-L1 or fusion protein ClyA-Flag-PD-L1-Fc), as shown in sequence listing 1 (SEQ ID NO.1~SEQ ID NO.2).

[0036] In the amino acid sequence shown in SEQ ID NO.1, positions 1-307 are bacterial ClyA, positions 308-315 are the tag, and positions 316-535 are human PD-L1.

[0037] In the amino acid sequence shown in SEQ ID NO.2, positions 1-307 are bacterial ClyA, positions 308-315 are the tag, positions 316-535 are human PD-L1, positions 536-539 are the linker peptide, and positions 540-771 are human Fc.

[0038] The DNA molecule shown in SEQ ID NO.3 encodes the fusion protein ClyA-Flag-PD-L1, and this DNA molecule is named the ClyA-Flag-mPD-L1 fusion gene. In the nucleotide sequence shown in SEQ ID NO.3, nucleotides 1-3 are the start codon, nucleotides 4-921 encode bacterial ClyA, nucleotides 922-945 encode the tag, nucleotides 946-1605 encode human PD-L1, and nucleotides 1606-1608 are the stop codon.

[0039] The DNA molecule shown in SEQ ID NO.4 encodes the fusion protein ClyA-Flag-PD-L1-Fc, and this DNA molecule is named the ClyA-Flag-mPD-L1-Fc fusion gene. In the nucleotide sequence shown in SEQ ID NO.4, nucleotides 1-3 are the start codon, nucleotides 4-921 encode bacterial ClyA, nucleotides 922-945 encode the tag, nucleotides 946-1605 encode human PD-L1, nucleotides 1606-1617 encode the linker peptide, nucleotides 1618-2313 encode human Fc, and nucleotides 2314-2316 are the stop codon.

[0040] The ClyA-Flag-mPD-L1 fusion gene and the ClyA-Flag-mPD-L1-Fc fusion gene fragment were obtained by whole-genome synthesis by Suzhou Genewise Biotechnology Co., Ltd.

[0041] (2) Rodent source

[0042] This invention provides a fusion protein (denoted as fusion protein ClyA-FLAG-ePD-L1 or fusion protein ClyA-FLAG-ePD-L1-mFc), as shown in sequence listing 1 (SEQ ID NO.5~SEQ ID NO.6). In the amino acid sequence shown in SEQ ID NO.5, positions 1-307 represent bacterial ClyA, positions 308-321 represent a tag, and positions 322-544 represent mouse PD-L1.

[0043] In the amino acid sequence shown in SEQ ID NO.6, positions 1-307 are bacterial ClyA, positions 308-321 are the tag, positions 322-544 are mouse PD-L1, positions 545-548 are the linker peptide, and positions 549-762 are mouse Fc.

[0044] The DNA molecule shown in SEQ ID NO.7 encodes the fusion protein ClyA-FLAG-ePD-L1, and this DNA molecule is named the ClyA-FLAG-ePD-L1 fusion gene. In the nucleotide sequence shown in SEQ ID NO.7, nucleotides 1-3 are the start codon, nucleotides 4-921 encode bacterial ClyA, nucleotides 922-963 encode the tag, nucleotides 964-1632 encode human PD-L1, and nucleotides 1633-1635 are the stop codon.

[0045] The DNA molecule shown in SEQ ID NO.8 encodes the fusion protein ClyA-FLAG-ePD-L1-mFc, and this DNA molecule is named the ClyA-FLAG-ePD-L1-mFc fusion gene. In the nucleotide sequence shown in SEQ ID NO.8, nucleotides 1-3 are the start codon, nucleotides 4-921 encode bacterial ClyA, nucleotides 922-963 encode the tag, nucleotides 964-1632 encode human PD-L1, nucleotides 1633-1644 encode the linker peptide, nucleotides 1645-2286 encode mouse Fc, and nucleotides 2287-2289 are the stop codon.

[0046] Among them, the ClyA-Flag-ePD-L1 fusion gene and the ClyA-Flag-ePD-L1-mFc fusion gene fragment were obtained by whole-genome synthesis by Suzhou Genewiz Biotechnology Co., Ltd.

[0047] II. Construction of Expression Vectors and Genetically Engineered Bacteria

[0048] 1. Engineered probiotics

[0049] This invention relates to the preparation of genetically engineered probiotics capable of secreting anti-inflammatory vesicles through engineered modification. The engineered probiotic modification scheme includes the following steps:

[0050] (1) Screening suitable probiotics for the chassis: The chassis bacteria selected in this study were probiotics, including Escherichia coli, Akkermansia, Bifidobacterium adolescentis, Bifidobacterium lactis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus curvatureii, Lactobacillus bulgaricus, Lactobacillus delbrueckii subsp. lactis, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus fermentum, and Lactobacillus roximatei. Lactobacillus oryzae, Lactobacillus plantarum, Lactobacillus salivarius, Lactobacillus curvatureus, Lactobacillus sakei, Streptococcus thermophilus, Lactococcus lactis subsp. lactis, Lactococcus lactis, Lactococcus lactis diacetyl subsp. lactis, Lactococcus lactis subsp. lactis, Propionibacterium fischeri subsp. Scheres, Propionibacterium propionate, Leuconostoc mesenteroides subsp. mesenteroides, Pediococcus lactis, Pediococcus pentosaceus, Bacillus coagulans, Staphylococcus calfii, Staphylococcus xylose, Staphylococcus carminatus, and Kluyveromyces maxkwai.

[0051] (2) The probiotic selected in this study is Nissle 1917.

[0052] (3) Removal of Nissle 1917 intrinsic plasmids

[0053] Using CRISPR / Cas9 tools and pCas and pTargetF as tool plasmids, pMUT1 and pMUT2 plasmids were knocked out to obtain EcN△pMUT1 / 2 strains. Cleavage sites were designed (see Table 1), and the target sequence on the pTargetF plasmid was replaced with our designed target sequence, resulting in tool plasmids pTargetF1 and pTargetF2 used to eliminate endogenous plasmids 1 and 2, respectively. The tool plasmid pCas was transformed into Nissle 1917 strain, cultured at 30°C, and electroporation competent cells were prepared. pTargetF1 was then transformed, plated onto kanamycin + spectinomycin double antibiotic LB agar plates, and cultured overnight at 30°C. Identification primers were designed on endogenous plasmid 1, and the next day, single clones and controls were used to confirm successful elimination. After successful elimination, the identified monoclonal clones were cultured overnight in LB broth containing 0.2 mM IPTG. The next day, they were plated or streaked onto kanamycin-resistant LB plates and identified using pTargetF plasmid identification primers. The single clone plus control was used to verify successful elimination of pTargetF1. After successful elimination of endogenous plasmid 1 and pTargetF1, single clones were picked, cultured at 30°C, and electrotransfer competent cells were prepared. pTargetF2 was then introduced, plated onto kanamycin + spectinomycin dual-antibody LB plates, and cultured overnight at 30°C. Identification primers were designed on endogenous plasmid 2, and the single clone plus control was used the next day to verify successful elimination. After successful elimination, the identified monoclonal clones were cultured overnight in LB broth containing 0.2 mM IPTG. The next day, they were plated or streaked onto kanamycin-resistant LB plates and identified using pTargetF plasmid identification primers. The single clone plus control was used to verify successful elimination of pTargetF2. After all the above endogenous plasmids have been successfully eliminated, the pCas plasmid needs to be eliminated. Select single clones and culture them at 37 degrees Celsius. Then, streak or plate them onto antibiotic-free LB plates and incubate them overnight at 30 degrees Celsius. Design primers on the tool plasmid pCas for identification. Combined with the positive control, the absence of electrophoretic bands in the PCR product indicates that the two endogenous plasmids and the tool plasmid of Nissle 1917 have been completely eliminated.

[0054] Table 1 shows the design of cleavage sites to eliminate endogenous plasmids pMUT1 and pMUT2.

[0055]

[0056] (4) Agarose gel electrophoresis to verify plasmid knockout

[0057] The plasmid knockout results were validated for strains EcN△pMUT1, EcN△pMUT2, and EcN△pMUT1 / 2. PCR amplification products were mixed with 6× loading buffer and loaded onto a 1.5% agarose gel. Agarose gel electrophoresis was performed in 1×TAE buffer at 100 V for 45 minutes. After electrophoresis, the agarose gel was stained with ethidium bromide for 15 minutes. The gel images were then observed and captured using a UV transilluminator to confirm the presence and size of the target DNA fragment. Primer sequences are detailed in List 2.

[0058] Table 2 Primer sequences for knocking out endogenous plasmids pMUT1 and pMUT2

[0059]

[0060] 2. Construction of expression vectors and genetically engineered bacteria (human origin)

[0061] The tool plasmid pCas was introduced into strain EcN△pMUT1 / 2 to obtain strain EcN△pMUT1 / 2-pCas. The target sequence (5'-3') was determined: cagaatcactgccaaaatcgagg contains a 20bp protospacer sequence (N20) and a 3bp PAM sequence. The tool plasmid N20.pTF containing the N20 sequence was constructed. The primer sequences are listed in List 3.

[0062] Table 3 Primer sequences for constructing the tool plasmid N20.pTF

[0063]

[0064] Repair templates HL and HR, fusion protein expression components, and linear plasmids were amplified separately to construct the tool plasmid PFCF.pTF, which contains ClyA-Flag-PD-L1-Fc or ClyA-Flag-PD-L1-Fc expression elements and a genome integration homologous template. Primer sequences are listed in List 4.

[0065] Table 4 Primer sequences for constructing PFCF.pTF

[0066]

[0067] Electroporation competent cells of strain EcNΔpMUT1 / 2-pCas were prepared, and then the PFCF.pTF plasmid was introduced by electroporation. After overnight culture in a plate, the genome editing results were verified by PCR. After successful verification, the tool plasmid of the cells was eliminated. Finally, the engineered strain EcNΔpMUT1 / 2-ClyA-Flag-PD-L1-Fc, which can express ClyA-Flag-PD-L1-Fc protein in the genome using arabinose, was obtained.

[0068] 3. Construction of expression vectors and genetically engineered bacteria (mouse-derived)

[0069] The ClyA-Flag-ePD-L1-mFc gene was cloned into the 5'Ncol and 3'NehI restriction sites of the pBAD vector (see...). Figure 1 The expression vector pBAD-ClyA-ePD-L1-mFc was inserted into the engineered strain EcNΔpMUT1 / 2 to obtain the engineered strain EcNΔpMUT1 / 2-ClyA-Flag-ePD-L1-mFc (EcN-ePD-L1-mFc). The strain was cultured in LB medium at 37°C and 220 rpm, with ampicillin (50 μg / ml) added as needed.

[0070] IV. In vitro experiments

[0071] 1. Preparation and characterization of OMVs

[0072] The recombinant engineered strain EcN-ePD-L1-mFc was cultured in LB medium containing ampicillin for 12 hours at 37°C and 220 rpm. When the bacterial optical density (OD600) reached 0.6 to 0.8, 2 g / L arabinose (Ara) was added to further induce protein expression, and the culture was continued at 37°C and 220 rpm for 6 hours. Subsequently, the culture was centrifuged at 4°C and 4000 rpm for 20 minutes, and the supernatant was filtered through a 0.22 μm vacuum filter (Wuxi Nais Biotechnology Co., Ltd., catalog number 343011), and then concentrated to 50 ml using a 50 kDa ultrafiltration membrane (Millipore; UFC905008). Next, the culture was filtered using a 0.22 μm disposable syringe filter. Finally, the culture was ultracentrifuged at 4°C and 150,000 xg for 3 hours, and the outer membrane vesicles (OMVs) were resuspended in 1 ml PBS and stored at -20°C. OMVs were extracted from EcN△pMUT1 / 2 strains using the same method. These strains were cultured in LB medium at 37°C and 220 rpm for 18 hours.

[0073] The concentration of extracted exosome proteins was determined using a BCA protein assay kit (Nanjing Novizan Biotechnology Co., Ltd., E112-02). TEM images of the EcN-ePD-L1-mFcOMVs and EcN△pMUT1 / 2 OMVs strains were obtained using a TEM microscope (TEcNai G2 spirit Biotwin). Particle size and potential data for the two OMVs were obtained using a nanoparticle size and Zeta potential analyzer (Zetasizer NanoZS90).

[0074] 2. Western Blot Analysis Western blotting analysis was performed on the expression of heterologous proteins in recombinant engineered strains EcN-ePD-L1-mFc and OMVs. Total protein was extracted from the engineered bacteria using a bacterial protein extraction kit (catalog number C600596) from Beijing Puyihua Technology Co., Ltd., according to the manufacturer's instructions. OMV proteins were mixed with the extracted exosome solution at a 4:1 ratio using 5× loading buffer and heated at 100°C for 15 minutes. Protein lysates of 30 μg from each sample were separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes. After blocking with 5% skim milk (Sangon Biotech; A600669-0250), the membranes were incubated with rabbit polyclonal anti-FLAG-tag antibody (Wuhan Sanying Biotechnology Co., Ltd., 20543-1-AP) and the corresponding secondary antibody. Immunoreactive proteins were detected using Bio-Rad's enhanced chemiluminescence reagent. Image analysis was performed using ImageJ software (version 1.8.0).

[0075] 3. Experimental Results

[0076] Select suitable chassis probiotics and use gene editing to make them more suitable for engineering modification. Figure 2 A), verifying the successful elimination of the endogenous dual plasmids in EcN△pMUT1 / 2 ( Figure 2 B). It was confirmed that knocking out the diplasmid had no effect on bacterial growth. Figure 2 C); Detection showed that EcNΔpMUT1 / 2 was significantly superior to wild-type EcN in inducing protein expression (C); Figure 2(D) Based on this, pBAD-ClyA-FLAG-ePD-L1 and pBAD-ClyA-FLAG-ePD-L1-mFc were inserted into EcNΔpMUT1 / 2 respectively. To verify whether PD-L1 was expressed in this engineered strain, the experimental results showed that the addition of the mFc fragment significantly increased PD-L1 expression. Therefore, ClyA-FLAG-ePD-L1-mFc was inserted into EcNΔpMUT1 / 2 to construct EcNΔpMUT1 / 2-ClyA-FLAG-ePD-L1-mFc (EcN-ePD-L1-mFc), and the expression of PD-L1 in its OMVs was detected. The experimental results showed that PD-L1 was also highly expressed in the OMVs. These results indicate that we have successfully constructed a high-expression extracellular region of PD-L1 and successfully expressed it in the OMVs (Figure 2E-F). Transmission electron microscopy (TEM) was used to analyze the morphology, zeta particle size, and potential of the OMVs of our target engineered strain. The results showed that the constructed OMVs all exhibited a bilayer structure, with uniform spherical morphology of approximately 100-150 nm in diameter and a potential of -20 mV, which was not significantly different from EcNΔpMUT1 / 2. Figure 2 GI).

[0077] Example 2: Bioactivity study of EcN-ePD-L1-mFc in mice

[0078] Mice: Female mice C57BL / 6 (6-8 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.; the mice were placed in a room with a temperature of 20-22°C, a light / dark cycle of 12 hours, and a humidity of 30-70%.

[0079] I. Mouse Experimental Protocol

[0080] 1. EcN-ePD-L1-mFc intestinal colonization and epithelial permeability assay

[0081] C57BL / 6 mice (n=5) were randomly divided into three groups. The intestinal colonization stability of the engineered bacteria was assessed via oral administration using Cy5.5-labeled EcN△pMUT1 / 2 and EcN-ePD-L1-mFc strains (both at a concentration of 1×10^13 cfu / ml). Mice received 200 μl of bacterial solution via gavage. Mice were sacrificed using an in vivo imaging system (IVIS) at different time points (3h, 6h, 12h, 24h), and the gastrointestinal tract was extracted for imaging analysis to observe the retention of the engineered bacteria in the colon.

[0082] After a 6-hour fast, mice were anesthetized and their colons were ligated. Different OMVs (50 μg) were then injected into the ligated intestinal lumen. Two hours later, the ligated colon was excised for immunofluorescence analysis. Cell nuclei were stained with DAPI (blue) and OMVs were labeled with anti-PD-L1 antibody (green).

[0083] 2. In vitro OMVs inhibit immune cell function

[0084] In 96-well plates, 100 μl of a mixture of CD3 (2 μg / ml), CD8 (0.4 μg / ml), and PBS was added to each well. The plates were sealed and incubated overnight at 4°C. Spleens from C57BL / 6 mice were ground, filtered, and the cells were resuspended and plated (5 × 10^5 / ml). 50 μl each of EcNΔpMUT1 / 2 OMVs and EcN-ePD-L1-mFc OMVs were added to each well, and flow cytometry analysis was performed (n=5).

[0085] 3. Safety and efficacy analysis of EcN-ePD-L1-mFc in the treatment of inflammatory bowel disease

[0086] All healthy female mice (n=50) weighing 20g were housed in a specific pathogen-free environment. Mice were randomly divided into five groups, and colitis conditions were simulated throughout the treatment using 2% DSS (w / v). The control group received PBS solution without DSS, while the other four groups received DSS-containing solutions and different engineered bacteria (EcNΔpMUT1 / 2, EcN-ePD-L1-mFc, and EcN-PD-L1-mFc+Ara) by gavage on days 0, 3, and 6, respectively, at a dose of 1×10^9 cfu. Following gavage, mice were administered a solution containing 20g / L Ara for 12 hours daily to induce protein expression. On day 12, mice (n=10 per group) were sacrificed, their weight was recorded, and blood was collected from the eyeballs of 50 mice. After standing at room temperature for 1 hour, the blood was centrifuged at 5000 rpm, and the supernatant was collected and subsequently frozen at -80℃ for cytokine assay. After opening the abdominal cavity, mesenteric lymph nodes were first collected and immersed in 1640 medium for dendritic cell (DC) maturation testing. The intestines (from the upper cecum to the anus) were collected and stored in centrifuge tubes. After all collections were completed, the colonic segment was removed for photography and stored in 1640 medium for HE staining, frozen sectioning, and flow cytometry analysis. The spleen and liver were weighed and stored after collection. One of each of the other major organs was collected for HE staining. The cecal contents were divided into two portions for 16S sequencing and short-chain fatty acid analysis.

[0087] Blood was collected from the eyeballs of 50 mice. After collection, the blood was allowed to stand at room temperature for 1 hour, centrifuged at 5000 rpm to collect the supernatant, and then stored at -80°C. Biochemical indicators such as ALT, AST, ALP, and UREA were measured within one month.

[0088] 4. Effects of EcN-ePD-L1-mFc on intestinal flora and metabolism in colitis mice.

[0089] Colonic contents from the aforementioned mice were collected and sequenced using 16S ribosomal RNA. The generated sequences were then subjected to OTU clustering analysis based on a 97% similarity level. Simultaneously, alpha and beta diversity of the samples were calculated, and abundance differences between populations were identified to reveal the composition and changes in the gut microbiota. Non-targeted GC-MS metabolomics was used to analyze the colonic contents samples to reveal changes in gut metabolomics.

[0090] II. Experimental Results

[0091] 1. EcN-ePD-L1-mFc can inhibit the function of immune cells and reduce their proliferative activity.

[0092] Oral administration of EcNΔpMUT1 / 2 and EcN-ePD-L1-mFc, followed by Ara induction, revealed that the bioluminescent signal of EcN-ePD-L1-mFc began to colonize the intestine via the gastric mucosa 6 hours after administration, with the cecum being the primary colonization site at this time point. At 12 hours after administration, colonization in the colon of the PD-L1 group was significantly higher than that of the control group. After 24 hours, the bioluminescent signal weakened due to bacterial metabolic clearance. Figure 3 A). To further verify the ability of OMVs to penetrate the intestinal epithelial barrier, mice were anesthetized and their colons were ligated. Different labeled OMVs were injected into the ligated intestinal lumen, and the colons were incubated at a constant temperature for 2 hours. The ligated colons were then excised for immunofluorescence analysis. Cell nuclei were labeled with DAPI (blue), and EcN-ePD-L1-mFc OMVs were labeled with anti-PD-L1 antibody (green). The results showed that the OMVs secreted by the engineered bacteria in the EcN-ePD-L1-mFc group could effectively penetrate the epithelial barrier. Figure 3 B). Spleens from C57 mice were ground and plated with cell suspensions. After co-incubation with OMVs of EcNΔpMUT1 / 2 and EcN-ePD-L1-mFc, flow cytometry analysis was performed. The results showed that compared to the control, the proportion of PD-L1+CD11c+ in the EcN-ePD-L1-mFc group was significantly increased, indicating that PD-L1 was significantly enriched and bound to DC cells. Simultaneously, regulatory T cells were activated. In CD4+ and CD8+ immune cells, Ki-67 levels were significantly reduced in the EcN-ePD-L1-mFc group, indicating that T cell proliferation was significantly inhibited. Furthermore, decreased expression of T-bet, which is closely related to the development and function of cytotoxic T cells, was observed. Decreased expression of RORrt, which is associated with Th17 cell formation, and decreased Th17 cell differentiation were also observed. All of these flow cytometry analyses were statistically significant. Figure 3D). These results indicate that the EcN-ePD-L1-mFc engineered bacteria can inhibit the function of immune cells and reduce their proliferative activity.

[0093] 2. Efficacy assessment of EcN-ePD-L1-mFc in treating IBD

[0094] C57BL / 6 mice were selected, weighed, and randomly divided into 5 groups: G1 (PBS group), G2 (DSS group), G3 (DSS + wild-type probiotic group), G4 (DSS + EcN-ePD-L1-mFc group), and G5 (DSS + EcN-ePD-L1-mFc + Ara group). Except for group G1, the other groups were fed 3% DSS solution throughout the experiment. Groups G3, G4, and G5 were administered wild-type and engineered probiotics via gavage on days 0, 3, 6, 9, and 12, respectively, with a bacterial load of 5 × 10⁻⁶ each time. 8 On the day of gavage, Ara was added to the drinking water of mice in group G5. Mice were sacrificed on day 13, and their body weight, colon length, liver and spleen weight, and histopathological findings in the colon were assessed. Figure 4 A). The results showed that from day 7, differences in body weight appeared among the subgroups. On day 12, the weight loss of mice in the EcN-ePD-L1-mFc+Ara treatment group was significantly inhibited, while the weight loss in the DSS and DSS+EcNΔpMUT1 / 2 groups was the most significant. Figure 4 B). The changes in colon length in each group were consistent with body weight. Specifically, the EcN-ePD-L1-mFc+Ara group showed the least significant shortening of colon length, while the DSS group showed the most significant shortening. There was a statistically significant difference in colon length between the DSS group and the EcN-ePD-L1-mFc+Ara group, and also a statistically significant difference between the DSS+EcN-ePD-L1-mFc and DSS+EcN-PD-L1-mFc+Ara groups (Figures 4C-D). This indicates that EcN-ePD-L1-mFc can alleviate IBD-related symptoms under Ara induction. Simultaneously, we dissected and analyzed the liver and spleen weights of mice in each subgroup, finding no statistically significant difference in liver and spleen weight between subgroups. Figure 4 EF). HE staining and immunohistochemical staining were performed on the colons of mice in each subgroup. The results showed that the DSS+EcN-ePD-L1-mFc+Ara group had significantly lower levels of CD3 (T cell marker), CD11c+ (DC cell marker), F4 / 80 (macrophage marker), and TNF-α (a cytokine secreted by monocytes and macrophages) than the control group, while PD-L1 expression was significantly higher than in other groups. All relevant statistical analyses were statistically significant. Figure 4The above results indicate that the engineered bacteria in the EcN-ePD-L1-mFcg+Ara group can treat colitis in mice, and mainly exert their effects by inhibiting the infiltration of various inflammatory cells, including T cells, DC cells, and macrophages.

[0095] 3. Mechanism of action of EcN-ePD-L1-mFc in the treatment of IBD

[0096] Flow cytometry analysis was performed on pericolonic lymph nodes collected from mice. The results showed that the number of CD69+ cells in the EcN-ePD-L1-mFc+Ara group was similar to that of the negative control, but significantly lower than that in the DSS, DSS+EcNΔpMUT1 / 2, and DSS+EcN-ePD-L1-mFc groups, indicating a trend of suppressed CD4+ T cell activation in the EcN-ePD-L1-mFc+Ara group. Simultaneously, a decrease in IL17 secretion from CD4+ T cells was observed, with a statistically significant difference between the DSS and EcN-ePD-L1-mFc+Ara groups. Meanwhile, the activation of naturally occurring regulatory T cells (CD4+Foxp3+) showed a sequentially increasing trend across the groups. Figure 5 AB). Immunohistochemical detection of intestinal mucosal barrier markers Claudin-1 and Occludin was performed on each of the above subgroups. The results showed that the expression of Claudin-1 and Occludin in the engineered bacteria of the EcN-ePD-L1-mFc+Ara group was significantly upregulated, indicating that the engineered bacteria can promote the repair of the intestinal mucosal barrier and inhibit the occurrence of inflammation. Figure 5 CD). Meanwhile, TUNEL apoptosis assays showed that apoptosis was significantly inhibited in the engineered bacteria of the EcN-ePD-L1-mFc+Ara group, and this was statistically significant. Figure 5 The results above show that the engineered bacteria in the EcN-ePD-L1-mFc+Ara group exert their inhibitory effect on enteritis by inhibiting IL17 expression, repairing the intestinal mucosal barrier, and inhibiting apoptosis.

[0097] 4. Effects of EcN-ePD-L1-mFc on intestinal flora and metabolism in colitis mice

[0098] Colonic contents were collected from the mice before sacrifice. 16S ribosomal RNA sequencing was used to reveal the composition and changes in the gut microbiota, and non-targeted GC-MS metabolomics was employed to analyze these changes. It was found that DSS-induced enteritis reduced the diversity of gut microbial species in mice, while treatment with engineered probiotics + Ara restored both the community and diversity of the gut microbiota. Figure 6A). Beta diversity analysis revealed significant differences in the gut microbiota species composition between the colitis model group and the EcN-ePD-L1-mFc+Ara treatment group, indicating that EcN-ePD-L1-mFc+Ara treatment caused significant changes in the gut microbiota species of colitis mice. Figure 6 B). In addition, analysis at the gut microbiota genus level revealed an increase in the abundance of harmful bacteria (Bacteroides) in the DSS-induced enteritis group, while the abundance of beneficial bacteria (Lactobacillus) was significantly restored and improved after EcN-ePD-L1-mFc+Ara treatment. Figure 6 C). Metabolomics analysis indicated that short-chain fatty acids (acetic acid and propionic acid) were decreased in the model group mice. After treatment with EcN-ePD-L1-mFc+Ara, the levels of short-chain fatty acids were restored to some extent, indicating that the treatment reduced intestinal inflammation and restored intestinal barrier function to some extent. Figure 6 D, E).

[0099] 5. Preliminary assessment of the safety of EcN-ePD-L1-mFc in treating IBD

[0100] HE sections were prepared from the heart, liver, spleen, lungs, and kidneys of the five groups of mice. The results showed no significant differences in histological morphology among the groups. Figure 7 A); Serum biochemical tests were performed on mice. The results showed no significant differences in important biochemical indicators such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), and urea (UREA). However, there was a significant difference in alkaline phosphatase (ALP) between the C2 and C5 groups, indicating that ALP reverts to an increase when enteritis is relieved. Figure 7 B).

[0101] Example 3: In vitro identification of EcN-humanPD-L1-mFc

[0102] This embodiment employs gene editing technology to edit the genes of EcN, introducing a foreign gene to induce human PD-L1-mFc expression in its outer membrane vesicles (OMVs). Bacterial OMVs were then collected, and the expression of human PD-L1 in OMVs and bacterial lysates was identified using Western blot with an anti-human PD-L1 antibody. The results showed that the experimental group's EcN lysates and OMVs highly expressed human PD-L1, while no human PD-L1 expression was observed in the control group's bacterial lysates and OMVs. This embodiment demonstrates that engineered EcN can heterologously express human PD-L1 protein (mFc). Figure 8 ).

[0103] SEQ ID NO.1:

[0104] MGMTEIVADKTVEVVKNAIETADGALDLYNKYLDQVIPWQTFDETIKELSRFKQEYSQAASVLVGDIKTLLMDSQDKYFEATQTVYEWCGVATQLLAAYILLFDEYNEKKASAQKDILIKVLDDGITKLNEAQKSLLVSSQSFNNASGKLLALDSQLTNDFSEKSSYFQSQVDKIRKEAYAGAAAGVVAGPFGLIISYSIAAGVVEGKLIPELKNKLKSVQNFFTTLSNTVKQANKDIDAAKLKLTTEIAAIGEIKTETTRFYVDYDDLMLSLLKEAAKKMINTCNEYQKRHGKKTLFEVPEVGGDYKDDDDKFTVTVPKDLYVVEYGSNMTIECKPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNER

[0105] SEQ ID NO.2:

[0106] 。

[0107] SEQ ID NO.3:

[0108]

[0109] SEQ ID NO.4:

[0110]

[0111] SEQ ID NO.5:

[0112] MGMTEIVADKTVEVVKNAIETADGALDLYNKYLDQVIPWQTFDETIKELSRFKQEYSQAASVLVGDIKTLLMDSQDKYFEATQTVYEWCGVATQLLAAYILLFDEYNEKKASAQKDILIKVLDDGITKLNEAQKSLLVSSQSFNNASGKLLALDSQLTNDFSEKSSYFQSQVDKIRKEAYAGAAAGVVAGPFGLIISYSIAAGVVEGKLIPELKNKLKSVQNFFTTLSNTVKQANKDIDAAKLKLTTEIAAIGEIKTETETTRFYVDYDDLMLSLLKEAAKKMINTCNEYQKRHGKKTLFEVPEVGGDYKDDDDKGGEFLEFTITAPKDLYVVEYGSNVTMECRFPVERELDLLALVVYWEKEDEQVIQFVAGEEDLKPQHSNFRGRASLPKDQLLKGNAALQITDVKLQDAGVYCCIISYGGADYKRITLKVNAPYRKINQRISVDPATSEHELICQAEGYPEAEVIWTNSDHQPVSGKRSVTTSRTEGMLLNVTSSLRVNATANDVFYCTFWRSQPGQNHTAELIIPELPATHPPQNRTHAS SEQ ID NO.6:

[0113] MGMTEIVADKTVEVVKNAIETADGALDLYNKYLDQVIPWQTFDETIKELSRFKQEYSQAASVLVGDIKTLLMDSQDKYFEATQTVYEWCGVATQLLAAYILLFDEYNEKKASAQKDILIKVLDDGITKLNEAQKSLLVSSQSFNNASGKLLALDSQLTNDFSEKSSYFQSQVDKIRKEAYAAGAAAGVAG PFGLIISYSIAAGVVEGKLIPELKNKLKSVQNFFTTLSNTVKQANKDIDAAKLKLTTEIAAIGEIKTETETTRFYVDYDDLMLSLLKEAAKKMINTCNEYQKRHGKKTLFEVPEVGGDYKDDDDKGGEFLEFTITAPKDLYVVEYGSNVTMECRFPVERELDLLALVVYWEKEDEQVIQFVAGEEDLKPQH SNFRGRASLPKDQLLKGNAALQITDVKLQDAGVYCCIISYGGADYKRITLKVNAPYRKINQRISVDPATSEHELICQAEGYPEAEVIWTNSDHQPVSGKRSVTTSRTEGMLLNVTSSLRVNATANDVFYCTFWRSQPGQNHTAELIIPELPATHPPQNRTHASGGGGMEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGKE

[0114] SEQ ID NO.7:

[0115]

[0116] SEQ ID NO.8:

[0117]

Claims

1. A fusion protein, characterized in that, The fusion protein is derived from the fusion of bacterial outer membrane vesicle protein and human PD-L1, or from the fusion of bacterial outer membrane vesicle protein, human PD-L1, and human Fc protein.

2. The fusion protein of claim 1, wherein, The bacterial outer membrane vesicle protein is bacterial ClyA, and the amino acid sequence of the fusion protein is any one of the following 1)-2): 1) An amino acid sequence as shown in SEQ ID NO.1 or SEQ ID NO.2; 2) An amino acid sequence that has the same function as the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2, obtained by replacing, deleting or inserting one, several or dozens of amino acids in the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.

2.

3. A fusion gene, characterized in that, The fusion gene is any one of the following 1)-3): 1) It has a nucleotide sequence as shown in SEQ ID NO.3 or SEQ ID NO.4; 2) Possesses a polynucleotide that can encode a protein sequence as shown in SEQ ID NO.1 or SEQ ID NO.2; 3) Has 75% or more identity with the DNA fragment defined in 1) or 2), and encodes a protein that is functionally equivalent to the protein shown in SEQ ID NO.1 or SEQ ID NO.

2.

4. An expression vector, characterized by, The expression vector includes a gene expressing the fusion protein of claim 1 and a basic vector.

5. A genetically engineered bacterium, characterized by, The genetically engineered bacteria include probiotics and the expression vector described in claim 4.

6. The genetically engineered bacteria according to claim 5, characterized in that, The probiotics include any one or more of the following: *Escherichia coli*, *Aktermannii*, *Bifidobacterium adolescentis*, *Bifidobacterium lactis*, *Bifidobacterium bifidum*, *Bifidobacterium breve*, *Bifidobacterium longum*, *Bifidobacterium infantis*, *Lactobacillus acidophilus*, *Lactobacillus curvatureii*, *Lactobacillus bulgaricus*, *Lactobacillus delbrueckii* subsp. *lactobacter*, *Lactobacillus gasseri*, *Lactobacillus helveticus*, *Lactobacillus casei*, *Lactobacillus paracasei*, *Lactobacillus rhamnosus*, *Lactobacillus fermentum*, *Lactobacillus reuteri*, *Lactobacillus plantarum*, *Lactobacillus salivarius*, *Lactobacillus curvatureii*, *Lactobacillus sakei*, *Streptococcus thermophilus*, *Lactococcus lactis* subsp. *lactobacter*, *Lactococcus lactis*, *Lactococcus lactis* subsp. *lactobacter*, *Lactococcus lactis* subsp. *lactobacter*, *Propionibacterium fischeri* subsp. *she*, *Propionibacterium propionate*, *Leuconostoc mesenteroides* subsp. *enteroides*, *Pediococcus lactis*, *Pediococcus pentosaceus*, *Bacillus coagulans*, *Staphylococcus calf*, *Staphylococcus xylose*, *Staphylococcus carminatis*, and *Klu 7. The use of the fusion protein of claims 1-2, the fusion gene of claim 3, the expression vector of claim 4, or the genetically engineered bacteria of any one of claims 5-6 in the preparation of drugs for digestive system diseases, systemic autoimmune diseases, intestinal aGVHD, or liver aGVHD.

8. Use according to claim 7, characterized in that, The digestive system diseases mentioned include inflammatory bowel disease.

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