Construction method of engineered lactic acid bacteria for constitutive expression of SodA protein and application of engineered lactic acid bacteria in treatment of IBD

Abstract

The invention provides a construction method of engineered lactic acid bacteria for constitutive expression of SodA protein and application of the engineered lactic acid bacteria in treatment of IBD. The invention provides a construction method of engineered lactic acid bacteria (KI (at) lSodA) for relieving IBD (Infectious Bronchitis Disease) constitutive expression SodA protein. A verification experiment result shows that the KI (at) lSodA highly expresses the SodA protein, and the oxidative stress management capability of the strain is improved; meanwhile, the KI (at) lSodA also has long-term use safety, the treatment of the KI (at) lSodA remarkably relieves the colitis symptoms and in-vivo inflammation conditions of mice in an acute stage, a recovery stage and a chronic state, and the KI (at) lSodA shows a strong intestinal microbial diversity recovery capability in all three experimental models, and particularly shows a remarkable repair effect in a recovery stage model. Therefore, the invention provides a new basis for IBD treatment.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and relates to construction of an engineered bacterium for treating inflammatory bowel disease (IBD), in particular to a method for constructing an engineered lactic acid bacterium constitutively expressing SodA protein and application of the engineered lactic acid bacterium in treating IBD. BACKGROUND

[0002] Inflammatory bowel disease (IBD) includes ulcerative colitis (UC) and Crohn's disease (CD), which is a kind of idiopathic intestinal inflammatory disease involving ileum, rectum and colon. The clinical manifestations are diarrhea, abdominal pain, and even bloody stool, and the disease is recurrent, which has a serious impact on the physical and mental health of patients. However, most of the current treatment methods mainly focus on inhibiting inflammation, rather than targeting the root cause of IBD, such as excessive production of reactive oxygen species (ROS), intestinal barrier dysfunction and intestinal microbiota imbalance. In addition, long-term use of these drugs often leads to serious adverse reactions. Therefore, there is an urgent need to develop new treatment strategies that target the root cause of IBD and minimize non-target effects in order to ultimately improve treatment outcomes.

[0003] More and more evidence links intestinal microbiota imbalance to the pathogenesis of IBD, which is characterized by excessive growth of commensal bacteria during initial inflammation, thereby exacerbating disease symptoms. Therefore, probiotics have become a potential treatment option due to their ability to restore intestinal microecosystem homeostasis. In recent years, there have been a growing number of engineered commensal / probiotic bacteria targeting inflammatory bowel disease, such as Escherichia coli Nissle 1917, Lactobacillus casei, etc. Existing design schemes can be roughly divided into two categories: one is to make the bacteria continuously express exogenous (human) proteins through genetic engineering technology, and the other is to construct special bacterial coatings (such as selenium attachment). The problems of these two types of design schemes include: technical complexity, and unstable reproducibility for ordinary skilled persons in the art; the strains have not been safety verified and approved, and the clinical conversion efficiency is low; the efficacy has only been verified in DSS-induced acute colitis in mice, and the role in the recovery and chronic stages of intestinal inflammation has not been explored.

[0004] SodA protein is a manganese superoxide dismutase, which is one of the key members of the superoxide dismutase family and plays an irreplaceable role in the process of resisting oxidative stress in organisms. In eukaryotes (including humans), SodA protein is specifically located in mitochondria, and since mitochondria is the main place where superoxide anions are generated, SodA protein is crucial to protect cells from oxidative damage from the inside. The main function of SodA is to catalyze the dismutation reaction of superoxide anion radicals to generate oxygen and hydrogen peroxide, and this reaction is the first and most important line of defense for organisms to defend against active oxygen toxicity.

[0005] Recombinating SodA and engineering strains to improve the activity of SodA and enhance the tolerance of strains to oxidative stress, prolong their service life and stability is one of the current research hotspots. However, whether SodA protein has an effect in IBD treatment and whether it can be used to treat long-term IBD after being recombinated with engineering strains have not been reported. SUMMARY

[0006] The present application aims at the above-mentioned problems, and provides a method for constructing an engineered lactic acid bacteria constitutively expressing SodA protein, and verifies its treatment of IBD.

[0007] The research idea of the present application is as follows: the present application first constructs an engineered lactic acid bacteria constitutively expressing lSodA protein (referred to as KI@lSodA), and confirms the expression efficiency of protein lSodA and the growth rate; then, safety verification is carried out through nitrate reductase detection experiment, amino acid decarboxylase detection experiment, indole experiment, K-B method (drug sensitive paper disc agar diffusion method) drug sensitive experiment and hemolysis experiment; further, the treatment effect of KI@lSodA in three DSS-induced enteritis models (acute phase, recovery phase and chronic phase) is verified, and the changes of intestinal flora of mice in the three enteritis models are analyzed.

[0008] The results showed that the transcriptional level of sodA in KI@lSodA was approximately 50 times that of wild-type Lactococcus lactis NZ9000. The recombinant strain exhibited a significantly prolonged logarithmic growth phase, higher maximum biomass, and improved oxidative stress management, indicating that SodA-mediated redox homeostasis supported sustained growth. Nitrate reductase, amino acid decarboxylase, indole, and hemolysis assays were all negative, and no significant antibiotic resistance was observed in the engineered strain. Histological analysis of long-term biosafety showed no significant changes in the spleen, intestine, colon, brain, or liver in any group, and blood factor indicators were normal, suggesting good safety. KI@lSodA treatment significantly alleviated colitis symptoms and inflammation in mice. The KI@lSodA mutant demonstrated a strong ability to restore intestinal microbial diversity in all three experimental models, especially showing a significant repair effect in the recovery phase model.

[0009] Based on the above research, the technical solution to be protected by this invention is as follows:

[0010] In a first aspect, this invention provides a method for constructing engineered lactic acid bacteria constitutively expressing the SodA protein. The core steps are as follows: primer pairs P1 and P2 are used to amplify the 5' upstream homologous arm of the target gene, and primer pairs P3 and P4 are used to amplify the 3' flanking region of the target gene; to connect the three fragments, primers P1, P2, P3, and P4 are designed to overlap with the target fragments, and each DNA fragment is connected by PCR-mediated overlap amplification. Subsequently, the target PCR product is transformed into Lactobacillus NZ9000 competent cells, and the homologous recombination mutant positive clone verified by PCR amplification and sequencing is named KI@lSodA.

[0011] As a preferred method, the transformation procedure for Lactobacillus NZ9000 is as follows: Lactobacillus NZ9000 colonies are cultured in M17 medium. When the cell absorbance (OD600) reaches 0.2-0.3, they are placed on ice and centrifuged at 4,000 ×g at 4°C. The cells are then suspended in sterile 0.5M sucrose solution for a certain period of time, and then centrifuged again at 4°C at 4,000 ×g. The supernatant is used to resuspend the cells for electroporation, with the parameters set as follows: 2.5 kV, 200 ohms, 5 ms.

[0012] Further optimization is achieved by the following method for culturing Lactobacillus NZ9000: Lactobacillus NZ9000 colonies are placed in M17 medium and cultured overnight at 32°C. Subsequently, the bacterial solution is inoculated into fresh M17 medium containing 1-2% glycine at a volume ratio of 1:100. The bacterial solution is placed on ice for 10 minutes and centrifuged for 10 minutes. The cells are suspended in sterile 0.5M sucrose solution for 30 minutes and centrifuged for 10 minutes.

[0013] As a preferred method, the positive clone of homologous recombination mutant is as follows: screen for targeted knockout Lactobacillus NZ9000 mutants by culturing on M17 agar plates containing antigens corresponding to the target gene resistance gene, and further confirm them by PCR or sequence detection.

[0014] When using PCR for identification, the colonies are identified by PCR using primers P5 and P6, and the gene sequences of P5 and P6 are shown in SEQ ID NO.5~6, respectively. When confirming by sequence detection, primers P7, P8 and P9 are used, and the gene sequences of P7~P9 are shown in SEQ ID NO.7~9, respectively.

[0015] The primer sequences are shown in Table 1 below.

[0016] Further optimization revealed that the 3' flanking region of the target gene contains a chloramphenicol resistance gene; therefore, a culture medium containing chloramphenicol was used for screening positive clones of homologous recombination mutants. Electroporation was used to insert a strong P32 promoter into the NZ9000 gene sequence to facilitate rapid and massive proliferation of recombinant bacteria within the host.

[0017] In a second aspect, this invention provides engineered lactic acid bacteria constitutively expressing SodA protein, constructed using the above method. Experimental results show that the recombinant bacteria highly express SodA protein, and SodA-mediated redox homeostasis supports its continuous growth. Simultaneously, its safety and therapeutic efficacy were also confirmed. In the recovery model, KI@lSodA administration significantly enriched *Ackermania* and *Ellerythrella*, mucodegrading commensal bacteria belonging to the Bacteroidetes family. Both strains are recognized as having anti-inflammatory potential and can intervene in the pathogenesis of IBD. Furthermore, the relative abundance of *Enterococcus*, a commensal bacteria associated with Crohn's disease, was significantly reduced in the KI@lSodA administration group.

[0018] Therefore, in a third aspect, the present invention provides the application of engineered lactic acid bacteria constitutively expressing SodA protein as described above in the preparation of intestinal flora conditioning agents; preferably in the preparation of therapeutic agents for inflammatory bowel disease.

[0019] In a fourth aspect, the present invention provides a therapeutic agent for inflammatory bowel disease, comprising an active component and pharmaceutically acceptable excipients, wherein the functional component is the engineered lactic acid bacteria constitutively expressing the SodA protein as described above.

[0020] The beneficial protections and effects of this invention are as follows:

[0021] This invention explores the application of SodA protein in the treatment of IBD and provides a method for constructing an engineered lactic acid bacteria (KI@lSodA) that constitutively expresses SodA protein to alleviate IBD. Results show that KI@lSodA highly expresses SodA protein, and the strain's ability to manage oxidative stress is improved. KI@lSodA also demonstrates long-term safety, and treatment with KI@lSodA significantly alleviated colitis symptoms and inflammation in mice during the acute, recovery, and chronic phases. It exhibited a strong ability to restore intestinal microbial diversity in all three experimental models, especially showing a significant repair effect in the recovery model. Therefore, this invention provides a new basis for IBD treatment. Attached Figure Description

[0022] Figure 1 The PCR validation and sequencing results are shown, verifying the successful construction of KI@lSodA.

[0023] Figure 2 The results show that SodA-mediated redox homeostasis supports continuous growth. From left to right, the comparisons of SodA protein expression levels, SodA protein bands, and growth curves between KI@lSodA and NZ9000 are shown.

[0024] Figure 3 The SEM and TEM images of KI@lSodA are shown;

[0025] Figure 4 The results of safety and antibiotic resistance tests for KI@lSodA were presented.

[0026] Figure 5 The study demonstrated the safety effects of KI@lSodA on organ tissues and blood factors.

[0027] Figure 6 The study compared the treatment process and weight loss reduction effects of KI@lSodA and NZ9000 in the acute phase of acute enteritis, the recovery phase of acute enteritis, and chronic colitis.

[0028] Figure 7 The comparison of the effects of KI@lSodA and NZ9000 treatment on increasing colon length is shown;

[0029] Figure 8 The comparison of the effects of KI@lSodA and NZ9000 treatment on improving endoscopic scores was shown;

[0030] Figure 9 The study compared the effects of KI@lSodA and NZ9000 treatments on reducing the Disease Activity Index (DAI).

[0031] Figure 10 The results of histopathological analysis (H&E staining) in the KI@lSodA and NZ9000 treatment groups are shown in comparison.

[0032] Figure 11 The comparison of the reduction in fibrosis between the KI@lSodA and NZ9000 treatment groups was shown;

[0033] Figure 12 The study compared the reduction in ROS levels in colon tissue between the KI@lSodA and NZ9000 treatment groups.

[0034] Figure 13 The results of flow cytometry and immunofluorescence assays of macrophage-related cells in colon tissue were compared between the KI@lSodA and NZ9000 treatment groups.

[0035] Figure 14 The results of flow cytometry and immunofluorescence assays of neutrophils in colon tissue were compared between the KI@lSodA and NZ9000 treatment groups.

[0036] Figure 15 The comparison of changes in inflammatory factors between the KI@lSodA and NZ9000 treatment groups was shown;

[0037] Figure 16 The results showed that the levels of Lactococcus lactis in the colon of mice treated with KI@lSodA and NZ9000 were both increased;

[0038] Figure 17 The results showed that the KI@lSodA mutant exhibited a strong ability to restore gut microbial diversity in all three experimental models, especially showing a significant repair effect in the recovery model;

[0039] Figure 18 The results of principal coordinate analysis (PCoA) and principal component analysis (PCA) are displayed;

[0040] Figure 19 The effects of KI@lSodA on different intestinal bacteria in mouse enteritis were shown. Detailed Implementation

[0041] The following embodiments further illustrate the present invention and should not be construed as limiting the invention. The embodiments do not include a detailed description of conventional methods, which are well known to those skilled in the art and described in numerous publications.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to this invention, and the preferred embodiments and materials described in the specific implementation are for illustrative purposes only.

[0043] Unless otherwise specified, the experimental methods described in the following examples are generally performed under standard conditions or as recommended by the manufacturer.

[0044] I. KI@lSodA Construction

[0045] The method for constructing the constitutively lSodA-expressing Lactococcus lactis subsp. cremoris NZ9000 genetically engineered bacterium (KI@lSodA) is as follows:

[0046] (1) Construction of recombinant plasmids: Three pairs of gene-specific primers (P1 and P2, P3 and P4, P5 and P6) were designed. P1 and P2 were used to amplify the 5' upstream homologous arm of the target gene, and P3 and P4 were used to amplify the 3' flanking region of the target gene containing the chloramphenicol resistance gene. To achieve ligation of the two fragments and the inserted sequence (SOD protein), primers P1, P2, P3, and P4 were designed to overlap with the target fragments. The DNA fragments were ligated by PCR-mediated overlap amplification, and the target PCR product was then transformed into Lactobacillus NZ9000 competent cells. The positive clone verified by PCR amplification and sequencing was named KI@lSodA. The primer sequences for SOD protein amplification are shown in Table 1.

[0047] (2) Transformation of Lactobacillus NZ9000: Lactobacillus NZ9000 colonies were cultured overnight at 32°C in M17 medium. Then, 1 mL of the bacterial culture was inoculated into 100 mL of fresh M17 medium containing 1-2% glycine. When the cell absorbance (OD600) reached 0.2-0.3, the cells were placed on ice for 10 minutes and collected by centrifugation at 4°C at 4,000 × g for 10 minutes. The cells were resuspended in sterile 0.5 M sucrose solution for 30 minutes and then centrifuged at 4°C at 4,000 × g for 10 minutes. Finally, the cells were resuspended in 2 mL of 0.5 M sucrose solution for electroporation. Lactococcus lactis NZ9000 was transformed using electroporation with the following parameters: 2.5 kV, 200 ohms, 5 ms.

[0048] (3) Confirmation of homologous recombination mutants: Targeted knockout Lactobacillus NZ9000 mutants were screened by culturing on M17 agar plates containing 17 μg / mL chloramphenicol. Colonies were identified by PCR using primers P5 and P6, or sequenced using primers P7, P8, and P9.

[0049] The primer sequences involved in the above steps, as well as the primer sequences involved in the following verification experiments, are shown in Table 1 below:

[0050] Table 1 Summary of oligonucleotide sequences involved in this invention

[0051]

[0052]

[0053] II. KI@lSodA Detection and Effect Verification

[0054] 1. Physical performance verification: The P32 strong promoter was inserted into the NZ9000 gene sequence by electroporation. The successful construction of the engineered bacteria KI@lSodA and the expression efficiency of the protein lSodA were confirmed by PCR, sequencing, qPCR and Western blotting (WB). The physical characterization of the engineered bacteria was observed by scanning electron microscopy and transmission electron microscopy. KI@lSodA was cultured at 37 degrees and its bacterial concentration was detected every hour and growth curves were plotted.

[0055] 2. Safety verification of engineered bacteria KI@lSodA: Nitrate reductase detection experiment, amino acid decarboxylase detection experiment, indole test, KB method (antibiotic susceptibility paper disk agar diffusion method) drug susceptibility test, and hemolysis test. The safety of NZ9000 and KI@lSodA lactic acid bacteria suspensions (10...) were tested by oral administration of... 8 CFU / mice, administered every other day for 42 days) to assess long-term biocompatibility. Blood samples were collected on day 42 for complete blood count and blood biochemistry analysis. Major organs (heart, liver, lung, spleen and kidney) were taken for H&E histological analysis.

[0056] 3. Verify the therapeutic effects of wild-type NZ9000 and KI@lSodA in three DSS-induced enteritis models: (1) Acute phase: 9-week-old mice were orally administered PBS, 100 µL of wild-type lactic acid bacteria NZ9000 or genetically engineered bacteria KI@lSodA every two days during 2.3% DSS exposure, and the severity of intestinal tissue inflammation was assessed on day 7; (2) Recovery phase: After DSS induction, mice were treated with PBS, 100 µL of NZ9000 or KI@lSodA every two days, and the healing status was assessed on day 17; (3) Chronic phase: Low-dose 1.8% DSS was administered for 42 consecutive days, and PBS, 100 µL of NZ9000 or KI@lSodA was orally administered every two days. CFU / mL of wild-type lactic acid bacteria NZ9000 or genetically engineered bacteria KI@lSodA was used to evaluate long-term protective effects. Mice were weighed daily during the period, and their pathological scores (DAI) were assessed. On the last day, colonic pathological changes were observed using colonoscopy, and the mice were then treated.

[0057] The length of the colon in treated mice was measured. The ROS level of colonic epithelial cells was detected by DCFH-DA method. The content of neutrophils and macrophages in the lamina propria of the colon was detected by flow cytometry. The expression levels of MPO and Occludin in the colon, as well as the number of macrophages and neutrophils, were detected by immunofluorescence. The degree of colonic cell apoptosis was detected by TUNEL assay. The levels of inflammatory factors IL-6, IL-10, IL-1β, TNF-α and TGF-β in mouse colonic tissue were detected by enzyme-linked immunosorbent assay (ELISA) and quantitative real-time PCR (qPCR) (primers used are shown in Table 1 above). The CALP level in mouse colonic feces was detected by ELISA to assess prognosis. Hematoxylin and eosin staining (H&E) was used to assess colonic pathological changes. Fluorescence in situ hybridization (FISH) was used to detect the content of Lactococcus lactis in colonic microbiota. Masson staining was used to assess the degree of colonic fibrosis in mice.

[0058] 4. Verify the regulatory effect of KI@lSodA on the structure and function of the gut microbiome in the IBD model: Fecal microbial 16S rRNA sequencing analysis revealed changes in the gut microbiota of mice in three enteritis models.

[0059] III. Experimental Results

[0060] 1. Construction and validation of KI@lSodA constitutively expressing lSodA protein: PCR validation and sequencing results show the successful construction of KI@lSodA. Figure 1qPCR showed that the transcriptional level of sodA was approximately 50 times that of wild-type Lactococcus lactis NZ9000. Furthermore, a clearly observed high-expression band of SodA protein was observed in KI@lSodA during the logarithmic growth phase. The growth curve showed that the initial proliferation rate of the KI@lSodA strain was slow, but its logarithmic growth phase was significantly prolonged (exceeding 30 hours in M17 medium) and its maximum biomass was higher. This indicates that SodA-mediated redox homeostasis supported sustained growth. Figure 2 Scanning electron microscopy (SEM) revealed a significant reduction in cell size; the KI@lSodA mutant exhibited higher cell surface roundness and more abundant budding structures, while transmission electron microscopy (TEM) showed a reduction in autophagy-like vesicles in the cytoplasm of the KI@lSodA mutant. Figure 3 This indicates an improved ability to manage oxidative stress. In summary, we have successfully constructed a *Lactococcus lactis* strain that consistently expresses the lSodA protein at high levels.

[0061] 2. Safety verification of KI@lSodA: Nitrate reductase detection, amino acid decarboxylase detection, indole test, and hemolysis test were all negative. Antibiotic resistance was assessed using the Kirby-Bauer assay, which showed no significant antibiotic resistance observed in the engineered strain. Figure 4 Histological analysis during long-term biosafety studies showed no significant changes in the spleen, intestines, colon, brain, or liver in any group. This safety profile was further confirmed by routine blood tests, including blood cell parameters such as white blood cells, red blood cells, hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin content, hemoglobin, and platelets, which showed no significant differences compared to the healthy control group mice. Similarly, liver and kidney function parameters, such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin (ALB), and blood urea nitrogen (BUN), were all within the normal range. Figure 5 ).

[0062] 3. Validation of KI@lSodA efficacy: The effects of NZ9000 and engineered bacteria KI@lSodA in the acute, recovery, and chronic colitis models induced by DSS: Treatment with both NZ9000 and KI@lSodA alleviated colitis symptoms in mice, including reducing weight loss. Figure 6 ), increase colon length ( Figure 7 ), improve endoscopy score ( Figure 8 ) and reduce the disease activity index (DAI) Figure 9The KI@lSodA group showed the most significant therapeutic effect. Histopathological analysis (H&E staining) showed that, compared with the PBS treatment group, the colonic structure of mice treated with KI@lSodA was close to normal, with intact epithelial layer, preserved crypt structure, and mild inflammatory infiltration. The NZ9000 treatment group also alleviated enteritis symptoms to some extent. Figure 10 Meanwhile, the degree of fibrosis decreased in the NZ9000 and KI@lSodA treatment groups. Figure 11 The DCFH-DA method showed that KI@lSodA treatment significantly reduced ROS levels in colonic tissue. Figure 12 Flow cytometry and immunofluorescence showed that KI@lSodA treatment reduced inflammatory cells, including macrophages and neutrophils, in mouse colon tissue, with a decrease in the proportion of M1 macrophages and an increase in the proportion of M2 macrophages. Figure 13 , Figure 14 Both ELISA and qPCR results showed that treatment with both bacteria reduced pro-inflammatory factors IL-6, IL-1β, and TNF-α in mouse colon tissue and increased anti-inflammatory factors IL-10 and TGF-β. The reduction in CALP in the protein treatment group also indicated a good prognosis. Figure 15 FISH results showed that the content of Lactococcus lactis in the colon of mice in the NZ9000 and KI@lSodA groups was increased. Figure 16 ).

[0063] 4. Analysis of Gut Microbiota Improvement: Analysis of the ACE index and Chao index revealed that the KI@lSodA mutant exhibited a strong ability to restore gut microbiota diversity in all three experimental models, especially showing a significant repair effect in the recovery phase model. Figure 17 The integrated analysis of principal coordinate analysis (PCoA) and principal component analysis (PCA) further confirmed that the therapeutic effect of KI@lSodA intervention was most significant in the recovery model, and significantly altered the abundance and diversity of the gut microbiota in the recovery IBD model. Figure 18 In the recovery model, KI@lSodA administration significantly enriched mucodegrading commensal bacteria belonging to the Bacteroidetes family—Ackermania and Ilex spp. Both strains are recognized as having anti-inflammatory potential and can intervene in the pathogenesis of IBD. In addition, the relative abundance of Enterococcus, a commensal bacteria associated with Crohn's disease, was significantly reduced in the KI@lSodA administration group. Figure 19 ).

[0064] The undescribed parts of this invention are the same as or implemented using existing technology. The applicant declares that this invention is illustrated through the above embodiments, but the invention is not limited to the above detailed methods, i.e., it does not mean that the invention must rely on the above detailed methods to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the product of this invention, additions of auxiliary components, and selection of specific methods all fall within the protection and disclosure scope of this invention.

Claims

1. A method for constructing an engineered lactic acid bacterium constitutively expressing a SodA protein, characterized in that, Comprising the following steps: The 5' upstream homologous arm of the target gene is amplified by primer pair P1 and P2, and the 3' flanking region of the target gene is amplified by primer pair P3 and P4; in order to connect the three fragments, P1, P2, P3 and P4 primers are designed to overlap with the target fragment, and each DNA fragment is connected by PCR-mediated overlapping amplification, then the target PCR product is transformed into Lactobacillus lactis NZ9000 competent cells, and the positive clone of homologous recombination mutant verified by PCR amplification and sequencing is named KI@lSodA, wherein the gene sequences of P1, P2, P3 and P4 are shown in SEQ ID NO. 1-4.

2. The construction method of claim 1, wherein, The transformation method of Lactobacillus lactis NZ9000 is as follows: Lactobacillus lactis NZ9000 colonies are cultured in M17 medium, when the O.D.600 cell absorbance reaches 0.2-0.3, they are placed on ice and centrifuged at 4,000 ×g at 4℃ to collect; then the cells are suspended in sterile 0.5M sucrose solution for a certain time, and then centrifuged at 4,000 ×g at 4℃, and the supernatant is used for resuspension of cells for electroporation, with parameters set as: 2.5 kilovolts, 200 ohms, 5 milliseconds.

3. The construction method of claim 2, wherein, The culture method of Lactobacillus lactis NZ9000 is as follows: Lactobacillus lactis NZ9000 colonies are placed in M17 medium and cultured at 32℃ overnight, then the bacterial solution is inoculated into fresh M17 medium containing 1-2% glycine at a volume ratio of 1:100; The time for placing the bacterial solution on ice is 10 minutes, and the centrifugation time is 10 minutes; The time for suspending the cells in sterile 0.5M sucrose solution is 30 minutes, and the centrifugation time is 10 minutes.

4. The construction method of claim 1, wherein, The confirmation method of the positive clone of homologous recombination mutant is as follows: Lactobacillus lactis NZ9000 mutants targeted for knockout are screened by culturing on M17 agar plates containing antigens corresponding to the resistance genes of the target gene, and further confirmed by PCR or sequence detection.

5. The construction method according to claim 4, characterized in that, When identified by PCR, the colonies are identified by PCR with P5 and P6 primers, and the gene sequences of P5 and P6 are shown in SEQ ID NO. 5-6; When confirmed by sequence detection, P7, P8 and P9 primers are used, and the gene sequences of P7-P9 are shown in SEQ ID NO. 7-9. The 3' flanking region of the target gene contains a chloramphenicol resistance gene; the electroporation method inserts a P32 strong promoter into the NZ9000 gene sequence.

6. The method of construction of claim 2, wherein, Obtained by the method of any one of claims 1-6.

7. An engineered lactic acid bacterium constitutively expressing a SodA protein, characterized in that, 8. The use of the engineered lactic acid bacteria constitutively expressing SodA protein of claim 7 in the preparation of an intestinal flora conditioner.

9. The use of the engineered lactic acid bacteria constitutively expressing SodA protein of claim 7 in the preparation of an inflammatory bowel disease treatment agent. A composition comprising an active ingredient and a pharmaceutically acceptable excipient, wherein the functional component is the engineered lactic acid bacteria constitutively expressing SodA protein of claim 7.

10. A therapeutic agent for inflammatory bowel disease, characterized in that, ​

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