EcNc engineering bacteria constitutively expressing MLH protein and application thereof in treatment of enteritis

Abstract

The application provides an EcNc engineering bacterium constitutively expressing MLH protein and application in treating intestinal inflammation, and belongs to the technical field of biological medicine. The application provides an EcNc engineering bacterium constitutively expressing MLH protein, taking Escherichia coli Nissle 1917 (EcN) as a chassis cell, and then introducing the reconstructed endogenous cryptic plasmid pMUT1 and / or pMUT2 after removing two hidden plasmids; the reconstruction is that a fusion protein MLH coding gene formed by using a flexible linker to connect SOD and CAT is inserted into pMUT1 and / or pMUT2. The application constructs an engineering bacterium constitutively expressing a fusion protein, and the engineering bacterium can express the fusion protein with double enzyme activities of SOD and CAT without antibiotic pressure and IPTG induction. The engineering bacterium constructed by the application can be used as an engineered microorganism for treating UC disease, for example, as a live bacterial drug.

Description

TECHNICAL FIELD

[0001] The application provides the technical field of biological medicine, and particularly relates to an EcNc engineering bacterium constitutively expressing MLH protein and application thereof in treatment of enteritis. BACKGROUND

[0002] Ulcerative colitis (UC) is a chronic inflammatory bowel disease (IBD) whose cause is not yet fully clear. The main clinical symptoms of UC include bloody diarrhea, abdominal pain, urgency and tenesmus, and may be accompanied by extraintestinal manifestations such as anemia, fever, weight loss and other systemic symptoms, which is a disease that seriously affects human health. Traditional chemical drugs for treating UC have the problems of incomplete treatment, unpredictable disease recurrence, and harm to the physical and mental health of patients. Chemical drugs can only relieve symptoms and cannot fundamentally cure UC, and have a great impact on other organs of the human body. Moreover, the current market gastrointestinal drugs for treating chronic colitis are one-sided and single, and can only repair the mucosa or kill pathogenic bacteria.

[0003] In recent years, emerging treatment strategies such as biological agents and fecal microbiota transplantation (FMT) have shown important value in the treatment of UC. Biological agents, in particular, have shown significant performance in relieving moderate to severe UC cases, but there is currently no live bacterial drug with significant effect that can be used to treat UC. SUMMARY

[0004] The application provides an EcNc engineering bacterium constitutively expressing MLH protein and application thereof in treatment of enteritis, and develops an engineering bacterium EcNc-MLH constitutively expressing fusion protein RtSOD-CAT (MLH), which can be used as a live biotherapeutic product (LBP) for the prevention and treatment of enteritis.

[0005] The application provides an EcNc engineering bacterium constitutively expressing MLH protein, which takes Escherichia coli Nissle1917 as a chassis cell and contains a modified endogenous cryptic plasmid pMUT1 and / or pMUT2.

[0006] The modification includes inserting a coding gene of MLH protein into the endogenous cryptic plasmid pMUT1 and / or pMUT2, and the MLH protein is a fusion protein formed by connecting SOD and CAT using a flexible linker.

[0007] In a preferred mode of the application, the SOD includes RtSOD with GenBank number WP_012843426.1, and the CAT includes FeCAT with GenBank number WP_012845258.1.

[0008] The application provides a construction method of the EcNc engineering bacterium, and comprises the following steps: (1) inserting a coding gene of SOD into endogenous cryptic plasmid pMUT1 and / or pMUT2, and constructing linearized pMUT1-SOD and / or pMUT2-SOD plasmids by a PCR method;

[0009] (2) inserting a coding gene of CAT into a prokaryotic expression vector, and constructing a linearized linker-CAT gene fragment by a PCR method;

[0010] (3) connecting the linearized pMUT1-SOD and / or pMUT2-SOD plasmid in step (1) with the linearized linker-CAT gene fragment in step (2), so as to construct a recombination vector pMUT1-MLH and / or pMUT2-MLH;

[0011] (4) transforming the recombination vector pMUT1-MLH and / or pMUT2-MLH in step (3) into Escherichia coli Nissle 1917 from which two endogenous cryptic plasmids pMUT1 and pMUT2 are removed, so as to construct the EcNc engineering bacterium.

[0012] In one preferred mode of the application, the primers used in the PCR method in step (1) comprise pMRtSOD-X-F and pMRtSOD-X-R, wherein the nucleotide sequence of pMRtSOD-X-F is shown in SEQ ID No. 1, and the nucleotide sequence of pMRtSOD-X-R is shown in SEQ ID No. 2.

[0013] In one preferred mode of the application, the primers used in the PCR method in step (2) comprise L-FeCAT-F and L-FeCAT-R, wherein the nucleotide sequence of L-FeCAT-F is shown in SEQ ID No. 3, and the nucleotide sequence of L-FeCAT-R is shown in SEQ ID No. 4.

[0014] The application further provides application of the EcNc engineering bacterium or the EcNc engineering bacterium constructed by the construction method in preparation of an antioxidant preparation.

[0015] The application further provides application of the EcNc engineering bacterium or the EcNc engineering bacterium constructed by the construction method in preparation of a medicine for preventing and / or treating enteritis.

[0016] In one preferred mode of the application, the type of enteritis comprises ulcerative colitis.

[0017] In one preferred mode of the application, the type of the medicine for preventing and / or treating enteritis comprises a live bacterium medicine.

[0018] The application further provides a medicine for preventing and / or treating enteritis, wherein the active ingredient comprises the EcNc engineering bacteria or the EcNc engineering bacteria constructed by the construction method, and further comprises a pharmaceutically acceptable adjuvant.

[0019] Beneficial effects: the application provides an EcNc engineering bacteria constitutively expressing MLH protein, which takes Escherichia coli Nissle 1917 (EcN) as a chassis cell, and then inserts the improved endogenous cryptic plasmid pMUT1 and / or pMUT2 into the chassis cell after removing two cryptic plasmids; wherein the improvement is that the fusion protein MLH gene formed by connecting SOD and CAT by a flexible linker is inserted into pMUT1 and / or pMUT2. The application constructs the engineering bacteria EcNc-MLH constitutively expressing the fusion protein RtSOD-CAT (MLH), and the engineering bacteria EcNc-MLH can express the SOD and CAT double-enzyme fusion protein without antibiotic pressure and IPTG induction, and has the enzyme activities of SOD and CAT, and is also relatively stable in the environment of pH 3-11 and artificial intestinal fluid, thereby further improving the application value of the EcN engineering bacteria strain.

[0020] The engineering bacteria EcNc-MLH constructed in the application can significantly reduce DSS-induced intestinal inflammation of mice, and promote the intestinal mucosal healing of UC mice. Biochemical analysis shows that the contents of SOD and CAT in intestinal tissues are significantly increased, the content of malondialdehyde (MDA) is significantly reduced, and the mRNA expression levels of inflammatory factors IL-6 and IL-1 beta are also reduced. The engineering bacteria EcNc-MLH can be used as an engineered microorganism for treating UC disease, such as a live biotherapeutic product (LBP) for preventing and treating enteritis, and has a good application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 PCR electrophoresis map for constructing plasmid pMUT1-MLH, wherein A: linker-FeCAT target fragment; B: linearized pMUT1-RtSOD vector;

[0022] Figure 2 Construction and protein expression identification results of EcNc-MLH engineering bacteria expressing MLH fusion protein, wherein A: schematic diagram of recombinant cryptic plasmid pMUT1-MLH and pMUT2-MLH; B: SDS-PAGE identification of EcN engineering bacteria constitutively expressing recombinant MLH fusion protein, band 1 represents no IPTG induction; band 2 represents IPTG induction;

[0023] Figure 3Figure for MLH stability test results, A: SOD and CAT enzyme of MLH high temperature resistance test; B: SOD and CAT enzyme of MLH acid and alkali resistance test; C: SOD and CAT enzyme of MLH artificial gastric juice resistance test; D: SOD and CAT enzyme of MLH artificial intestinal juice resistance test;

[0024] Figure 4 Figure for EcNc-MLH engineering strain for the treatment of UC mice, A: changes in body weight of mice; B: changes in DAI of mice; C: macroscopic changes in colon length of mice; D: statistical analysis of colon length of mice; E: macroscopic changes in spleen size of mice; F: statistical analysis of spleen size of mice; G: HE staining of colon of mice;

[0025] Figure 5 Figure for the influence of EcN engineering strain on the oxidative stress level and cytokines of UC mice, A: MDA content; B: CAT content; C: SOD content; D: IL-1β level; E: IL-6 level. DETAILED DESCRIPTION

[0026] The application provides an EcNc engineering strain constitutively expressing MLH protein, which is based on Escherichia coli Nissle1917 and comprises an improved endogenous cryptic plasmid pMUT1 and / or pMUT2.

[0027] The improvement comprises inserting a coding gene of MLH protein into the endogenous cryptic plasmid pMUT1 and / or pMUT2, wherein the MLH protein is a fusion protein of SOD and CAT connected by a flexible linker.

[0028] In a preferred mode of the application, the SOD comprises RtSOD, and the GenBank number is WP_012843426.1; the CAT comprises FeCAT, and the GenBank number is WP_012845258.1. In an embodiment of the application, in order to express the fusion protein of RtSOD and CAT, the CAT gene is inserted into the downstream of the RtSOD gene sequence and connected by a sequence coding for a flexible linker peptide (linker), which is named as MLH. The linker in the application is a flexible peptide GGGGS, which is introduced by a primer L-FeCAT-F.

[0029] The application provides a construction method of the above-mentioned EcNc engineering strain, comprising the following steps: (1) inserting a coding gene of SOD into the endogenous cryptic plasmid pMUT1 and / or pMUT2, and constructing linearized pMUT1-SOD and / or pMUT2-SOD plasmids by a PCR method;

[0030] (2) The coding gene of CAT is inserted into a prokaryotic expression vector, and a linearized linker-CAT gene fragment is constructed by using a PCR method;

[0031] (3) The linearized pMUT1-SOD and / or pMUT2-SOD plasmids in step (1) are connected with the linearized linker-CAT gene fragment in step (2) respectively, and recombinant vectors pMUT1-MLH and / or pMUT2-MLH are obtained;

[0032] (4) The recombinant vectors pMUT1-MLH and / or pMUT2-MLH in step (3) are respectively transformed into Escherichia coli Nissle 1917 competent cells from which two endogenous cryptic plasmids pMUT1 and pMUT2 are removed, and EcNc engineering bacteria are obtained.

[0033] In one preferred mode of the present application, the primers used in the PCR method in step (1) include pMRtSOD-X-F and pMRtSOD-X-R, wherein the nucleotide sequence of pMRtSOD-X-F is shown in SEQ ID No. 1, and the nucleotide sequence of pMRtSOD-X-R is shown in SEQ ID No. 2. In the PCR of the present application, pMUT1-RtSOD and / or pMUT2-RtSOD, which contain the RtSOD gene, are used as templates, and pMUT1-RtSOD and / or pMUT2-RtSOD are linearized by reverse PCR using pMRtSOD-X-F and pMRtSOD-X-R. The construction method of the template of the present application preferably includes first extracting the pMUT1 cryptic plasmid from wild-type EcN cells, and then linearizing and amplifying the plasmid by primers pMUT1-F / pMUT1-R. Then, the RtSOD-KanR fragment is amplified by primers pMUT1-RtSOD-F / pMUT1-RtSOD-R using the plasmid pGEX-4T-1-M-RtSOD as a template, which is synthesized by a biological company by inserting the RtSOD gene (WP_012843426.1) into the downstream GTATTC site of the pGEX-4T-1 plasmid operon, and then obtaining by mutating the operon (the nucleotide sequence includes mutating AGCGG to GATCC). The linearized pMUT1 and the RtSOD-KanR fragment are connected by the seamless cloning technology to obtain the recombinant plasmid pMUT1-RtSOD. The recombinant plasmid pMUT2-RtSOD is also constructed by the same method, wherein the primers used in the PCR amplification include pMUT2-F / pMUT2-R and pMUT2-RtSOD-F / pMUT2-RtSOD-R. The program of the reverse PCR of the present application includes first pre-denaturation at 98℃ for 2 min, then denaturation at 98℃ for 10 s, annealing at 60℃ for 30 s, extension at 72℃ for 1.5 min, 35 cycles, and finally extension at 72℃ for 10 min.

[0034] In the present application, the nucleotides of CAT are codon-optimized according to the codon bias of E. coli, and then inserted into the pET-28a(+) vector. The open reading frame (ORF) of FeCAT is synthesized by gene synthesis and inserted between the Nco I and EcoR I sites of the pET-28a(+) plasmid to generate the recombinant plasmid pET28a-FeCAT.

[0035] The PCR method in step (2) of the application comprises using the recombinant plasmid pET28a-FeCAT as a template, and using L-FeCAT-F and L-FeCAT-R as primers for PCR amplification to obtain a target fragment linker-FeCAT containing a flexible connecting peptide and CAT, which also carries a homologous arm. The nucleotide sequence of L-FeCAT-F is shown in SEQ ID No. 3, and the nucleotide sequence of L-FeCAT-R is shown in SEQ ID No. 4.

[0036] The linearized pMUT1-RtSOD or pMUT2-RtSOD and the linker-FeCAT fragment are connected to obtain recombinant vectors pMUT1-MLH and pMUT2-MLH containing FeCAT and RtSOD fusion genes.

[0037] In the embodiment of the application, the two endogenous cryptic plasmids pMUT1 and pMUT2 of EcN are removed by using the plasmid incompatibility principle to obtain EcNc cells without cryptic plasmids. The method for removing plasmids has been disclosed in the article (Lin Q, Jiang Z, Zhong B, Chen JQ, Lv ZB, Nie ZM. Unveiling the impact of cryptic plasmids curing on Escherichia coli Nissle 1917: massive increase in Ag43c expression. AMB Express, 2024, 14(1): 48).

[0038] The successfully constructed pMUT1-MLH and pMUT2-MLH plasmids are transformed into EcNc competent cells to obtain EcNc-pMUT1-MLH and EcNc-pMUT2-MLH engineering strains. In an embodiment of the application, the transformation is performed by using the method of electroporation, and SDS-PAGE electrophoresis shows that both engineering strains have constitutive expression of MLH protein without IPTG induction. It is also found that the expression amount of EcNc-pMUT2-MLH engineering strain is less than that of EcNc-pMUT1-MLH.

[0039] The primer sequences used in the application are shown in Table 1.

[0040] Table 1: Primers used

[0041]

[0042]

[0043] The application further provides application of the EcNc engineering bacterium or the EcNc engineering bacterium constructed by the construction method in preparation of an antioxidant preparation.

[0044] The EcNc-pMUT1-MLH and EcNc-pMUT2-MLH engineering strains have significant SOD and CAT activities, wherein the total number of colonies in the EcNc-pMUT1-MLH is about 7.5*10 10 When the CFU is 1*10, the SOD activity is about 600 U / mL, and the CAT activity is about 15000 U / mL, and the EcNc-pMUT2-MLH can be used as an antioxidant preparation.

[0045] The application further provides application of the EcNc engineering bacterium or the EcNc engineering bacterium constructed by the construction method in preparation of a medicine for preventing and / or treating enteritis.

[0046] In the embodiment of the application, the EcNc engineering bacterium is used to gavage an animal model, which can relieve the UC symptoms of mice induced by DSS to a certain extent, relieve the symptoms of weight loss, diarrhea and blood in stool, improve the DAI score, reduce the shortening of the colon length, reduce the damage of the colon tissue, improve the oxidative stress index and reduce the expression of inflammatory factors in the colon tissue.

[0047] The EcNc engineering bacterium can stably express the RtSOD and FeCAT double-enzyme fusion protein MLH in a constitutive manner without IPTG induction and antibiotic pressure, the EcNc engineering bacterium is rich in the high-stable SOD / CAT double-enzyme fusion protein and has SOD and CAT enzyme activities, the EcNc engineering strain has good protection and treatment effects on UC mice, and the treatment effect is stronger than that of the wild EcN strain, and the EcNc engineering bacterium can be used as an LBP live bacterium medicine for prevention and treatment of UC.

[0048] The application further provides a medicine for preventing and / or treating enteritis, wherein the active ingredient comprises the EcNc engineering bacterium or the EcNc engineering bacterium constructed by the construction method, and further comprises a pharmaceutically acceptable adjuvant.

[0049] In order to further illustrate the application, the EcNc engineering bacterium for constitutively expressing the MLH protein and the application thereof in treatment of enteritis are described in detail in combination with the embodiments, but they should not be understood as limiting the protection scope of the application.

[0050] The materials used in the embodiments of the present application are common materials in the art, such as EcN recombinant endogenous cryptic plasmids pMUT1-RtSOD and pMUT2-RtSOD containing RtSOD gene (GenBank No. WP_012843426.1) constructed and preserved by the laboratory, which can constitutively express RtSOD protein. The open reading frame (ORF) of FeCAT (GenBank No. WP_012845258.1) was synthesized by Suzhou Jinyuzhi Biological Technology Co., Ltd. and inserted into the pET28a plasmid to generate the recombinant plasmid pET28a-FeCAT.

[0051] The two endogenous cryptic plasmids pMUT1 and pMUT2 of EcN were removed by using the principle of plasmid incompatibility to obtain EcNc cells without cryptic plasmids. This method has been disclosed in the article (Lin Q, Jiang Z, Zhong B, Chen JQ, Lv ZB, Nie ZM. Unveiling the impact of cryptic plasmids curing on Escherichia coli Nissle 1917: massive increase in Ag43c expression. AMB Express, 2024, 14(1): 48).

[0052] C57BL / 6 mice (8 weeks old) were purchased from Jisui Yekang, and the qualified certificate number was B202401030434. The mice were raised in individually ventilated cages, six per cage, in a standard 12-hour light-dark cycle (light from 8:00 am to 8:00 pm; dark from 8:00 pm to 8:00 am), with a temperature control at 26℃ and a relative humidity of 40-70%. The feed and water were supplied adequately. The animal experiment was approved by the Zhejiang University of Technology Experimental Animal Ethics Committee (acceptance number: 20231224-01).

[0053] GraphPad Prism software (version 8.0) was used for statistical analysis and graphical representation in the embodiments of the present application. The differences between groups were evaluated by t-test (n≥3). In the statistical significance analysis, p<0.05 indicates a significant difference, p<0.01 indicates a very significant difference, and p<0.001 indicates an extremely significant difference, respectively marked by *p<0.05, **p<0.01, ***p<0.001, and each row mean with SEM.

[0054] Example 1

[0055] 1. Construction of EcNc-MLH engineering strain and protein expression

[0056] Linearized pMUT1-RtSOD and linearized pMUT2-RtSOD were obtained by linear amplification with primers pMRtSOD-X-F and pMRtSOD-X-R, respectively, using pMUT1-RtSOD and pMUT2-RtSOD as templates. Figure 1 In the middle B);

[0057] The target fragment linker-FeCAT containing a flexible linker peptide and CAT was amplified with primers L-FeCAT-F and L-FeCAT-R using plasmid pET28a-FeCAT as a template, while the homologous arm was also added. Figure 1 In the middle A);

[0058] Linearized pMUT1-RtSOD and FeCAT fragment were ligated by ClonExpress II One-Step Cloning Kit (Nanjing Huada Biological Technology Co., Ltd.) to construct recombinant plasmid pMUT1-MLH. Linearized pMUT2-RtSOD and FeCAT fragment were ligated by the same method to construct recombinant plasmid pMUT2-MLH. Figure 2 In the middle A).

[0059] Recombinant plasmids pMUT1-MLH and pMUT2-MLH were transformed into DH5α competent cells, respectively, and confirmed by sequencing. The strains grown under antibiotic pressure were sent to Yikang Biological Technology Co., Ltd. for sequencing and were consistent with the expected, indicating that the plasmid construction was successful.

[0060] The above constructed plasmids pMUT1-MLH and pMUT2-MLH were sequentially electroporated into EcNc competent cells to obtain EcN engineering strain EcNc-MLH containing two recombinant cryptic plasmids pMUT1-MLH and pMUT2-MLH. The constitutive expression level of MLH protein in EcN engineering strain was analyzed by SDS-PAGE.

[0061] The SDS-PAGE electrophoresis results are shown in Figure 2 In the middle B), both engineering strains had constitutive expression of MLH protein without IPTG induction. It was also found that the expression level of EcNc-pMUT2-MLH engineering strain was less than that of EcNc-pMUT1-MLH. The engineering strain EcNc-pMUT1-MLH with high expression was named EcNc-MLH and used for subsequent research. The total number of colonies in each milliliter was about 7.5×10 10 CFU, the SOD activity of EcNc-MLH was about 600 U / mL, and the CAT activity was about 15000 U / mL.

[0062] 2. Stability identification of MLH protein

[0063] 2.1 Thermal stability of MLH: EcNc-MLH engineering strain was cultured for 16 hours, centrifuged at 12,000 rpm for 5 minutes, and the cells were collected, ultrasonically treated, and the supernatant was collected as the crude enzyme solution of MLH. Then, the crude enzyme solution was heated in a metal bath at 80°C for 0, 20, 40, 60, 80, 100, and 120 minutes, and the SOD and CAT enzyme activities were detected at each time point, respectively. The SOD enzyme activity was determined using the Marklund method (National Standard: GBT 5009.171); the CAT enzyme activity was determined using a kit. Each group included three parallel samples. The relative enzyme activity at each heating time was calculated based on the SOD enzyme activity without heating as 100%.

[0064] The results are shown in Table A and Table B. Figure 3 As shown in Table A, it was found that the SOD and CAT enzyme activities of MLH still had more than 20% activity after heating at 80°C for 20 min.

[0065] 2.2 Acid and alkali tolerance of MLH crude enzyme solution: The crude enzyme solution was diluted 10 times with a series of pH buffers (pH 2-12), heated in a metal bath at 37°C for 30 min, centrifuged, and the supernatant was taken for SOD and CAT enzyme activity determination. Each group included three parallel samples. The relative SOD and CAT enzyme activities after treatment with different pH buffers were calculated based on the enzyme activity at pH 7 as 100%.

[0066] The results are shown in Table A and Table B. Figure 3 As shown in Table B, MLH maintained relatively stable SOD enzyme activity at pH 4-11, still maintained more than 80% SOD enzyme activity at pH 3, SOD enzyme activity was significantly reduced at pH 2, RtSOD still maintained more than 60% SOD enzyme activity at pH 12, and the CAT enzyme activity of MLH was relatively stable at pH 2-10, decreased to 70% of the enzyme activity at pH 11, and decreased significantly at pH 12, indicating that MLH has good high-temperature resistance and acid and alkali resistance.

[0067] 2.3 Artificial gastric juice / artificial intestinal juice tolerance of MLH crude enzyme solution: The crude enzyme solution was diluted 10 times with artificial gastric juice / artificial intestinal juice, heated in a metal bath at 37°C for 0 min, 20 min, 40 min, 60 min, 80 min, 100 min, 120 min, 140 min, 160 min, and 180 min, respectively, and the SOD and CAT enzyme activities at each artificial gastric juice / artificial intestinal juice treatment time point were detected. Each group included three parallel samples. The SOD and CAT enzyme activities at 0 min were taken as 100%.

[0068] The results are shown in Table A and Table B. Figure 3As shown in C and D, the activity of MLH decreased to about 15% within 20 min, and further decreased to complete disappearance after 2 h, while the activity of CAT enzyme decreased sharply to about 25% within 20 min, and further decreased to complete disappearance after 1 h. Figure 3 As shown in C, it is indicated that the MLH protein has poor resistance to gastric juice, but the live bacteria medicine of the application can protect the MLH from damage by gastric juice. In order to more accurately evaluate the oral stability, the artificial intestinal fluid resistance test was simulated in the human intestinal environment. The activity of MLH remained more than 70% within 3 h, and the SOD activity could be maintained at 90% in the first 1.5 h, while the activity of CAT enzyme slowly decreased to about 70% within 3 h, and the CAT activity could be maintained at 90% in the first 1.5 h, showing excellent stability. Figure 3 As shown in D, it is indicated that the MLH has high intestinal fluid stability, and can retain high SOD and CAT enzyme activity after oral administration to the intestine, and can exert better antioxidant effect.

[0069] 3. Evaluation of EcNc-MLH engineering bacteria in the treatment of UC

[0070] 3.1 Establishment of mouse UC model and administration scheme

[0071] C57BL / 6 mice (8 weeks old) were adaptively fed for one week, during which all groups were normally given water and sufficient feed, and the light was kept on for 12 hours. After the adaptation period, the mice were randomly divided into groups, 6 in each group, and a total of 4 groups were set: a blank control group (Control), a model group (DSS), a 10 9 CFU / mL EcN group, a 10 9 CFU / mL EcNc-MLH group (SOD: 8 U / mL, CAT: 200 U / mL). The EcN group and the EcNc-MLH group used 1xPBS as a bacteria protective agent for dissolution.

[0072] Administration scheme: three days before modeling, pre-administration was started, the Control and DSS groups were given 200 μL of PBS by gavage on D0 and D2 days, the EcN group was given 200 μL of EcN bacterial solution by gavage on D0 and D2 days. At the beginning of modeling, the drinking water of the DSS group, the EcN group and the EcNc-MLH group was replaced with 2.5% DSS solution, and the Control group kept drinking water. During the modeling period, D4, D6 and D8 were given drugs by gavage once. After the modeling was completed, the 2.5% DSS solution was replaced with drinking water, the last administration was given on D10 day, and all the mice were sacrificed on D15 day. The mice were dissected, the colon of the mice was taken, and the distal colon part was fixed with 4% paraformaldehyde, then sent to Sivier Biological Company for paraffin embedding, frozen sectioning and HE staining. The colon was placed in a-80°C refrigerator.

[0073] 3.2 Disease Activity Index (DAI) score

[0074] The mice in each experimental group were further assessed using the DAI (Defecation Artery Identification and Assessment) system. Throughout the experiment, the mice were weighed at fixed times each day, their weight was recorded, and the characteristics of their stool, fecal blood loss, and activity level were observed. The DAI score was the sum of the scores for weight loss, stool characteristics, and fecal blood loss, and was determined according to the standards in Table 2.

[0075] Table 2 DAI Scoring Table

[0076] Score Weight loss (%) Stool consistency Blood in stool 0 0 Normal Normal 1 1-5 Slightly loose stools Small amount 2 6-10 Loose stools Clearly visible 3 11-15 Diarrhea Heavy bleeding 4 ＞15

[0077] Mice were grouped according to their body weight and given the drug. Throughout the experiment, the mice's condition was observed and their weight was weighed daily. The results are as follows: Figure 4 As shown in Figure A, the overall body weight of mice in the Control group remained stable and showed a slow upward trend; while the body weight of mice in the DSS group remained stable from days 0 to 7, began to decline on day 8, and continued to decrease even after stopping 2.5% DSS on day 10, only starting to recover on day 14; the body weight of mice in the ECN group began to decline slowly on day 11, and the weight loss on day 13 after drug withdrawal was significantly less than that of the DSS group. The body weight of the MLH engineered bacteria group rebounded rapidly on day 14, suggesting that the MLH engineered bacteria had colonized the mouse intestines and were exerting their effects.

[0078] During the experiment, mice in the Control group had shiny fur and normal activity levels; in the later stages of the experiment, mice in the DSS group had dull fur, reduced activity, and mostly curled up and lay still. Mice in the Control group had normal fecal shape and consistency, and no bloody stools were observed; mice in the DSS group began to have increasingly loose stools on day 6, and started experiencing bloody stools on day 7, which worsened as the experiment progressed, peaking on day 12, and gradually subsiding on day 13. Compared to the DSS group, all experimental groups showed significant weight gain and improved bloody stools on day 13 after drug withdrawal. Therefore, a DAI score was calculated based on the changes in body weight and fecal characteristics of each group. Figure 4 (B) The DAI scores of the MLH engineered bacteria group and the EcN group were lower than those of the DSS group, and the DAI score of the MLH engineered bacteria group was also lower than that of the EcN group, indicating that both the MLH engineered bacteria and the EcN wild-type strain have certain therapeutic effects on enteritis, and the MLH engineered bacteria have a better therapeutic effect than the EcN wild-type bacteria.

[0079] After each group of mice completed drug administration, they were sacrificed, and their colons were collected. The colon length of each group of mice was measured. Figure 4The colon length of the Control group was longer, without hyperemia and edema, and the feces in the intestinal cavity were normal. The colon length of the DSS group was shorter, with hyperemia and edema, and the feces in the intestinal cavity were mucous. The colon length of each administration group was slightly longer than that of the DSS group, among which the wild EcN and the MLH engineering bacteria were significantly different from the DSS group, but the MLH colon was longer than the EcN group, as shown in FIG. 2B. Figure 4 As shown in FIG. 2C, the Control group had normal intestinal morphology, and the feces in the intestinal cavity were normal. The DSS group had intestinal mucosal erosion, and the feces in the intestinal cavity were mucous. The MLH engineering bacteria group had a certain intestinal morphology, and the feces in the intestinal cavity were normal, with reduced bleeding. The size of the mouse spleen can well reflect the damage to the mouse body. The spleen of the Control group was normal size, and the spleen of the DSS group was significantly larger. The size of the spleen of the MLH group was significantly smaller than that of the DSS group, indicating that the damage to the mouse body of the MLH group was significantly lower than that of the DSS group (FIG. 2D). Figure 4

[0080] To observe the development of colitis, the distal colon tissues collected above were observed by HE staining. The colon muscle layer of the Control group was complete, the mucosal layer of the goblet cells was complete, the crypt structure was complete, the intestinal glands were regular, the villi were arranged in order and were close and clear, and no inflammatory cell infiltration was observed. The colon of the DSS group showed inflammatory reaction, manifested as mucosal erosion, reduced goblet cells, dissolved and missing crypts and intestinal glands, severe submucosal edema, and obvious inflammatory cell infiltration. Compared with the DSS group, the wild EcN and the MLH engineering bacteria group effectively reduced the inflammatory cell infiltration, reduced the mucosal epithelial necrosis, and restored the crypt structure. The effect of the MLH engineering bacteria group was more obvious (FIG. 2E and FIG. 2F). Figure 4

[0081] 3.3 Determination of MDA / SOD / CAT oxidative stress indicators

[0082] SOD determination: at the end of the animal experiment, the mouse colon tissues were taken, washed with physiological saline, ice-bathed homogenized with SOD sample preparation liquid, and then centrifuged at 12000g for 3 min. The total SOD activity detection kit (WST-8 method) (Bi Yun Tian Biotechnology Co., Ltd., Shanghai, Beijing) was used to detect the SOD enzyme activity.

[0083] MDA determination: 0.1 g of tissue was weighed, 1 mL of MDA extraction solution was added for homogenization, and then centrifuged at 8000g at 4°C for 10 min. The supernatant was detected according to the malondialdehyde (MDA) content detection kit (Beijing Solabio Technology Co., Ltd., Beijing, China) to detect the MDA content.

[0084] CAT determination: 0.1 g of tissue was weighed, 1 mL of CAT extraction solution was added for ice-bath homogenization, and then centrifuged at 8000g at 4°C for 10 min. The supernatant was detected according to the catalase (CAT) activity detection kit (Beijing Solabio Technology Co., Ltd., Beijing, China) to detect the CAT content.

[0085] The results are shown in Table 2.​​Figure 5 As shown in Figs. 2A, 2B and 2C, compared with the Control group, the SOD and CAT activities of the DSS group decreased, and the MDA content increased, indicating that a certain degree of oxidative stress occurred. These injuries were alleviated after administration in each group, and the SOD and CAT activities of the EcNc-MLH group were significantly different from those of the DSS group. The results suggest that EcNc-MLH has a certain antioxidant effect.

[0086] 3.4 Determination of inflammatory factors

[0087] To a pre-cooled grinding tube, 1 mL of RNA extraction reagent and three 3 mm grinding beads were added. The colon tissue (5-20 mg) was placed in the tube and homogenized using a tissue grinder until no visible fragments were left. Then, centrifugation was performed at 12,000 rpm at 4°C for 10 minutes, and the supernatant was collected. Total RNA was extracted using a Direct-zol RNA Microprep kit (Beijing Genstone Biotechnology Co., Ltd., Beijing, China) and reverse transcribed using an Evo M-MLV Plus cDNA synthesis kit (AGBio, Hunan, China) to obtain cDNA. The primers for the target genes IL-6, IL-1β and the reference gene GAPDH were designed using the qPCR primer design tool of NCBI. The primer sequences are shown in Table 1. The primers were diluted to 10 μM with double-distilled water and used according to the instructions of the qPCR MasterMix enzyme. The qPCR experiment was performed.

[0088] The results are shown in Figs. 3A, 3B and 3C. Figure 5 As shown in Figs. 3A, 3B and 3C, compared with the Control group, the gene expression levels of IL-6 / IL-1β in the colon tissue of the DSS group increased significantly. However, compared with the DSS group, the mRNA levels of IL-6 / IL-1β decreased after administration, and the EcNc-MLH group had a significant difference from the DSS group. Therefore, the EcNc-MLH engineered strain can reduce the inflammatory response of the colon tissue caused by DSS and alleviate the damage to the colon tissue.

[0089] Although the above embodiments have made a detailed description of the present application, it is only a part of the embodiments of the present application, not all the embodiments, and other embodiments can be obtained according to the present embodiments without creativity, which are within the scope of protection of the present application.

Claims

1. An EcNc engineered bacterium constitutively expressing MLH protein, characterized in that, The chassis cells were Escherichia coli Nissle 1917 and contained the modified endogenous cryptogenic plasmids pMUT1 and / or pMUT2. The modification involves inserting the gene encoding the MLH protein into the endogenous cryptic plasmids pMUT1 and / or pMUT2. The MLH protein is a fusion protein formed by linking SOD and CAT using a flexible linker. The SOD is RtSOD, with GenBank number WP_012843426.1; the CAT is FeCAT, with GenBank number WP_012845258.

1.

2. The method for constructing the EcNc engineered bacteria according to claim 1, characterized in that, Includes the following steps: (1) Insert the SOD coding gene into the endogenous cryptic plasmids pMUT1 and / or pMUT2, and construct linearized pMUT1-SOD and / or pMUT2-SOD plasmids using PCR. (2) Insert the CAT coding gene into a prokaryotic expression vector and construct a linearized linker-CAT gene fragment using PCR. (3) The linearized pMUT1-SOD and / or pMUT2-SOD plasmids described in step (1) are respectively linked to the linearized linker-CAT gene fragment described in step (2) to construct the recombinant vectors pMUT1-MLH and / or pMUT2-MLH. (4) Transform the recombinant vectors pMUT1-MLH and / or pMUT2-MLH described in step (3) into Escherichia coli Nissle 1917 in which the two endogenous cryptogenic plasmids pMUT1 and pMUT2 have been removed, respectively, to construct the EcNc engineered bacteria.

3. The construction method according to claim 2, characterized in that, The primers used in the PCR method described in step (1) include pMRtSOD-XF and pMRtSOD-XR, wherein the nucleotide sequence of pMRtSOD-XF is shown in SEQ ID No.1 and the nucleotide sequence of pMRtSOD-XR is shown in SEQ ID No.

2.

4. The construction method according to claim 2, characterized in that, The primers used in the PCR method described in step (2) include L-FeCAT-F and L-FeCAT-R, wherein the nucleotide sequence of L-FeCAT-F is shown in SEQ ID No. 3, and the nucleotide sequence of L-FeCAT-R is shown in SEQ ID No.

4.

5. The use of the EcNc engineered bacteria of claim 1 or the EcNc engineered bacteria constructed using the construction method of any one of claims 2 to 4 in the preparation of antioxidant agents.

6. The use of the EcNc engineered bacteria of claim 1 or the EcNc engineered bacteria constructed using the construction method of any one of claims 2 to 4 in the preparation of drugs for the prevention and / or treatment of ulcerative colitis.

7. The application according to claim 6, characterized in that, The types of drugs mentioned for the prevention and / or treatment of enteritis include live bacteria drugs.

8. A medicament for the prevention and / or treatment of ulcerative colitis, characterized in that, The active ingredient includes the EcNc engineered bacteria as described in claim 1 or the EcNc engineered bacteria constructed using any one of the construction methods described in claims 2 to 4, and also includes pharmaceutically acceptable excipients.

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