EcNc engineering bacterium for constitutive expression of MLH protein and application of EcNc engineering bacterium in treatment of enteritis

Abstract

The invention provides an EcNc engineering bacterium for constitutive expression of MLH protein and application of the EcNc engineering bacterium in treatment of enteritis, and belongs to the technical field of biological medicine. The invention provides an EcNc engineering bacterium for constitutive expression of MLH protein, which is characterized in that Escherichia coli Nissle 1917 (EcN) is taken as a chassis cell, two concealed plasmids are removed, and then the EcNc engineering bacterium is transferred into modified endogenous concealed plasmids pMUT1 and / or pMUT2; according to the modification, a fusion protein MLH coding gene formed by connecting SOD and CAT through a flexible linker is inserted into pMUT1 and / or pMUT2. According to the invention, an engineering bacterium for constitutive expression of the fusion protein is constructed, and the engineering bacterium can express the fusion protein with SOD and CAT double enzyme activities without antibiotic pressure and IPTG (isopropyl-beta-d-thiogalactoside) induction. The engineering bacterium constructed by the invention can be used as an engineering microorganism for treating UC diseases, such as a viable bacterium medicine.

Description

Technical Field

[0001] The present invention provides the field of biomedicine technology, and specifically relates to an EcNc engineered bacterium constitutively expressing MLH protein and its application in treating enteritis. Background Art

[0002] Ulcerative colitis (UC) is a chronic inflammatory bowel disease (IBD) whose etiology is not yet fully understood. 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. It is a disease that seriously affects human health. Traditional chemical drugs are not thorough in the treatment of UC, and the condition is erratic, causing harm to the patient's physical and mental health. Chemical drugs can only relieve symptoms, but cannot fundamentally cure UC, and have a great impact on other organs of the human body. In addition, the gastrointestinal drugs currently on the market are relatively one-sided and single in the treatment of chronic colitis. They can only repair the mucosa but cannot kill pathogens, or only kill pathogens.

[0003] In recent years, emerging treatment strategies such as biological agents and fecal microbial transplantation (FMT) have shown important value in the treatment of UC. Biological agents are particularly effective in relieving moderate to severe UC patients, but there is currently no effective live bacterial agent available for the treatment of UC. Summary of the invention

[0004] The present invention provides an EcNc engineered bacterium constitutively expressing MLH protein and its application in treating enteritis. An engineered bacterium EcNc-MLH constitutively expressing fusion protein RtSOD-CAT (MLH) is developed, which can be used as live biotherapeutic products (LBP) for the prevention and treatment of enteritis.

[0005] The present invention provides an EcNc engineered bacterium constitutively expressing MLH protein, which uses Escherichia coli Nissle1917 as a chassis cell and contains modified endogenous cryptic plasmids pMUT1 and / or pMUT2;

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

[0007] In a preferred embodiment of the present invention, the SOD includes RtSOD, with GenBank number WP_012843426.1; the CAT includes FeCAT, with GenBank number WP_012845258.1.

[0008] The present invention provides a method for constructing the above-mentioned EcNc engineered bacteria, comprising the following steps: (1) inserting the SOD coding gene into the endogenous cryptic plasmid pMUT1 and / or pMUT2, and constructing a linearized pMUT1-SOD and / or pMUT2-SOD plasmid using a PCR method;

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

[0010] (3) connecting the linearized pMUT1-SOD and / or pMUT2-SOD plasmids described in step (1) with the linearized linker-CAT gene fragments described in step (2) to construct recombinant vectors pMUT1-MLH and / or pMUT2-MLH;

[0011] (4) The recombinant vectors pMUT1-MLH and / or pMUT2-MLH described in step (3) are respectively transformed into Escherichia coli Nissle 1917 from which the two endogenous cryptic plasmids pMUT1 and pMUT2 have been removed, thereby constructing EcNc engineered bacteria.

[0012] In a preferred embodiment of the present invention, the primers used in the PCR method in step (1) include pMRtSOD-XF and pMRtSOD-XR, wherein the nucleotide sequence of pMRtSOD-XF is shown as SEQ ID No.1, and the nucleotide sequence of pMRtSOD-XR is shown as SEQ ID No.2.

[0013] In a preferred embodiment of the present invention, the primers used in the PCR method of 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.

[0014] The present invention also provides the use of the EcNc engineered bacteria or the EcNc engineered bacteria constructed by the above construction method in the preparation of antioxidant preparations.

[0015] The present invention also provides the use of the EcNc engineered bacteria or the EcNc engineered bacteria constructed by the above construction method in the preparation of drugs for preventing and / or treating enteritis.

[0016] In a preferred embodiment of the present invention, the type of enteritis includes ulcerative colitis.

[0017] In a preferred embodiment of the present invention, the types of drugs for preventing and / or treating enteritis include live bacteria drugs.

[0018] The present invention also provides a drug for preventing and / or treating enteritis, wherein the active ingredients include the EcNc engineered bacteria or the EcNc engineered bacteria constructed by the above construction method, and also include pharmaceutically acceptable excipients.

[0019] Beneficial effects: The present invention provides an EcNc engineered bacterium constitutively expressing MLH protein, which uses Escherichia coli Nissle 1917 (EcN) as a chassis cell, removes two cryptic plasmids and then transfers them into the modified endogenous cryptic plasmids pMUT1 and / or pMUT2; wherein the modification is to insert the fusion protein MLH gene formed by connecting SOD and CAT using a flexible linker into pMUT1 and / or pMUT2. The present invention constructs an engineered bacterium EcNc-MLH constitutively expressing the fusion protein RtSOD-CAT (MLH), and the engineered bacterium EcNc-MLH can express the SOD and CAT dual enzyme fusion protein without antibiotic pressure and IPTG induction, and has the enzyme activity of SOD and CAT, and remains relatively stable in an environment of pH 3 to 11 and in artificial intestinal fluid, further improving the application value of the EcN engineered strain.

[0020] The engineered bacteria EcNc-MLH constructed by the present invention can significantly reduce DSS-induced intestinal inflammation in mice and promote the healing of intestinal mucosa in UC mice. Biochemical analysis showed that the content of SOD and CAT in intestinal tissue increased significantly, the content of malondialdehyde (MDA) decreased significantly, and the mRNA expression level of inflammatory factors IL-6 and IL-1β also decreased. The engineered bacteria EcNc-MLH of the present invention can be used as an engineered microorganism for the treatment of UC diseases, such as live biotherapeutic products (LBP) for the prevention and treatment of enteritis, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the PCR electrophoresis diagram of the construction of plasmid pMUT1-MLH, in which A: linker-FeCAT target fragment; B: linearized pMUT1-RtSOD vector;

[0022] Figure 2 The figure shows the construction of EcNc-MLH engineering bacteria expressing MLH fusion protein and the results of protein expression identification. A: Schematic diagram of recombinant cryptic plasmids pMUT1-MLH and pMUT2-MLH; B: SDS-PAGE identification of EcN engineering strains constitutively expressing recombinant MLH fusion protein, band 1 indicates no IPTG induction; band 2 indicates IPTG induction;

[0023] Figure 3The results of the MLH stability test are shown in Figure A: high temperature resistance test of MLH's SOD and CAT enzymes; B: acid and alkali resistance test of MLH's SOD and CAT enzymes; C: artificial gastric juice resistance test of MLH's SOD and CAT enzymes; D: artificial intestinal juice resistance test of MLH's SOD and CAT enzymes;

[0024] Figure 4 The results of the treatment evaluation of mouse UC by the EcNc-MLH engineered strain are shown in Figure A: changes in mouse weight; B: changes in mouse DAI; C: macroscopic changes in mouse colon length; D: statistical analysis of mouse colon length; E: macroscopic changes in mouse spleen size; F: statistical analysis of mouse spleen size; G: HE staining of mouse colon;

[0025] Figure 5 This is a graph showing the effects of the EcN engineered strain on the oxidative stress levels and cytokines in UC mice. In the graph, A: MDA content; B: CAT content; C: SOD content; D: IL-1β level; E: IL-6 level. DETAILED DESCRIPTION

[0026] The present invention provides an EcNc engineered bacterium constitutively expressing MLH protein, which uses Escherichia coli Nissle1917 as a chassis cell and contains modified endogenous cryptic plasmids pMUT1 and / or pMUT2;

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

[0028] In a preferred embodiment of the present invention, the SOD includes RtSOD, with a GenBank number of WP_012843426.1; the CAT includes FeCAT, with a GenBank number of WP_012845258.1. In one embodiment of the present invention, in order to express a fusion protein of RtSOD and CAT, the CAT gene is inserted downstream of the RtSOD gene sequence and connected by a sequence encoding a flexible linker, named MLH. The linker of the present invention is a flexible peptide GGGGS, which is introduced by the primer L-FeCAT-F.

[0029] The present invention provides a method for constructing the above-mentioned EcNc engineered bacteria, comprising the following steps: (1) inserting the SOD coding gene into the endogenous cryptic plasmid pMUT1 and / or pMUT2, and constructing a linearized pMUT1-SOD and / or pMUT2-SOD plasmid using a PCR method;

[0030] (2) inserting the coding gene of CAT into a prokaryotic expression vector and constructing a linearized linker-CAT gene fragment using PCR;

[0031] (3) connecting the linearized pMUT1-SOD and / or pMUT2-SOD plasmids described in step (1) with the linearized linker-CAT gene fragments described in step (2) to construct recombinant vectors pMUT1-MLH and / or pMUT2-MLH;

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

[0033] In a preferred embodiment of the present invention, the primers used in the PCR method 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. When the PCR is performed in the present invention, the EcN recombinant endogenous cryptic plasmid pMUT1-RtSOD and / or pMUT2-RtSOD containing the RtSOD gene is used as a template, and pMRtSOD-XF and pMRtSOD-XR are used to linearize pMUT1-RtSOD and / or pMUT2-RtSOD by reverse PCR. The template construction method of the present invention preferably includes first extracting the pMUT1 cryptic plasmid from wild-type EcN cells, and performing linear amplification using primers pMUT1-F / pMUT1-R. Then, the RtSOD-KanR fragment was amplified using the plasmid pGEX-4T-1-M-RtSOD as a template by using primers pMUT1-RtSOD-F / pMUT1-RtSOD-R. The pGEX-4T-1-M-RtSOD plasmid was synthesized by a biological company and the RtSOD gene (WP_012843426.1) was inserted into the GTATTC site downstream of the pGEX-4T-1 plasmid operon, and obtained by mutating the operon (nucleotide sequence including mutation from AGCGG to GATCC). The linearized pMUT1 and the RtSOD-KanR fragment were connected by seamless cloning technology to obtain the recombinant plasmid pMUT1-RtSOD. The recombinant plasmid pMUT2-RtSOD was constructed by the same method as above, wherein the primers used for PCR amplification included pMUT2-F / pMUT2-R and pMUT2-RtSOD-F / pMUT2-RtSOD-R. The reverse PCR procedure of the present invention includes first pre-denaturation at 98°C for 2 minutes, then denaturation at 98°C for 10 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 1.5 minutes, 35 cycles, and finally 72°C for 10 minutes.

[0034] The present invention performs codon optimization on the nucleotide of CAT according to the codon preference of Escherichia coli, and synthesizes and inserts it into the pET-28a(+) vector. The open reading frame (ORF) of FeCAT is gene synthesized 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 described in step (2) of the present invention comprises using the recombinant plasmid pET28a-FeCAT as a template, using L-FeCAT-F and L-FeCAT-R as primers for PCR amplification, and obtaining a target fragment linker-FeCAT containing a flexible linker peptide and CAT, and carrying homology arms. 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 present invention connects the linearized pMUT1-RtSOD or pMUT2-RtSOD and the linker-FeCAT fragment to obtain recombinant vectors pMUT1-MLH and pMUT2-MLH containing the FeCAT and RtSOD fusion genes.

[0037] In an embodiment of the present invention, the two endogenous cryptic plasmids pMUT1 and pMUT2 of EcN are removed by the plasmid incompatibility principle to obtain EcNc cells free of cryptic plasmids, wherein the method for removing the plasmid has been disclosed in the article (LinQ, 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 Ag43cexpression. AMB Express, 2024, 14(1): 48).

[0038] The present invention transforms the successfully constructed pMUT1-MLH and pMUT2-MLH plasmids into EcNc competent cells respectively to obtain EcNc-pMUT1-MLH and EcNc-pMUT2-MLH engineered strains. In one embodiment of the present invention, the transformation is performed by electroporation, and SDS-PAGE electrophoresis shows that both engineered strains have constitutive expression of MLH protein without IPTG induction, and it is found that the expression amount of EcNc-pMUT2-MLH engineered bacteria is less than that of EcNc-pMUT1-MLH.

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

[0040] Table 1 Primers used

[0041]

[0042]

[0043] The present invention also provides the use of the EcNc engineered bacteria or the EcNc engineered bacteria constructed by the above construction method in the preparation of antioxidant preparations.

[0044] The EcNc-pMUT1-MLH and EcNc-pMUT2-MLH engineered strains of the present invention both have significant SOD and CAT activities, wherein the total number of colonies per ml of EcNc-pMUT1-MLH is approximately 7.5×10 10 At CFU, the SOD activity is about 600U / mL and the CAT activity is about 15000U / mL, which can be used as an antioxidant preparation.

[0045] The present invention also provides the use of the EcNc engineered bacteria or the EcNc engineered bacteria constructed by the above construction method in the preparation of drugs for preventing and / or treating enteritis.

[0046] In the embodiment of the present invention, the EcNc engineered bacteria gavage animal model can alleviate the DSS-induced UC symptoms in mice to a certain extent, which is manifested in relieving weight loss and diarrhea and bloody stool symptoms, improving DAI scores, reducing the shortening of colon length, reducing colon tissue damage, improving oxidative stress indicators, and reducing the expression of inflammatory factors in colon tissue.

[0047] The EcNc engineered bacteria of the present invention can constitutively and stably express the RtSOD and FeCAT dual-enzyme fusion protein MLH without IPTG induction and antibiotic pressure, and the EcNc engineered bacteria are rich in highly stable SOD / CAT dual-enzyme fusion protein and have both SOD and CAT enzyme activities; the EcNc engineered strain has good protective and therapeutic effects on UC mice, and the therapeutic effect is stronger than that of the wild EcN strain, and can be used as a LBP live bacteria drug for the prevention and treatment of UC.

[0048] The present invention also provides a drug for preventing and / or treating enteritis, wherein the active ingredients include the EcNc engineered bacteria or the EcNc engineered bacteria constructed by the above construction method, and also include pharmaceutically acceptable excipients.

[0049] To further illustrate the present invention, the EcNc engineered bacteria constitutively expressing MLH protein and its application in treating enteritis provided by the present invention are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0050] Unless otherwise specified, the materials used in the embodiments of the present invention are all common materials in the art, such as EcN recombinant endogenous cryptic plasmids pMUT1-RtSOD and pMUT2-RtSOD containing the RtSOD gene (GenBank No. WP_012843426.1) constructed and preserved by this laboratory, which can constitutively express the RtSOD protein. Suzhou Jinweizhi Biotechnology Co., Ltd. was commissioned to synthesize the open reading frame (ORF) of FeCAT (GenBank No. WP_012845258.1) and insert it 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 the plasmid incompatibility principle to obtain EcNc cells without cryptic plasmids. This method has been published in the article (Lin Q, Jiang Z, Zhong B, Chen JQ, LvZB, 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 Jicui Yaokang, certificate number: B202401030434. Mice were housed in individually ventilated cages, six per cage, and placed in a standard 12-h light-dark cycle (light from 8:00 am to 8:00 pm; dark from 8:00 pm to 8:00 am), with temperature controlled at 26°C and relative humidity of 40-70%. Feed and water were adequately supplied. This animal experiment was approved by the Experimental Animal Ethics Committee of Zhejiang Sci-Tech University (acceptance number: 20231224-01).

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

[0054] Example 1

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

[0056] The plasmids pMUT1-RtSOD and pMUT2-RtSOD were used as templates, and the linearized pMUT1-RtSOD and pMUT2-RtSOD were obtained by PCR with primers pMRtSOD-XF and pMRtSOD-XR. Figure 1 Middle B);

[0057] Using plasmid pET28a-FeCAT as template, primers L-FeCAT-F and L-FeCAT-R were used to amplify the target fragment linker-FeCAT containing flexible linker peptide and CAT, and homology arms ( Figure 1 Middle A);

[0058] The linearized pMUT1-RtSOD was connected to the FeCAT fragment using the ClonExpress II one-step cloning kit (Nanjing Huada Biotechnology Co., Ltd.) to construct the recombinant plasmid pMUT1-MLH. Using the same method, the linearized pMUT2-RtSOD was connected to the FeCAT fragment to construct the recombinant plasmid pMUT2-MLH ( Figure 2 (A).

[0059] The recombinant plasmids pMUT1-MLH and pMUT2-MLH were transformed into DH5α competent cells and confirmed by sequencing. The strains grown under antibiotic pressure were sent to Youkang Bio for sequencing and were consistent with expectations, indicating that the plasmid construction was successful.

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

[0061] The results of SDS-PAGE electrophoresis are as follows Figure 2 As shown in B, both engineered bacteria expressed MLH protein constitutively without IPTG induction. It was also found that the expression level of EcNc-pMUT2-MLH engineered bacteria was less than that of EcNc-pMUT1-MLH. The engineered bacteria EcNc-pMUT1-MLH with high expression level was named EcNc-MLH for subsequent research. The total number of colonies per milliliter was about 7.5×10 10 At CFU, the SOD activity of EcNc-MLH was about 600U / mL, and the CAT activity was about 15000U / mL.

[0062] 2. Stability identification of MLH protein

[0063] 2.1 Thermal stability of MLH: The EcNc-MLH engineered strain was cultured for 16 hours, centrifuged at 12,000 rpm for 5 minutes, the cells were collected, ultrasonicated, and the supernatant was collected as the crude enzyme solution of MLH. Subsequently, 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. The SOD enzyme activity was determined using the Marklund method (national standard: GBT5009.171); the CAT enzyme activity was determined using a kit. Each group included three parallel samples. The relative enzyme activity of each heating time was calculated with the unheated SOD enzyme activity as 100%.

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

[0065] 2.2 Acid-base 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 37°C metal bath for 30 min, centrifuged, and the supernatant was taken for SOD and CAT enzyme activity determination. Each group included three parallel samples. The enzyme activity at pH 7 was taken as 100%, and the relative SOD and CAT enzyme activities after treatment with different pH buffers were calculated.

[0066] The results are as follows Figure 3 As shown in B, MLH maintains a relatively stable SOD enzyme activity at pH 4-11, and still maintains more than 80% of the SOD enzyme activity at pH 3, and the SOD enzyme activity decreases significantly at pH 2. At pH 12, RtSOD still maintains more than 60% of the SOD enzyme activity, while the CAT enzyme activity of MLH remains relatively stable at pH 2-10, decreases to 70% of the enzyme activity at pH 11, and decreases significantly at pH 12, indicating that MLH has good resistance to high temperature and acid and alkali.

[0067] 2.3 Tolerance of MLH crude enzyme solution to artificial gastric juice / artificial intestinal juice: After the crude enzyme solution was diluted 10 times with artificial gastric juice / artificial intestinal juice, it was heated in a 37°C metal bath for 0 min, 20 min, 40 min, 60 min, 80 min, 100 min, 120 min, 140 min, 160 min, and 180 min, 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 of treatment were 100%.

[0068] The results are as follows Figure 3As shown in C and D, the activity of MLH decreased to about 15% within 20 minutes, and further decreased to completely disappear after 2 hours, while the activity of CAT enzyme decreased sharply to about 25% within 20 minutes, and further decreased to completely disappear after 1 hour ( Figure 3 C). This shows that MLH protein has poor gastric juice tolerance, but the present invention uses live bacteria to protect MLH from gastric juice. In order to more accurately evaluate oral stability, the human intestinal environment was simulated to conduct an artificial intestinal juice tolerance test. MLH still retains more than 70% of its activity within 3 hours, and can maintain 90% of SOD activity in the first 1.5 hours, while CAT enzyme activity slowly decreases to about 70% within 3 hours, while 90% of CAT activity can be maintained in the first 1.5 hours, showing excellent stability ( Figure 3 D). This indicates that MLH has a high intestinal fluid stability and can retain a high level of SOD and CAT enzyme activities when taken orally into the intestine, thus exerting a better antioxidant effect.

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

[0070] 3.1 Establishment of mouse UC model and dosing regimen

[0071] C57BL / 6 mice (8 weeks old) were fed adaptively for one week, during which all groups had normal drinking water, adequate feed, and 12 hours of light. After the adaptation period, the mice were randomly divided into 4 groups, with 6 mice in each group: blank control group (Control), model group (DSS), 10 9 CFU / mL EcN group, 10 9 CFU / mL EcNc-MLH group (SOD: 8U / mL, CAT: 200U / mL). Both EcN group and EcNc-MLH group were dissolved using 1×PBS as a bacterial protective agent.

[0072] Dosage scheme: Pre-drug administration began three days before modeling. The Control and DSS groups were gavaged with 200 μL PBS on D0 and D2, and the EcN group was gavaged with 200 μL EcN bacterial solution on D0 and D2. At the beginning of modeling, the drinking water of the DSS group, EcN group, and EcNc-MLH group was replaced with 2.5% DSS solution, and the Control group maintained drinking water. During the modeling period, the drug was gavaged once on D4, D6, and D8. After the modeling was completed, the 2.5% DSS solution was replaced with drinking water, and the last drug was administered on D10, and all mice were killed on the evening of D15. The mice were dissected, the mouse colon was taken, and the distal colon part was fixed with 4% paraformaldehyde and sent to Sevier Bio for paraffin embedding, frozen sectioning, and HE staining. The colon was placed in a -80℃ refrigerator.

[0073] 3.2 Disease Activity Index (DAI) score

[0074] The above experimental groups of mice were further scored for DAI. During the entire experiment, the mice were weighed at a fixed time every day, the body weight was recorded, and the stool characteristics, blood in the stool, and activity status of the mice in each group were observed. The DAI score is the sum of the scores of weight loss, stool characteristics, and blood in the stool, and the DAI score is calculated according to the standards in Table 2.

[0075] Table 2 DAI score table

[0076] score Weight loss (%) Stool characteristics Blood in the 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] The mice were divided into groups and given medication according to their body weight. Throughout the experiment, the mice were observed and weighed every day. Figure 4 As shown in A, the overall weight of mice in the Control group was stable and slowly increased; while the weight of mice in the DSS group was stable from D0 to D7, and began to decrease on D8. After stopping drinking 2.5% DSS on D10, the weight continued to decrease and began to recover on D14. Among them, the weight of the ECN group began to decrease slowly on D11, and the weight loss on D13 after drug withdrawal was significantly less than that of the DSS group. The weight of the MLH engineered bacteria group recovered rapidly on D14, which suggests that the MLH engineered bacteria have colonized the mouse intestines and played a role.

[0078] During the experiment, the mice in the Contro group had shiny hair and normal activity; in the later stage of the experiment, the mice in the DSS group had dull hair, reduced activity, and curled up and lay still. The stool morphology and consistency of the mice in the Control group were normal, and there was no blood in the stool; the stool of the mice in the DSS group began to become thinner on D6, and blood in the stool began to appear on D7. The symptoms worsened as the experiment progressed, and finally reached a peak on D12. The blood in the stool began to gradually ease on D13; compared with the DSS group, on D13 after drug withdrawal, the weight of each experimental group increased significantly, and the blood in the stool improved. Therefore, the DAI score was calculated based on the weight changes and stool characteristics of each group of mice ( Figure 4 (B) The DAI scores of the MLH engineered bacteria group and EcN were lower than those of the DSS group, and the DAI score of the MLH engineered bacteria was also lower than that of the EcN group, indicating that both the MLH engineered bacteria and the EcN wild strain had a certain therapeutic effect on enteritis, and the MLH engineered bacteria had a better therapeutic effect than the EcN wild strain.

[0079] After the administration of each group, the mice were killed, the colons were collected, and the length of the colons of the mice in each group was measured. Figure 4As shown in C. The colon length of mice in the control group was longer, without congestion and edema, and the residual feces in the intestinal cavity were normal in shape; the colon length of the DSS group was shortened, accompanied by congestion and edema, and the feces in the intestinal cavity were mucous; the colon length of each drug-treated group was slightly longer than that of the DSS group, among which the wild EcN, MLH engineered bacteria and DSS groups had significant differences, but the MLH colon was longer than that of the EcN group, as shown in Figure 4 As shown in D, the feces in the intestinal cavity of each drug-treated group had a certain shape and bleeding was reduced. The spleen size of mice can well reflect the damage to the mouse body. The spleen of the mice in the Control group was normal in size, the spleen of the DSS group was obviously enlarged, and the spleen size of the MLH group was significantly smaller than that of the DSS group. It can be concluded that the damage to the body of mice in the MLH group was significantly lower than that in the DSS group ( Figure 4 E and F).

[0080] In order to observe the development of colon inflammation, HE staining was used to observe the distal colon tissue collected above. The control group mice had intact colon muscle layer, intact mucosal goblet cells, intact crypt structure and regular intestinal glands, neatly arranged villi with clear boundaries, and no inflammatory cell infiltration; the DSS group mice showed an inflammatory reaction in the colon, manifested as mucosal erosion, reduced goblet cells, partial dissolution and loss of crypts and intestinal glands, severe edema of the submucosal layer, and obvious inflammatory cell infiltration; compared with the DSS group, the wild EcN and MLH engineered bacteria groups effectively reduced inflammatory cell infiltration, reduced mucosal epithelial necrosis, and restored the crypt structure. Among them, the MLH engineered bacteria group had a more obvious effect ( Figure 4 Middle G).

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

[0082] SOD determination: After the animal experiment, the mouse colon tissue was taken, washed with physiological saline, homogenized with SOD sample preparation solution in an ice bath, and then centrifuged at 12000g for 3 minutes. The SOD enzyme activity was detected using the total SOD activity detection kit (WST-8 method) (Shanghai Biyuntian Biotechnology Co., Ltd., Shanghai, Beijing).

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

[0084] CAT assay: 0.1 g of tissue was weighed, 1 mL of CAT extract was added for ice bath homogenization, centrifuged at 8000 g for 10 min at 4 °C, and the supernatant was used to detect the CAT content according to the catalase (CAT) activity detection kit (Beijing Solebow Technology Co., Ltd., Beijing, China).

[0085] The results are as follows Figure 5 As shown in A, B and C, compared with the Control group, the SOD and CAT activities in the DSS group decreased, while the MDA content increased, indicating a certain degree of oxidative stress. These injuries showed a trend of alleviation after drug administration in each group, and the SOD and CAT activities in the EcNc-MLH group showed significant differences compared with the DSS group. The results suggest that EcNc-MLH has a certain antioxidant effect.

[0086] 3.4 Determination of inflammatory factors

[0087] Add 1 mL of RNA extraction reagent and three 3 mm grinding beads to a pre-cooled grinding tube. Place colon tissue (5-20 mg) in a tube and homogenize using a tissue grinder until there are no visible fragments. Then centrifuge at 12,000 rpm and 4°C for 10 minutes and collect the supernatant. Total RNA was extracted using the Direct-zol RNA Microprep Kit (Beijing Jianshi Biotechnology Co., Ltd., Beijing, China) and reverse transcribed using the Evo M-MLVPlus cDNA Synthesis Kit (AGBio, Hunan, China) to obtain cDNA. Primers for the target genes IL-6, IL-1β and the reference gene GAPDH were designed using the NCBI qPCR primer design tool. The primer sequences are shown in Table 1. Primers were diluted to 10 μM using double distilled water and used, and qPCR experiments were performed according to the qPCRMasterMix enzyme instructions.

[0088] The results are as follows Figure 5 As shown in D and E, compared with the Control group, the gene expression level of IL-6 / IL-1β in the colon tissue of mice in the DSS group increased significantly. However, compared with the DSS group, the mRNA level of IL-6 / IL-1β decreased after administration, and there was a significant difference between the EcNc-MLH group and the DSS group. It can be seen that the EcNc-MLH engineered strain can reduce the inflammatory response of colon tissue caused by DSS and alleviate the damage of colon tissue.

[0089] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An EcNc engineered bacterium constitutively expressing MLH protein, characterized in that: Using E. coli Nissle1917 as the chassis cell, and containing the modified endogenous cryptic plasmid pMUT1 and / or pMUT2; The transformation comprises inserting the coding gene of MLH protein into the endogenous cryptic plasmid pMUT1 and / or pMUT2, wherein the MLH protein is a fusion protein formed by connecting SOD and CAT by a flexible linker.

2. The EcNc engineered bacteria according to claim 1, characterized in that: The SOD includes RtSOD, with GenBank number WP_012843426.1; the CAT includes FeCAT, with GenBank number WP_012845258.

1.

3. The method for constructing the EcNc engineered bacteria according to claim 1 or 2, characterized in that: The following steps are involved: (1) inserting the coding gene of SOD into the endogenous cryptic plasmid pMUT1 and / or pMUT2, and constructing linearized pMUT1-SOD and / or pMUT2-SOD plasmids by PCR; (2) inserting the coding gene of CAT into a prokaryotic expression vector and constructing a linearized linker-CAT gene fragment using PCR; (3) connecting the linearized pMUT1-SOD and / or pMUT2-SOD plasmids described in step (1) with the linearized linker-CAT gene fragments described in step (2) to construct recombinant vectors pMUT1-MLH and / or pMUT2-MLH; (4) The recombinant vectors pMUT1-MLH and / or pMUT2-MLH described in step (3) are respectively transformed into Escherichia coli Nissle 1917 from which the two endogenous cryptic plasmids pMUT1 and pMUT2 have been removed, thereby constructing EcNc engineered bacteria.

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

2.

5. The construction method according to claim 3, characterized in that: The primers used in the PCR method of 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.

6. Use of the EcNc engineered bacteria according to claim 1 or 2 or the EcNc engineered bacteria constructed by the construction method according to any one of claims 3 to 5 in the preparation of antioxidant preparations.

7. Use of the EcNc engineered bacteria according to claim 1 or 2 or the EcNc engineered bacteria constructed by the construction method according to any one of claims 3 to 5 in the preparation of drugs for preventing and / or treating enteritis.

8. The use according to claim 7, characterized in that: The types of inflammatory bowel disease include ulcerative colitis.

9. The use according to claim 7, characterized in that: The types of drugs for preventing and / or treating enteritis include live bacteria drugs.

10. A drug for preventing and / or treating enteritis, characterized in that: The active ingredient comprises the EcNc engineered bacteria according to claim 1 or 2 or the EcNc engineered bacteria constructed by the construction method according to any one of claims 3 to 5, and also comprises pharmaceutically acceptable excipients.

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