Engineering probiotics for targeted therapy of inflammation as well as construction method and application of engineering probiotics

Abstract

The invention discloses an engineering probiotic for targeted treatment of inflammation and a construction method and application thereof, and the construction method of the engineering probiotic for targeted treatment of inflammation comprises the following steps: step 1, sequentially connecting pelB, a plurality of RGD peptides, a plurality of AAVV-KPV peptides and HIS to a homologous arm through PCR, carrying out seamless cloning, and then inserting into a pBAD33 plasmid to construct a recombinant plasmid; 2, transferring the plasmids obtained in the step 1 into competent E.coliDH5alpha through a heat shock method, verifying by adopting bacterial colony PCR and a double enzyme digestion method, and performing sequencing verification to determine whether the recombinant plasmids are successfully constructed or not; and step 3, under the condition that the recombinant plasmid is successfully constructed, transferring the recombinant plasmid into competent EcN by utilizing a heat shock method, performing screening treatment by adopting a chloramphenicol-resistant LB solid plate, culturing a bacterial colony obtained by screening in an LB liquid culture medium containing chloramphenicol, and performing centrifugal collection and storage to obtain the target engineering probiotics. The invention realizes the characteristics of targeting an inflammation site and treating inflammation.

Description

Technical Field

[0001] The present invention relates to the field of engineering bacteria, and in particular to an engineering probiotic for targeted treatment of inflammation and a construction method and application thereof. Background Art

[0002] At present, probiotics are generally used to treat inflammatory diseases, such as enteritis, hepatitis, pneumonia, arthritis, neuroinflammation, and wound inflammation. Non-steroidal anti-inflammatory drugs, immunosuppressants, etc. are mostly used clinically for treatment, but long-term use is prone to side effects such as drug resistance, and the production of anti-inflammatory substances expressed by probiotics is low, which makes the efficacy in shortening the course of the disease limited. When the target protein of the engineered live bacteria is secreted in large quantities, it often forms inclusion bodies and cannot exert its effect outside the cell. Moreover, even natural microbial secretory proteins are often dispersed in the intestinal cavity and cannot accurately target the site of enteritis. Importantly, the target protein of the engineered live bacteria is often unable to recognize the physiological state of the intestine and cannot respond to enteritis to exert its effect.

[0003] The invention patent with the existing patent publication number CN111514316A discloses an inflammation-targeted and microenvironment-responsive nanosystem, preparation method and application, the nanosystem includes a targeting functional molecule, a nanocarrier, and a core crystal; the targeting functional molecule is a CD163 targeting binding short peptide, and the short peptide is connected to the polyethylene glycol on the surface of the nanoparticle by covalent linkage. Although the adopted short peptide nanosystem has high stability, tissue penetration, biocompatibility and low immunogenicity, the preparation process of the short peptide nanosystem is complicated, costly and single in function. Therefore, it is urgent to study new solutions to enhance the effect of probiotics in targeting inflammatory sites, responding to inflammation and treating inflammation. Summary of the invention

[0004] The main purpose of the present invention is to provide an engineered probiotic for targeted treatment of inflammation and a construction method and application thereof, aiming to solve the technical problem that existing anti-inflammatory drugs are not universal.

[0005] To achieve the above-mentioned purpose, the present invention provides an engineered probiotic for targeted treatment of inflammation. The engineered probiotic has the Latin name of Escherichia coli Nissle 1917MY-1, which is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with the deposit number of CGMCC NO.31540 and the deposit date of August 5, 2024.

[0006] In addition, to achieve the above purpose, the present invention also provides a method for constructing the above-mentioned engineered probiotics for targeted treatment of inflammation, characterized in that the method comprises the following steps:

[0007] Step 1, the mRNA sequences of pelB, multiple RGD peptides, multiple AAPV-KPV peptides and 6*HIS were synthesized, and then the homology arms were connected in sequence by PCR, and then inserted into the multiple cloning site of the pBAD33 plasmid after seamless cloning to construct a recombinant plasmid;

[0008] Step 2, the plasmid obtained in step 1 is transformed into competent E. coli DH5α by heat shock method, and after verification by colony PCR and double enzyme digestion method, sequencing verification is performed to determine whether the recombinant plasmid is successfully constructed;

[0009] Step 3, under the condition that the recombinant plasmid is successfully constructed, the recombinant plasmid is transferred into the competent EcN by the heat shock method, and the chloramphenicol-resistant LB solid plate is used for screening. The screened colonies are cultured in LB liquid culture medium containing chloramphenicol, and collected and preserved by centrifugation to obtain the target engineered probiotics.

[0010] Optionally, the recombinant plasmid is Pbad33-pelB-RGD-AAPV-KPV-HIS, and the gene sequence of the recombinant plasmid is shown in SEQ ID NO.10.

[0011] Optionally, step 3 further includes performing Western blot protein verification on the obtained target engineered probiotics, and detecting the expression level by ELISA.

[0012] In addition, to achieve the above objectives, the present invention also provides a pharmaceutical composition, which includes the above-mentioned engineered probiotics for targeted treatment of inflammation.

[0013] Optionally, the pharmaceutical composition further comprises a pharmaceutically or pharmacologically acceptable carrier.

[0014] In addition, to achieve the above-mentioned purpose, the present invention also provides a kit, which includes the above-mentioned engineered probiotics for targeted treatment of inflammation or the above-mentioned pharmaceutical composition.

[0015] In addition, to achieve the above objectives, the present invention also provides the use of the above-mentioned engineered probiotics for targeted treatment of inflammation, the above-mentioned pharmaceutical composition or the above-mentioned kit in the preparation of in vivo antioxidant and anti-inflammatory drugs or in vitro dressing antioxidant drugs or health products.

[0016] Optionally, the dosage form of the medicine or health product is tablets, capsules, soft capsules, granules, pills, oral liquids, emulsions or dry suspensions.

[0017] Beneficial effects:

[0018] The engineered probiotics of the present invention are synthesized by using mRNA sequences of pelB, multiple RGD peptides, multiple AAPV-KPV peptides and 6*HIS, and then connected to homology arms by PCR in sequence, and inserted into the multiple cloning site of pBAD33 plasmid after seamless cloning to obtain a recombinant plasmid, and the final expression vector is constructed to achieve transformation EcN, thereby obtaining a new engineered probiotic for targeted treatment of inflammation. The engineered probiotics can produce the target protein in situ continuously under the condition of normal bacterial growth, and the produced target protein does not need to form inclusion bodies but is directly secreted outside the cell, and the secretory protein of the engineered bacteria has the characteristics of targeting the inflammatory site and treating inflammation. In addition, due to the stability of the multi-copy ultrashort peptide, the efficacy of the target protein is improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a schematic diagram of the design of proteins in the recombinant plasmid; Figure 2 I is the recombinant plasmid map; Figure 3 The mechanism of action of MY EcN bacteria; Figure 4 This is a schematic diagram of the results of enzyme digestion identification of the recombinant plasmid; Figure 5 Colony PCR for the constructed MY EcN bacteria; Figure 6 Compare the results of the PCR product of the recombinant colony with the expected DNA sequence; Figure 7 It is the protein electrophoresis diagram; Figure 8 Modeling and weight fluctuations during treatment for different groups of experimental mice; Fig. 9 The colon lengths of experimental mice after treatment in different groups; Fig.10 Fecal occult blood test for different groups of experimental mice; Fig.11 H&E staining of cross-sections of colon tissues of experimental mice after treatment in different groups; Fig.12 Figure 2 shows histological evaluation of colon tissues of mice in different groups; Fig.13 H&E staining of heart, liver, spleen, lung and kidney tissue sections of experimental mice after treatment in different groups; Fig.14 The spleen size and weight distribution of experimental mice after different groups of treatment; Fig.15 The changes of respiratory metabolism indicators of experimental mice in different groups after treatment within 24 hours (night and day); Fig.16 The total changes of respiratory metabolism indicators at night and during the day in experimental mice after treatment in different groups; Fig.17 This is a graph of the disease activity index of experimental mice after treatment in different groups.

[0020] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0021] It should be understood that the specific embodiments described herein are only used to explain the present invention, and are not used to limit the present invention.

[0022] Example 1

[0023] Experimental animals

[0024] C56BL / 6j mice, 23 g ± 2 g, male, 5 mice per group, were purchased from Hunan Slake Jingda Experimental Animal Co., Ltd., production license number: SCXK (Xiang) 2016-0002, and were uniformly raised in the experimental animal center of our college, with a temperature of 23 ° C ± 2 ° C, humidity of 55% ± 5%, 12 h day and night cycle lighting, caged, free food and water. All animal experiments met the requirements of the local experimental animal ethics review committee.

[0025] Experimental reagents

[0026] LB medium (g / L): 5 g yeast extract; 5 g sodium chloride; 10 g tryptone; 1000 mL distilled water; pH 7.0.

[0027] Table 1 shows the main reagents used in the experiment, and Table 2 shows the primer sequences used.

[0028] Table 1 Main experimental reagents

[0029]

[0030]

[0031] Table 2 shows the primer sequences used

[0032]

[0033] Experimental methods

[0034] Strains, plasmids and recombinant gene sequences

[0035] The strains of E. coli DH5α, strain E. coli Nissle1917, strain pTD103luxl-EcN, and plasmid pBAD33 used in this experiment were stored in the research group. The gene sequence of plasmid pBAD33 is shown in SEQ ID NO.3

[0036] PelB was obtained from the National Center for Biotechnology Information (NCBI) website, and its sequence is shown in SEQ ID NO.4. The specific gene sequence is:

[0037] Atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggcc.

[0038] The gene sequences of multiple RGD peptides, multiple AAPV, KPV peptides and HIS are shown in Table 3 below.

[0039] Table 3 - RGD peptide, multiple AAPV-KPV peptides and HIS gene sequences

[0040]

[0041] Construction method of engineered bacteria MY EcN

[0042] Step 1: synthesize the mRNA sequences of pelB, multiple RGD peptides, multiple AAPV-KPV peptides and 6*HIS, connect the homology arms by PCR, connect the homology arms by PCR, and insert them into the multiple cloning site of pBAD33 plasmid after seamless cloning. The seamless cloning steps are as follows: Follow the instructions for the Seamless Cloning Kit.

[0043] Step 2: The plasmid obtained in step 1 is transformed into competent E. coli DH5α by heat shock method, and then verified by colony PCR and double enzyme digestion method, and then sequenced to determine whether the recombinant plasmid is successfully constructed. And the design of the protein in the recombinant plasmid is as follows: Figure 1 As shown, the inserted sequence is shown as SEQ ID NO.8, and the amino acid corresponding to the inserted sequence is shown as SEQ ID NO.9.

[0044] Recombinant plasmid map Figure 2 As shown, and the sequence of the recombinant plasmid is shown as SEQ ID NO.10.

[0045] Furthermore, in step 2, the step of determining whether the recombinant plasmid is successfully constructed is to transform the constructed plasmid into the competent E. coli, and use MY-F1 and MY-R1 in Table 2 as primers, and the colonies obtained by the above transformation as templates to PCR amplify the target gene fragment, and identify whether the plasmid is successfully transferred by colony PCR identification. Specifically, the integrity of the constructed target plasmid is verified by agarose gel electrophoresis, such as Figure 4 As shown, HindIII+NcoI double enzyme digestion identification was performed, lane 1 was HindIII+NcoI double enzyme digestion identification, lane 2 was the original plasmid electrophoresis (integrity identification), M in the figure is a marker, and the two fragments after electrophoresis are consistent with expectations, proving that the plasmid was successfully constructed.

[0046] After the recombinant plasmid is successfully constructed and transferred into E. coli EcN, it is plated on a medium containing chloramphenicol for preliminary screening, and colony PCR is performed on the grown bacteria. Figure 5The colony PCR of the constructed MY EcN bacteria is shown in the figure. Lanes 1 and 2 are ECN, lanes 3 and 4 are P-ECN (EcN bacteria transferred into the empty vector Pbad33), and lanes 5 and 6 are MYECN. Among them, EcN without the pbad33 vector does not have any bands, and the agarose gel electrophoresis band positions of EcN containing the pbad33 empty vector and EcN containing the recombinant vector are consistent with expectations, indicating that the recombinant bacteria MY EcN was successfully constructed. In addition, the target product of the recombinant colony PCR can be compared with the expected DNA sequence, and the results are shown in Figure 6 As shown, the sequence above the dark part is the target product of the recombinant colony PCR, and the sequence below is the expected DNA sequence. It can be seen that the consistency after comparison of the two sequences using DNAMAN is 100%, and it is confirmed again that the recombinant bacteria MY EcN was successfully constructed.

[0047] Step 3, under the condition that the recombinant plasmid is successfully constructed, the recombinant plasmid is transferred into the competent EcN by heat shock method, and the chloramphenicol resistance LB solid plate is used for screening treatment, the colonies screened are cultured in LB liquid medium containing chloramphenicol, and collected and preserved by centrifugation, and the target bacterial liquid is sequenced and verified, and Western blot protein verification is performed. Specifically, the size of the recombinant protein is 16.9 kda, and the expression of the protein is verified by western blot experiment, such as Figure 7 As shown in the figure, the protein electrophoresis shows that the size of the target protein is consistent with the expected value, indicating that the target protein can be expressed in the recombinant bacteria.

[0048] The expression level was detected by ELISA to obtain the engineered probiotics for the targeted treatment of colitis. The target bacterial solution was subjected to colony PCR, sequencing and Western blot to identify protein expression and confirm the successful transformation. The target protein was named MY and the engineered bacteria after transformation were named MY EcN. The mechanism of action of MY EcN bacteria is as follows Figure 3 In addition, the Latin name of the engineered probiotic is Escherichia coli Nissle 1917MY-1, which is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with a deposit number of CGMCC NO.31540 and a deposit date of August 5, 2024.

[0049] Furthermore, in order to illustrate the therapeutic effect of the prepared MYEcN on intestinal inflammation and damage in UC mice, the following is a specific experimental description.

[0050] Example 2

[0051] Experimental animals

[0052] C56BL / 6j mice, 23 g ± 2 g, male, 5 mice per group, were purchased from Hunan Slake Jingda Experimental Animal Co., Ltd., production license number: SCXK (Xiang) 2016-0002, and were uniformly raised in the local experimental animal center, with a temperature of 23 ° C ± 2 ° C, humidity of 55% ± 5%, 12 h day and night cycle lighting, caged, free food and water. All animal experiments met the requirements of the local experimental animal ethics review committee.

[0053] Experimental instruments

[0054] Table 4 shows the main instruments used in the experiment, and Table 5 shows the main reagents used in the experiment.

[0055] Table 4 Main instruments used

[0056]

[0057] Table 5 Main reagents used

[0058]

[0059] Experimental methods

[0060] Establishment of a mouse model of ulcerative colitis

[0061] C56BL / 6j mice, male, weighing 23g±2g, were randomly divided into groups of 8 mice, with a total of 4 groups, namely PBS group (blank group), dextran sodium phosphate DSS group, P-EcN group, and MY EcN group. The establishment of the ulcerative colitis model refers to the existing technology. After the adaptation period, the drinking water of all mice except the PBS group was replaced with 3% DSS for 0-7 days. From the 8th day, all groups of mice were gavaged with the corresponding preparations, as shown in the following figure. Figure 8 As shown in the figure, it can be observed that after gavage of the engineered probiotic MY EcN in the experimental group, the increase in body weight was significantly higher than that in the other groups. Therefore, it is shown that the engineered probiotic MYEcN can alleviate the weight loss caused by ulcerative colitis.

[0062] Recording of mouse biochemical indicators and sample collection

[0063] During the experiment, the weight of the mice was recorded daily. After the experiment, the length of the colon of each mouse was recorded and photographed. Fig. 9 The above figure shows the actual length. Fig. 9 The figure below is the column corresponding to the figure above. The length of the colon can reflect the severity of colitis. The shorter the colon, the more severe the inflammation. From the figure, we can know that the colon length of the modeling group (DSS group) is significantly shorter than that of the normal group (PBS group), and the colon length of the treatment group (MY EcN group) is significantly longer than that of other modeling groups. Therefore, it is shown that the engineered probiotic MY EcN can significantly improve the shortening of the colon caused by colitis.

[0064] Fecal occult blood test

[0065] The concentration of o-tolidine is generally prepared at 0.5-1g / L. (1% o-tolidine reagent is required, prepared with a mixture of glacial acetic acid and anhydrous ethanol), mix the mouse feces from different experimental groups with 200μL of normal saline. Centrifuge the mixed fecal sample for 10 minutes and take the supernatant. Take 100μL of supernatant and put it in a new EP tube, add 100μL of o-tolidine reagent, and mix well. Add 10μl of hydrogen peroxide and observe the color change. If blue staining appears, it means that there is occult blood in the feces. The darker the color, the more serious the occult blood in the feces.

[0066] By observing the fecal changes of mice in different experimental groups from 1 to 16 days, Fig.10 As shown in the figure, the modeling group (DSS group) is significantly darker than the normal group (PBS group), indicating that the modeling is successful. The treatment group (MYEcN group) has a significant change in color compared to other modeling groups, and as the observation time increases, the color of the blood in the stool gradually becomes lighter. It can be seen that the engineered probiotic MYEcN can significantly improve the symptoms of fecal occult blood caused by colitis.

[0067] Pathological analysis

[0068] The colon samples were washed twice with saline, immersed in 1 mL of tissue fixative solution containing 4% paraformaldehyde, and the tissues were embedded in paraffin. 10 μm paraffin sections were used for H&E staining observation, and the H&E staining results were analyzed and evaluated. H&E staining scoring criteria: The total histological score is expressed as E+I, with a total score of 16 points. The specific scoring criteria are shown in Table 6.

[0069] Table 6 DSS colitis histological grading scheme

[0070]

[0071]

[0072] Colon histopathological analysis

[0073] Specific methods: The colon tissues of different experimental groups were fixed with 4% paraformaldehyde, embedded in paraffin hearts, cut into 4 μm thick sections, stained with hematoxylin and eosin (H&E), and observed under an upright optical microscope.

[0074] The images were further analyzed to assess intestinal tissue damage and colonic edema. Fig.11As shown in the figure, after H&E staining, the colon tissue of the normal group (PBS group) can be seen in the figure. DSS-induced colitis caused colon ulcers in the DSS group mice to spread to the mucosa and submucosal layer, inflammatory cell infiltration was seen in the mucosal layer, and significant crypt damage was observed. No obvious colon ulcers were observed in the engineered bacteria MY EcN group, a small amount of inflammatory cell infiltration was seen in the mucosal layer, while a large amount of inflammatory cell infiltration was seen in the empty EcN group. This shows that the engineered bacteria can significantly alleviate the colon tissue damage caused by DSS.

[0075] Furthermore, the colon tissues of mice in different groups were evaluated histologically. Fig.12 As shown, the colon tissue of mice treated with the engineered probiotic MYEcN showed significant therapeutic effects, with reduced histopathological scores, regular interdigitating crypt structure, intact epithelium, and reduced inflammatory cell infiltration, similar to healthy colon.

[0076] Histopathological analysis of heart, liver, spleen, lung and kidney

[0077] In vivo biocompatibility experiment: H&E staining of heart, liver, spleen, lung and kidney tissues was performed in the normal group and all the modeling groups with DSS added to drinking water. Fig.13 As shown, the heart, liver, spleen, lung and kidney tissues and cell structures of all groups were normal, indicating that the engineered probiotics would not cause lesions in other tissues and had good biocompatibility.

[0078] Furthermore, the size and weight of the spleen can reflect the severity of inflammation to a certain extent. The larger and heavier the spleen, the more severe the inflammation. Then the spleen size and weight of the experimental mice after different groups of treatment were measured, such as Fig.14 As shown, Fig.14 The above picture shows the shape of the spleen under different treatments. Fig.14 The figure below shows the corresponding weight distribution of the spleen. It can be seen from the figure that the spleen size and weight of the engineered probiotic MY EcN group are close to those of the normal group, indicating that the engineered probiotic MYEcN can significantly alleviate intestinal inflammation caused by DSS.

[0079] Respiratory metabolism experiment

[0080] Based on the fact that intestinal diseases affect the host's energy metabolism, studies have shown that patients with inflammatory bowel disease (IBD) suffer from weight loss, malnutrition and several other metabolic changes that affect their quality of life, that is, the body weight, respiratory exchange ratio (RER), food intake and body fat content of mice with acute inflammation all decrease. Mice with inflammation will change their metabolism, which can be used as a measurement indicator of response or adaptation to severe colon inflammation. The severity of the disease can then be reflected by the maximum oxygen consumption and carbon dioxide production. Furthermore, by measuring the changes in respiratory exchange rate, energy consumption, oxygen absorption and carbon dioxide exhalation of experimental mice after treatment in different groups within 24 hours (night and day), the specific measurement results are as follows Fig.15 As shown, Fig.15 a is the change in respiratory exchange rate, Fig.15 b is the change in energy consumption, Fig.15 c is the oxygen absorption, Fig.15 d is the exhaled carbon dioxide volume. It can be seen from the figure that the various respiratory metabolic indicators of the engineered bacteria MYEcN group are closer to the normal group (PBS group), indicating that the engineered bacteria MY EcN has the ability to significantly restore the respiratory metabolism of mice with colitis.

[0081] Furthermore, the total changes of respiratory metabolic indicators at night and during the day in the experimental mice after treatment in different groups were measured. Fig.16 As shown, Fig.16 b is the respiratory exchange rate during the day and night, Fig.16 a is the total respiratory exchange rate between night and day, Fig.16 d is the oxygen absorption during the day and night, Fig.16 c is the total oxygen absorption during the night and day, Fig.16 f is the amount of carbon dioxide exhaled during the day and night, Fig.16 e is the total amount of carbon dioxide exhaled during the night and day, Fig.16 h is the energy consumption during the day and night, Fig.16 a is the total energy consumption during the night and day. It can be seen from the figure that the respiratory metabolism indicators of the engineered probiotics MY EcN group are closer to the normal group (PBS group) both during the day and at night. This further indicates that the engineered bacteria MYEcN has the ability to significantly restore the respiratory metabolism of mice with colitis.

[0082] Disease Activity Index (DAI score) of mice

[0083] The disease activity index of the experimental mice in different groups after treatment was measured, that is, the percentage of weight loss, stool characteristics and the degree of fecal occult blood were measured. The specific standards and scores are shown in Table 7.

[0084] Table 7 - Disease Activity Index (DAI) parameters and their associated scores

[0085]

[0086] According to the scoring scheme in Table 7, we get Fig.17 The disease activity index of mice described in the , DAI score of mice treated with the engineered probiotic MY EcN was significantly reduced on day 15 compared with that of the colitis mice in the DSS group.

[0087] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or system including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or system. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or system including the element.

[0088] The serial numbers of the above embodiments of the present invention are only for description and do not represent the advantages or disadvantages of the embodiments.

[0089] The above are only preferred embodiments of the present invention, and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. An engineered probiotic for targeted treatment of inflammation, characterized in that: The Latin name of the engineered probiotic is Escherichia coli Nissle 1917MY-1, and it is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with a deposit number of CGMCC NO.31540.

2. A method for constructing an engineered probiotic for targeted treatment of inflammation as claimed in claim 1, characterized in that: The method comprises the following steps: Step 1, the mRNA sequences of pelB, multiple RGD peptides, multiple AAPV-KPV peptides and 6*HIS were synthesized, and then the homology arms were connected in sequence by PCR, and then inserted into the multiple cloning site of the pBAD33 plasmid after seamless cloning to construct a recombinant plasmid; Step 2, the plasmid obtained in step 1 is transformed into competent E. coli DH5α by heat shock method, and after verification by colony PCR and double enzyme digestion method, sequencing verification is performed to determine whether the recombinant plasmid is successfully constructed; Step 3, under the condition that the recombinant plasmid is successfully constructed, the recombinant plasmid is transferred into the competent EcN by the heat shock method, and the chloramphenicol-resistant LB solid plate is used for screening. The screened colonies are cultured in LB liquid culture medium containing chloramphenicol, and collected and preserved by centrifugation to obtain the target engineered probiotics.

3. The method for constructing an engineered probiotic for targeted treatment of inflammation according to claim 2, characterized in that: The recombinant plasmid is Pbad33-pelB-RGD-AAPV-KPV-HIS, and the gene sequence of the recombinant plasmid is shown in SEQ ID NO.

10.

4. The method for constructing an engineered probiotic for targeted treatment of inflammation according to claim 2, characterized in that: The step 3 further includes performing Western blot protein verification on the obtained target engineered probiotics.

5. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the engineered probiotics for targeted treatment of inflammation according to claim 1.

6. The pharmaceutical composition according to claim 5, characterized in that The pharmaceutical composition also includes a pharmaceutically or pharmacologically acceptable carrier.

7. A kit, characterized in that: The kit comprises the engineered probiotics for targeted treatment of inflammation according to claim 1 or the pharmaceutical composition according to any one of claims 5-6.

8. Use of the engineered probiotics for targeted treatment of inflammation according to claim 1, the pharmaceutical composition according to any one of claims 5 to 6, or the kit according to claim 7 in the preparation of in vivo antioxidant and anti-inflammatory drugs or in vitro dressing antioxidant drugs or health products.

9. The use according to claim 8, characterized in that: The dosage form of the medicine or health product is tablet, capsule, soft capsule, granule, pill, oral liquid, emulsion or dry suspension.

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