Enterobacter carrying CD176 antigen and use thereof

Abstract

Provided in the present invention are an Enterobacter carrying a CD176 antigen and the use thereof. The Enterobacter has a 16S rRNA gene sequence having 99% identity to the 16S rRNA gene sequence of Escherichia coli species, can express a CD176 antigen and can induce CD176 antibody production by means of immunization, and is deposited in China General Microbiological Culture Collection Center with the deposit number of CGMCC No. 27745. The bacterium can be used as an antigen for inducing an immune response targeting CD176, can be used as an engineered bacterium, and has broad applications.

Description

Enterobacterium carrying CD176 antigen and application thereof Technical Field

[0001] The present invention relates to an enterobacterium carrying a CD176 antigen and an application thereof, belonging to the technical field of microorganisms. Background Art

[0002] The CD176 carbohydrate antigen (also known as Thomsen-Friedenreich antigen (TF; Galb1-3GalNAca1-R) is a polysaccharide structure. Studies have shown that CD176 is a malignancy-associated carbohydrate antigen expressed on the cell surfaces of various malignant tumors, including breast, intestinal, gastric, lung, ovarian, and renal cancers, lymphomas, and leukemias. However, normal tissues and cells do not express CD176 because it is masked and cannot be recognized by antibodies or lymphocytes. CD176 plays a crucial role in the development and progression of malignant tumors. CD176 expressed on the surface of tumor cells acts as an adhesion molecule and participates in liver metastasis. CD176 expressed on tumor cells may also participate in the attachment of tumor cells to vascular walls, thereby contributing to hematogenous metastasis. CD176 is also expressed on various malignant tumor stem cells. Antibodies against CD176 can induce apoptosis in CD176-positive tumor cells in vitro and in vivo and block their adhesion to vascular endothelial cells and hepatocytes. Antibody therapy experiments have demonstrated that treatment with CD176 antibodies has a significant therapeutic effect in CD176-positive tumor-bearing mice: it reduces liver and hematogenous metastasis of tumor cells and significantly prolongs the survival of experimental mice. Active immunization against CD176 (TF antigen) to increase CD176 antibody levels has shown promising therapeutic results in both experimental animals and patients. CD176 antigens and antibodies have practical applications in both basic oncology research and clinical practice. However, chemical synthesis and biological extraction of CD176 glycoproteins require high technical requirements, resulting in high production costs and low yields, making them very expensive.

[0003] Some specialized bacterial variants possess specific polysaccharide macromolecular structures that are similar or identical to those found in humans and animals. These specific bacterial polysaccharide macromolecules are antigenic and can act as antigens to induce corresponding immune responses, such as antibody production, in humans and animals. For example, some bacteria possess human blood group antigens A or B on their surfaces. Bacteria carrying these antigens, when present in the human intestine or infecting humans, can induce the production of antibodies against these antigens. A small number of specialized bacterial variants possess the CD176 saccharide antigen on their surfaces. These bacteria can induce the production of CD176 antibodies in humans and animals. If specialized bacteria possessing or capable of producing the CD176 saccharide antigen are identified, they could be used as CD176 antigens to induce anti-CD176 immune responses, such as the induction and preparation of CD176 antibodies. This would have broad application prospects in tumor research, diagnosis, and treatment. Technical Solutions

[0004] The present invention provides an Enterobacterium carrying CD176 antigen and application thereof, which can effectively solve the above problems.

[0005] The present invention is achieved in that:

[0006] The invention relates to Escherichia coli JH166, which can express CD176 antigen and induce CD176 antibody through immunization, and has a deposit number of CGMCC No. 27745.

[0007] In some embodiments, the 16S rRNA gene sequence of Escherichia coli JH166 is at least 99.86% identical to the 16S rRNA gene sequence of Escherichia coli.

[0008] A bacterial component is one or more of the live bacteria, inactivated bacteria, lysate, precipitate after lysis, and culture medium of the above-mentioned Escherichia coli.

[0009] A food, health product, food additive or experimental material comprising the above-mentioned bacterial component.

[0010] A use of the above-mentioned live Escherichia coli bacteria in the preparation of engineered bacteria.

[0011] The invention relates to a use of Escherichia coli in the preparation of an anti-tumor drug or an anti-tumor vaccine, wherein the bacterium is the above-mentioned Escherichia coli.

[0012] An anti-tumor drug or anti-tumor vaccine comprises the above-mentioned Escherichia coli or the above-mentioned bacterial component.

[0013] A method for preparing CD176 antibodies comprises immunizing humans and animals with the above-mentioned Escherichia coli or one or more of the above-mentioned bacterial components to induce the production of CD176 antibodies.

[0014] A CD176 antibody prepared by the above method.

[0015] A method for preparing CD176 antigen comprises culturing the above-mentioned Escherichia coli and extracting CD176 antigen from the Escherichia coli, a lysate of the Escherichia coli, a precipitate after lysis, and a culture medium after cultivation. Beneficial effects

[0016] The beneficial effects of the present invention are:

[0017] The bacteria of the present invention can be repeatedly subcultured for more than 40 generations and can still stably and highly express the CD176 antigen. The antigen can be immune-induced to produce CD176 antibodies and can be used to prepare CD176 antibodies for tumor research, diagnosis and treatment, with broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 is a diagram of the colony morphology of the bacterial strain JH166 provided in Example 1 of the present invention. A, bacterial strain JH166 grown in solid E. coli chromogenic medium; B, bacterial strain JH166 grown in solid meat extract peptone medium; C, bacterial strain JH166 grown in MacConkey medium.

[0020] Figure 2 shows the expression of the CD176 antigen in the JH166 bacterial strain provided in Example 1 of the present invention after 40 repeated subcultures. P2-P40 represent the number of passages of JH166 bacterial strain; E. coli (a standard E. coli strain) does not express the CD176 antigen; the experimental group is the test group; the control group is a blank control without the addition of the primary antibody.

[0021] FIG3 is a partial BLAST comparison result of the 16S rRNA gene sequence of the bacterial strain JH166 provided in Example 3 of the present invention.

[0022] Figure 4 shows the detection results of five pathogenic E. coli genes in bacterial strain JH166, provided in Example 4 of the present invention. M: DNA marker; Sample 166: bacterial strain JH166. EPEC-escV and STEC / EHEC-stx2: positive controls; Blank: negative control.

[0023] Figure 5 is a genome map of bacterial strain JH166 provided in Example 5 of the present invention. The outermost circle represents the genomic sequence coordinates. From outside to inside, the following are the gene function annotation results (including COG (KOG) annotation information depending on the project), ncRNA, and genomic GC content: GC content is calculated using a window of (chromosome length / 1000) bp and a step size of (chromosome length / 1000) bp. Inward blue indicates that the GC content of the region is lower than the genome-wide average GC content, while outward red indicates the opposite. Higher peaks indicate greater differences from the average GC content. Genomic GC skew value: Window of (chromosome length / 1000) bp and a step size of (chromosome length / 1000) bp. The specific algorithm is GC / G+C. Inward green indicates that the G content of the region is lower than the C content, while outward orange indicates the opposite.

[0024] Figure 6 is a diagram showing the distribution of pathogen PHI phenotypic mutation types in the gene function annotation of bacterial strain JH166 provided in Example 5 of the present invention. Note: The horizontal axis represents the phenotypic mutation type, and the vertical axis represents the number of annotated genes.

[0025] Figure 7 shows the DNA fingerprint of bacterial strain JH166 provided in Example 6 of the present invention. M: DNA marker (M1, 250-10,000 bp; M2, 50-1,000 bp); 166: bacterial strain JH166. ERIC-PCR results show a JH166-specific band pattern between 250 and 2,500 bp; REP-PCR results show a JH166-specific band pattern between 250 and 1,000 bp; and BOX-PCR results show a JH166-specific band pattern below 1,000 bp.

[0026] Figure 8 shows the growth of heat-inactivated bacterial strain JH166, as provided in Example 7 of the present invention. A: No bacterial growth was observed after heat-inactivation of strain JH166 and inoculation into LB medium for 48 hours. B: Good bacterial growth was observed after inoculation of heat-inactivated strain JH166 into LB medium for 16 hours.

[0027] Figure 9 is a graph showing the weight change trend of mice after injection of heat-killed bacteria JH166, as provided in Example 9.1 of the present invention. Note: 1-5 are the experimental mouse numbers.

[0028] Figure 10 is a graph showing the weight change trend of mice after oral administration (or gavage) of live bacteria JH166 as provided in Example 9.2 of the present invention. Note: 1-5 are the experimental mouse numbers.

[0029] Figure 11 is a graph showing the weight change trend of mice after oral administration of heat-killed bacteria JH166 as provided in Example 9.3 of the present invention. Note: 1-5 are the experimental mouse numbers.

[0030] Figure 12 is a graph showing the normal weight change trend of mice without the addition of bacteria JH166, as provided in Example 9.4 of the present invention. Note: 1-5 are the experimental mouse numbers.

[0031] Figure 13 shows histological photographs of selected organs from mice treated with heat-killed bacteria, live bacteria administered orally (gavage), and heat-killed JH166 bacteria administered orally, as provided in Example 9 of the present invention, as well as a control group without added bacteria. No pathological changes were observed in the relevant organs. Note: 1-4 are mice treated with heat-killed bacteria; 5-8 are mice treated with live bacteria administered orally; 9-12 are mice treated with heat-killed bacteria administered orally; 13-16 are mice treated with normal control groups. Modes for Carrying Out the Invention

[0032] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments.

[0033] An embodiment of the present invention provides an Escherichia coli, whose 16S rRNA gene sequence is at least 99.86% identical to the 16S rRNA gene sequence of Escherichia coli species, can express CD176 antigen and can induce immune production of CD176 antibodies.

[0034] In some embodiments, the bacteria are deposited in the China General Microbiological Culture Collection Center with the accession number CGMCC No. 27745. The bacteria are isolated from feces of healthy adults.

[0035] In some embodiments, the E. coli does not contain the pathogenic E. coli genes escV, stx2, lt, invE, and aggR.

[0036] In some embodiments, the Escherichia coli does not contain the drug resistance genes blaTEM, NDM-1, tetA, MCR-1 and blaNDM.

[0037] In some embodiments, the E. coli does not express human blood group antigens A and B.

[0038] The embodiment of the present invention provides a bacterial component, which is one or more of the whole bacteria, bacterial lysate, precipitate after lysis, and culture medium after culture of the above-mentioned bacteria. Whole bacteria include all components of bacteria, specifically including bacterial cytoplasm, cell wall, cell membrane, pili, flagella, lipopolysaccharide (LPS), etc.; the lysate after bacterial lysis includes bacterial proteins and polypeptides, polysaccharides, glycoproteins, bacterial metabolites, etc.; the precipitate after bacterial lysis includes most components of bacteria, specifically including cell wall, cell membrane, pili, flagella, lipopolysaccharide, etc., and the culture medium after bacterial culture includes bacterial extracellular vesicles, bacteria-related exosomes, bacterial metabolites, etc. These bacterial components all contain CD176 antigens and can also be used for immune induction to produce CD176 antibodies. The whole bacteria include live bacteria and / or inactivated bacteria of the above-mentioned bacteria. The inactivated bacteria are obtained by inactivating the above-mentioned bacteria by conventional inactivation methods.

[0039] The present invention provides a composition comprising the aforementioned bacterial component. The bacterial component can be mixed with other components as needed to form a composition. The bacterial component in the composition contains CD176 antigen and can induce the production of CD176 antibodies, thus having a wide range of uses.

[0040] In some embodiments, the composition is a food, a health product, or a food additive. The bacterial or cell components are highly safe and meet food safety standards. The bacterial components can be added to some food-acceptable carriers or excipients to prepare the bacterial components into food, a health product, or a food additive.

[0041] An experimental material comprising the aforementioned bacterial component, which carries the CD176 antigen and can induce the production of CD176 antibodies, and can be used as an experimental material in tumor research, diagnosis, and treatment.

[0042] The use of the live E. coli bacteria mentioned above in the preparation of engineered bacteria. The bacteria mentioned above can be further improved into engineered bacteria by genetic engineering methods, such as implanting target genes, thereby improving their various properties and expanding their applications.

[0043] An anti-tumor vaccine comprises the live or inactivated Escherichia coli bacteria mentioned above. The live or inactivated bacteria can induce the body to produce CD176 antibodies and has high safety, and can be used as a vaccine to prevent tumors.

[0044] An anti-tumor drug comprising the aforementioned Escherichia coli and / or the aforementioned bacterial component. The aforementioned live or inactivated bacteria can induce the body to produce CD176 antibodies, which can be used to treat a variety of malignant tumors, such as breast cancer, intestinal cancer, lung cancer, leukemia, etc.

[0045] In some embodiments, the anti-tumor drug is prepared by adding an effective dose of the above-mentioned Escherichia coli and / or the above-mentioned bacterial components to pharmaceutically acceptable carriers and excipients to form tablets, capsules, granules or injections.

[0046] The invention relates to a use of Escherichia coli in the preparation of an anti-tumor drug, wherein the bacteria is the above-mentioned Escherichia coli. The CD176 antigen is extracted from the above-mentioned Escherichia coli as an anti-tumor drug, or the above-mentioned Escherichia coli is directly used as an anti-tumor drug.

[0047] The invention discloses an Escherichia coli component for use in preparing foods, health products, food additives, and experimental materials. The bacterial component is the aforementioned bacterial component. The Escherichia coli can induce the production of CD176 antibodies, which have anti-tumor effects and are highly safe. The Escherichia coli can be used in preparing foods, health products, food additives, and experimental materials.

[0048] The invention relates to the use of Escherichia coli in the preparation of an anti-tumor vaccine, wherein the bacteria are the live Escherichia coli or inactivated bacteria.

[0049] An embodiment of the present invention provides a method for preparing CD176 antibodies, which comprises immunizing humans and animals with the aforementioned bacteria or one or more of the aforementioned bacterial components to induce the production of CD176 antibodies, and then extracting the CD176 antibodies.

[0050] An embodiment of the present invention provides a CD176 antibody prepared using the above method.

[0051] An embodiment of the present invention provides a method for preparing CD176 antigen, which comprises culturing the aforementioned bacteria and extracting CD176 antigen from the bacteria, bacterial lysate, precipitate after lysis, and culture medium after culturing.

[0052] In some embodiments, the culturing of the Escherichia coli is performed for 1-50 generations.

[0053] An embodiment of the present invention provides a CD176 antigen prepared by the above method.

[0054] A method for identifying the above-mentioned bacteria comprises selecting three sets of primers, performing PCR amplification, and analyzing the amplified products by gel electrophoresis imaging. If a unique and specific DNA fingerprint is obtained, it is the above-mentioned bacteria.

[0055] Example 1 Isolation, purification, and identification of bacterial strain JH166

[0056] 1) Bacterial strain isolation and purification

[0057] Bacterial strain JH166 was isolated from the feces of a healthy adult and purified as a monoclonal strain using a chromogenic coliform medium (solid-state medium). The monoclonal bacterial strain was then expanded and cultured in liquid meat extract peptone medium (Luria-Bertani medium, LB medium).

[0058] 2) Detection of bacterial strains expressing CD176 antigen

[0059] 1) The bacteria were fixed with 4% formaldehyde, and the expression of CD176 antigen in the whole bacteria was detected by cell enzyme-linked immunosorbent assay (Cell-ELISA).

[0060] 2) Monoclonal bacterial strains were expanded and cultured in liquid LB medium. Bacterial components were lysed with bacterial lysis buffer, and CD176 antigen expression in the bacterial lysate was detected by solid-phase enzyme-linked immunosorbent assay (ELISA).

[0061] The biological reagents used in the cell-based ELISA and solid-phase ELISA assays included an anti-CD176 monoclonal antibody (clone A78-G / A7, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), catalase-conjugated goat anti-mouse IgM (EMD Millipore Corporation, Temecula, CA, USA), and catalase-conjugated peanut agglutinin (Sigma-Aldrich, Saint Louis, MO, USA). The results are shown in Table 1. These two assays confirmed that the selected strain JH166 expressed the CD176 antigen.

[0062] Table 1. Expression of CD176 antigen in JH166 bacteria and control bacteria

[0063] Bacterial strain CD176 antigen JH166 bacterial strain complete whole bacteria + + bacterial lysis lysis buffer + standard Escherichia coli bacterial strain complete whole bacteria - bacterial lysis lysis buffer -

[0064] Note: +++, CD176 antigen detected by enzyme-linked immunosorbent assay, optical density (OD) value >0.2; +, OD value 0.05-0.1; -, OD value <0.05.

[0065] 3) Colony characteristics of bacterial strain JH166

[0066] As shown in Figure 1A , the bacterial strain JH166 was streaked onto a chromogenic solid medium (Qingdao Haibo Biotechnology Co., Ltd.) for 12–18 h, and the isolated monoclonal colonies appeared as blue, round, slightly convex colonies with a diameter of approximately 1–3 mm.

[0067] As shown in Figure 1B , after culturing bacterial strain JH166 in solid meat extract peptone medium (LB medium) for 18 h, round, slightly convex, white colonies with a diameter of 1–3 mm appeared;

[0068] As shown in Figure 1C , after culturing bacterial strain JH166 in MacConkey medium (Guangdong Huankai Microbiology Technology Co., Ltd.) for 18 h, round, slightly convex, red colonies with a diameter of 1–3 mm appeared.

[0069] 4) Microscopic morphology of bacterial strain JH166

[0070] JH166 bacterial smear, Gram staining, and microscopic observation showed that it was a Gram-negative bacillus with a typical rod shape.

[0071] 5) Biochemical identification of bacterial strain JH166

[0072] The cultured JH166 bacteria were identified using the Escherichia coli IMVC biochemical identification kit (Guangdong Huankai Microbiology Technology Co., Ltd.), and the results are shown in Table 2.

[0073] Table 2. Biochemical identification results of bacterial strain JH166

[0074] Determination of reaction substrate results (+ for positive, - for negative) Indigo matrix-MR (methyl red) + VP-citrate-

[0075] After strain JH166 was cultured in a glucose-peptone water culture medium, the addition of methyl red indicator solution turned red, indicating that the bacteria decomposed glucose to produce pyruvate, which was then decomposed to produce formic acid, acetic acid, lactic acid, etc., lowering the pH of the culture medium to below 4.2. The MR test was positive, and the VP test showed that the culture medium did not change color and the result was negative, indicating that this bacterium decomposed glucose without a pyruvate decarboxylation step and that a series of subsequent chemical reactions would not occur.

[0076] Citrate Utilization Test Results: The bromothymol blue indicator in the culture medium does not change color, indicating a negative result. Indole Test: After incubation, indole reagent is added dropwise, and the culture medium does not change color, indicating a negative result.

[0077] Based on the above results, it was confirmed that strain JH166 was an atypical Escherichia coli.

[0078] 6) Identification of bacterial strain JH166 stably expressing CD176 antigen

[0079] The bacterial strain JH166, which highly expresses the CD176 antigen, was repeatedly subcultured. Gram staining was performed every 10 passages to examine the morphology of JH166, and bacterial expression of the CD176 antigen was assessed using a cell-based enzyme-linked immunosorbent assay (ELISA). The results showed that strain JH166 maintained stable, high-level expression of the CD176 antigen after 40 passages (see Figure 2). The cell-based ELISA is a semi-quantitative assay, and optical density (OD) values ​​vary between batches. To minimize the impact of experimental variation on the final results, each experiment was repeated three times for the semi-quantitative assay, and the average value was calculated. Despite the variation in OD values ​​between batches in the semi-quantitative analysis, the three replicates showed consistent positive (JH166) and negative (E. coli, negative control) results in the qualitative analysis. The control group consisted of a blank control without the addition of the primary antibody.

[0080] 7) Comparison of CD176 antigens in various components of bacterial strain JH166

[0081] The monoclonal bacterial strain JH166 was expanded in liquid LB medium to obtain intact whole bacteria. A portion of the intact bacteria was lysed with bacterial lysis buffer. The lysate (supernatant after centrifugation), the pellet (cement after centrifugation), and the culture medium (supernatant after centrifugation) were collected. The expression of the CD176 antigen by various components of the JH166 bacterial strain was assessed using cell-based enzyme-linked immunosorbent assays and solid-phase enzyme-linked immunosorbent assays. Intact whole bacteria, lysate, pellet, culture medium, and pre-culture medium from a strain not expressing CD176 (standard Escherichia coli) were used as negative controls. The biological reagents used in the cell-based ELISA and solid-phase ELISA assays included a monoclonal antibody against CD176 (clone A78-G / A7, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), catalase-conjugated goat anti-mouse IgM (EMD Millipore Corporation, Temecula, CA, USA), and catalase-conjugated peanut lectin (Sigma-Aldrich, Saint Louis, MO, USA). The results are shown in Table 3.

[0082] Table 3. Comparison of CD176 antigen in various components of JH166 bacteria

[0083] CD176 antigen of each bacterial component intact whole bacteria large amount+++ bacterial lysis solution small amount+ bacterial lysis precipitate medium amount++ bacterial culture medium very small amount (+)

[0084] Note: +++Enzyme-linked immunosorbent assay was used to detect CD176 antigen, with an optical density (OD) value >0.2; ++, OD value 0.1-0.2; +, OD value 0.05-0.1; (+), OD value 0.04-0.05.

[0085] The results showed that CD176 antigen was present in intact whole bacteria, lysate after bacterial lysis, sediment after bacterial lysis, and culture medium after bacterial culture. Intact whole bacteria include all bacterial components, specifically bacterial cytoplasm, cell wall, cell membrane, pili, flagella, and lipopolysaccharide (LPS). Lysate after bacterial lysis includes bacterial proteins and peptides, polysaccharides, glycoproteins, and bacterial metabolites. The sediment after bacterial lysis includes most bacterial components, specifically cell wall, cell membrane, pili, flagella, and LPS. The culture medium after bacterial culture includes bacterial extracellular vesicles, bacterial-associated exosomes, and bacterial metabolites. In other words, all of these components may contain CD176 antigen.

[0086] Example 2 Detection of Human Blood Group Antigens A and B in Bacterial Strain JH166

[0087] Monoclonal bacterial strain JH166 was expanded in liquid LB medium to obtain intact whole bacteria. A portion of the intact bacteria was lysed with bacterial lysis buffer. The lysate (supernatant after centrifugation), the pellet (cement after centrifugation), and the culture medium (supernatant after centrifugation) were collected. Cell-based enzyme-linked immunosorbent assay (ELISA) and solid-phase ELISA were used to assay the expression of human blood group antigens A and B in various components of strain JH166. Intact whole bacteria, lysate, and pellet from a bacterial strain expressing human blood group antigens A and B served as positive controls. Intact whole bacteria, lysate, pellet from a bacterial strain not expressing human blood group antigens A and B served as negative controls. The biological reagents used in the cell-based ELISA and solid-phase ELISA assays included monoclonal antibodies against human blood group antigens A and B (both antibodies were mouse monoclonal antibodies, IgM; Changchun Broad Biotechnology Co., Ltd.) and catalase-labeled goat anti-mouse IgM (EMD Millipore Corporation, Temecula, CA, USA). Experiments showed that whole JH166 bacterial strains did not express human blood group antigens A and B. Furthermore, ELISA analysis of the lysate and precipitate of JH166 bacteria in bacterial lysis buffer revealed that these lysates and precipitates also did not express human blood group antigens A and B.

[0088] Example 3 Molecular Biological Identification of Bacterial Strain JH166

[0089]

[0090] The bacterial 16S rRNA gene sequence was sequenced using BLAST comparisons at NCBI (a DNA sequence database maintained by the National Center for Biotechnology Information) (http: / / www.ncbi.nlm.nih.gov / BLAST / ). The results showed that strain JH166 was systematically classified as: Bacteria; Proteobacteria; Gammaproteobacteria; Enterobacteriaceae; Escherichia; Escherichia coli (see Figure 3). Its 16S rRNA gene sequence shared at least 99.86% identity with the 16S rRNA gene sequence of Escherichia coli.

[0091] Example 4 Pathogenic Escherichia coli Gene Detection

[0092] To determine whether E. coli strain JH166 is pathogenic, the five most important pathogenic E. coli genes were tested. Strain JH166 was inoculated onto solid LB medium and cultured at 37°C for 10-30 hours. A single colony was then inoculated into liquid LB medium and cultured at 37°C for 20 hours. Deoxyribonucleic acid (DNA) was extracted from the cultured bacteria. The DNA sample was submitted to a nationally recognized third-party testing agency, which conducted pathogenic E. coli gene testing in accordance with the National Food Safety Standard: Food Microbiology Examination, Diarrhea-Causing Escherichia coli Examination (GB4789.6-2016).

[0093] Using the molecular biological method of polymerase chain reaction (PCR), five pathogenic Escherichia coli genes were detected: escV (protein secretion regulator gene, gene encoding LEE-encoded type Ⅲ secretion system factor), stx2 (Shiga toxin II gene, Shiga toxin two gene), lt (heat-labile enterotoxin gene, heat-labile enterotoxin), invE (invasive plasmid regulator gene, invasive plasmid regulator), and aggR (aggregative adhesive fimbriae regulator gene, aggregative adhesive fimbriae regulator). The PCR primer sequences used were: escV, 5'-ATTCTGGCTCTCTTCTTCTTTATGGCTG-3' (SEQ ID NO:4), 5'-CGTCCCCTTTTACAAACTTCATCGC-3' (SEQ ID NO:5); stx2, 5'-GTTTTGACCATCTTCGTCTGATTATTGAG-3' (SEQ ID NO:6), 5'-AGCGTAAGGCTTCTGCTGTGAC-3' (SEQ ID NO:7); lt, 5'-GAACAGGAGGTTTCTGCGTTAGGTG-3' (SEQ ID NO:8), 5'-CTT TCAATGGCTTTTTTTTGGGAGTC-3' (SEQ ID NO:9); invE, 5'-CGATAGATGGCGAGAAATTATATCCCG-3' (SEQ ID NO:10), 5'-CGATCAAGAATCCCTAACAGA AGA ATCAC-3' (SEQ ID NO:11); aggR, 5'-ACGCAGAGTTGCCTGATAAAG-3' (SEQ ID NO:12), 5'-AATACAGAATCG TCAGCATCAGC-3' (SEQ ID NO:13).

[0094] The test results showed that all five genes were negative, indicating that the JH166 bacterial strain did not contain pathogenic E. coli genes, as shown in Figure 4. The results indicate that the bacterial strain JH166 is not pathogenic E. coli.

[0095] Example 5 Whole genome sequencing analysis of bacterial strain JH166

[0096] Strain JH166 was inoculated onto solid LB medium and cultured at 37°C for 20 hours. A single colony was then inoculated into liquid LB medium and cultured at 37°C for 20 hours. Genomic DNA was extracted using a bacterial DNA extraction kit. Genomic DNA was analyzed by gel electrophoresis. DNA that met the required quality and quantity was sent to a third party for whole-genome sequencing and bioinformatics analysis. The whole-genome sequencing results of strain JH166 were systematically analyzed and compared with data from internationally recognized authoritative DNA libraries.

[0097] 1) Whole-genome sequencing analysis of bacterial strain JH166 confirmed the strain's lineage. Analysis of the average nucleic acid similarity of the whole-genome sequencing of bacterial strain JH166 revealed that strain JH166 was systematically classified as Escherichia coli (see Table 4).

[0098] Table 4. Statistics of average nucleic acid similarity analysis results of whole genome sequencing of bacterial strain JH166

[0099] NameTop-hit strainSimilarity (%)Completeness (%)Query Length (bp)Top-hit taxonJH166SB11199.86991441Escherichia coli

[0100] Note: Name: genome number; Top-hit strain: strain with the highest similarity; Similarity: similarity; Completeness: gene completeness; Query Length: search matching length; Top-hit taxon: most likely classification.

[0101] 2) Whole genome sequencing analysis of bacterial strain JH166 and bacterial completion map

[0102] Escherichia coli is easy to culture, grows quickly, is safe, and amenable to various genetic manipulations, making it an ideal engineering bacterium. The entire genome of E. coli strain JH166 has been sequenced, and the results have been systematically analyzed to produce a complete genome map (see Figure 5). This strain has a clear genetic background and a complete genome, making it suitable for use as an engineering bacterium.

[0103] 3) Pathogenicity gene analysis based on whole genome sequencing results of bacterial strain JH166.

[0104] Potential pathogenic genes were analyzed using multiple databases, including the Pathogen Host Interactions Database (PHI), the Virulence Factors of Pathogenic Bacteria (VFDB), the Gene Ontology (GO), and the Kyoto Encyclopedia of Genes and Genomes (KEGG). Whole-genotype PHI annotation (Figure 6) shows that a minority of key pathogenic genes belong to the fourth category, Increased pathogenicity (Hypervirulence). The majority of genes belong to the Reduced virulence and Unaffected pathogenicity categories, meaning genes with reduced or no effect on pathogenicity. VFDB (Virulence Factors of Pathogenic Bacteria) results revealed a total of 162 virulence factors. No pathogenic E. coli genes or specific pathogenicity genes were identified across all these databases. In Example 4, the five pathogenic Escherichia coli genes (escV, stx2, lt, invE, aggR) detected by molecular biological methods did not appear in the bacterial whole genome sequencing results.

[0105] 4) Whole genome sequencing of bacterial strain JH166 was used to analyze drug resistance-related genes.

[0106] Resistance gene detection was performed using the Antibiotic Resistance Genes Data Base (ARDB) and the Comprehensive Antibiotic Research Database (CARD). Annotations from these databases revealed resistance-associated genes and provided information on the mechanisms and types of antimicrobial resistance. JH166 has three main resistance-associated gene groups: 1) altered antimicrobial target sites, such as baca and arna; 2) antimicrobial inactivation, such as bl1_ec; and 3) antimicrobial efflux pumps, such as emrd, acrb, mdtk, mdth, mdtg, mdfa, and mdtm. These resistance genes in JH166 are primarily members of the major facilitator superfamily (MFS). Some important and common drug-resistant genes, such as β-lactam resistance gene blaTEM, multidrug resistance gene NDM-1, tetracycline resistance gene tetA, polymyxin resistance gene MCR-1, carbapenem resistance gene blaNDM, etc., did not appear in JH166 bacterial beads.

[0107] Example 6 Establishing DNA Fingerprinting of Bacterial Strain JH166

[0108] Bacterial DNA fingerprinting uses specific primers to randomly amplify polymorphic DNA from repetitive sequences in non-coding regions of the bacterial genome (these sequences are highly conserved throughout evolution). This yields distinct DNA fragments. Gel electrophoresis of the amplified products yields multiple DNA bands, which form a characteristic polymorphic pattern (similar to a barcode). Specific bacterial strains exhibit a unique gel electrophoresis imaging pattern, known as a DNA fingerprint. Analysis of the DNA fingerprint of a specific bacterial strain can establish a method for identifying and authenticating that specific bacterial strain. In this example, a DNA fingerprint of bacterial strain JH166 was established using three PCR detection methods.

[0109] (1) ERIC (Enterobacterial Repetitive Intergenic Consensus)-PCR fingerprinting analysis technology. Primers were designed based on the 126 bp repetitive intergenic consensus sequence in the non-coding region of Enterobacteriaceae. The designed amplification primers are: ERIC1 sequence: 5'-ATG TAA GCT CCT GGG GAT TCA C-3' (SEQ ID NO:14); ERIC2 sequence: 5'-AAG TAA GTG ACT GGG GTG AGC G-3' (SEQ ID NO:15). The ERIC-PCR reaction conditions are: pre-denaturation at 95°C for 7 minutes; denaturation at 94°C for 1 minute, annealing at 52°C for 1 minute, extension at 65°C for 8 minutes, 35 cycles; and final end extension at 65°C for 16 minutes.

[0110] (2) REP (Repetitive sequence)-PCR fingerprint analysis technology. This method designs primers based on the 38 bp repetitive extragenic palindromic structural element of bacteria. The designed amplification primers are: Rep1R-I sequence: 5'-III ICG ICG ICA TCI GGC-3' (I is inosine) (SEQ ID NO:16); Rep2 RI sequence: 5'-ICG ICTT ATC IGG CCT AC-3' (I is inosine) (SEQ ID NO:17). The REP-PCR reaction conditions are: pre-denaturation at 94°C for 1 minute; denaturation at 95°C for 7 minutes, annealing at 40°C for 1 minute, extension at 65°C for 8 minutes, 35 cycles; and final end extension at 65°C for 16 minutes.

[0111] (3) BOX-PCR fingerprint analysis technology. Primers were designed based on the BOX insertion factor (154 bp in size, consisting of subunits such as box A (57 bp), box B (43 bp), and box C (50 bp) with different conserved sequences). BOX-PCR can complete DNA polymorphism analysis of a large number of strains with only a single primer. The designed primer is: BOXA1R sequence: 5'-CTA CGG CAA GGC GAC GCT GAC G-3' (SEQ ID NO:18). The BOX-PCR reaction conditions are: pre-denaturation at 95°C for 7 minutes; denaturation at 94°C for 1 minute, annealing at 53°C for 1 minute, extension at 65°C for 8 minutes, 35 cycles; and final end extension at 65°C for 16 minutes.

[0112] Strain JH166 was inoculated onto solid LB medium and cultured at 37°C for 18 hours. A single colony was inoculated into liquid culture and cultured at 37°C for 18 hours. Deoxyribonucleic acid (DNA) was extracted from the cultured bacteria and amplified by PCR using the three primer sets described above. The amplified products were analyzed by gel electrophoresis imaging to obtain unique and specific DNA fragments, i.e., fragments specific to strain JH166, thus obtaining a DNA fingerprint unique to strain JH166.

[0113] The results are shown in Figure 7. ERIC-PCR results showed a JH166-specific band pattern between 250 and 2500 bp; REP-PCR results showed a JH166-specific band pattern between 250 and 1000 bp; and BOX-PCR results showed a JH166-specific band pattern below 1000 bp. DNA markers (M1, 250-10,000 bp; M2, 50-1000 bp) were used.

[0114] The strain JH166 was deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms with the deposit number CGMCC No. 27745 and the deposit date of June 29, 2023. The address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0115] Example 7 Heat inactivation experiment of bacteria JH166

[0116] After an overnight culture at 37°C in a shaker, JH166 bacteria were heat-treated at 100°C for 10 minutes or 56°C for 30 minutes. After heat treatment, JH166 bacteria were plated onto solid meat extract peptone medium and incubated at 37°C for 48-72 hours. No bacterial colonies grew, as shown in Figure 8. This indicates that heat treatment at 100°C for 10 minutes or 56°C for 30 minutes completely inactivated JH166 bacteria, preventing their growth.

[0117] Example 8 Immunization Experiment of Mice with Injection and Oral Administration of Bacteria JH166

[0118] 1. Immunization Experiment of Mice with Heat-killed JH166

[0119] 1) Preparation of heat-killed JH166 bacteria

[0120] Bacterial strain JH166 was cultured overnight at 37°C in a shaking incubator, boiled in water for 30 minutes, and then centrifuged (5000 rpm for 15 minutes) to obtain heat-inactivated strains. Bacterial quantification was performed by turbidimetry.

[0121] 2) Immunization of mice with heat-killed JH166

[0122] Prepare 5 female Kunming mice, weighing 20-25 grams. Intraperitoneally inject heat-killed bacteria JH166 once a week for three consecutive weeks, with a bacterial dose of 5×108 cfu (colony forming unit) each time. One week after the second immunization, blood was collected from the tail to prepare serum. Thereafter, blood was collected from the tail every 10 to 14 days to prepare serum. Two weeks after the third immunization, the fourth injection was performed, and then blood was collected from the tail every 10-14 days, and serum was prepared 4 times to observe the changes in antibody concentration after injection immunization. Two months after the fourth immunization, the fifth injection was performed, and then the mice were anesthetized after one week of observation, and whole blood was collected from the heart to prepare serum. The collected serum was subjected to antibody analysis, and the level of CD176 antibodies in mouse serum was detected by solid-phase enzyme-linked immunosorbent assay (ELISA). The biological reagents used included Asialoglycophorin, TFa-PAA, sialidase-treated human type O red blood cell protein lysate, catalase-labeled goat anti-mouse IgM (m-chain specific), and catalase-labeled goat anti-mouse IgG (g-chain specific). It can be seen that after two immunizations, mice began to express high levels of CD176. Antibody levels decreased after cessation of immunization, but further injection of bacterial JH166 rapidly induced CD176 antibody production. The results are shown in Table 5.

[0123] Table 5-1. CD176 antibody expression in mice immunized with heat-killed JH166

[0124]

[0125] Note: Serum samples were collected before the first injection (day 1), 7 days after the second injection (total day 13), and 8 days after the third injection (total day 21). Relative CD176 antibody levels were calculated on days 10 (total day 37), 28 (total day 56), and 42 (total day 70) after the fourth injection, and the percentage change relative to the pre-immunization CD176 antibody level was determined at each time point. Increases between 10% and 30% are indicated by (+), between 30% and 50% by (++), and greater than 50% by (+++). Percent change values ​​less than 10% are considered negative (-).

[0126] Table 5-2. CD176 antibody expression in mice after secondary immunization with heat-killed JH166

[0127]

[0128] Note: Serum samples were collected on days 7 (98th day overall) and 13 (103rd day overall) after the fifth injection to calculate relative CD176 antibody levels. The percentage change relative to the pre-immunization CD176 antibody level at each time point was determined. Increases between 10% and 30% were indicated by (+), between 30% and 50% by (++), and greater than 50% by (+++). Percent change values ​​below 10% were considered negative (-).

[0129] 2. Oral immunization experiment of mice with live JH166 bacteria (gavage)

[0130] 1) Preparation of oral live bacteria JH166

[0131] JH166 bacteria were cultured overnight at 37°C in a shaker and then centrifuged (5000 rpm, 15 minutes) to obtain viable strains. Bacterial quantification was performed by turbidimetry.

[0132] 2) Oral immunization of mice with live bacteria JH166

[0133] Prepare five female Kunming mice weighing approximately 25 grams. Prepare an oral gavage solution containing 0.2 ml of 5 × 108 cfu of live bacterial suspension per mouse. House the mice in separate cages. Blood was collected from the tail one week before the oral gavage experiment.

[0134] The day before gavage, the mice were housed in separate cages and fasted for 24 hours. They were deprived of water 1 hour before gavage. They were gavaged with 0.3 ml of 3% NaHCO3 and observed for 30 minutes before proceeding to the next step. Each mouse was gavaged once a week for 3 consecutive weeks. The status of the mice was observed and recorded every day after each gavage. After three weeks (22 days), the mice were killed and whole blood was taken from the heart to prepare serum. The CD176 antibody level of the mice was detected by solid-phase enzyme-linked immunosorbent assay (ELISA). The method and reagents used were the same as those in Example 8 (1). The results are shown in Table 6. As can be seen from Table 6, oral administration of live bacteria JH166 can induce the production of CD176 antibodies in mice and significantly increase the expression level of CD176 antibodies in the body.

[0135] Table 6. CD176 antibody expression in mice after oral immunization with live JH166

[0136]

[0137] Note: CD176 antibody levels were detected by enzyme-linked immunosorbent assay. “++” indicates strongly positive, “+” indicates weakly positive, and “-” indicates negative.

[0138] 3. Oral immunization of mice with heat-killed JH166

[0139] 1) Preparation of heat-killed JH166 bacteria

[0140] Bacterial strain JH166 was cultured overnight at 37°C in a shaking incubator, boiled in water for 30 minutes, and then centrifuged (5000 rpm for 15 minutes) to obtain heat-inactivated strains. Bacterial quantification was performed by turbidimetry.

[0141] 2) Oral immunization of mice with heat-killed bacteria JH166

[0142] 5 female Kunming mice, 20-25 g in size, were fed 4 g of mouse feed per adult mouse per day. 1×10 10 CFU-killed bacteria were used as special feed for 14 consecutive weeks (98 days). The mental state, diet, hair, and urine and feces of the orally immunized mice were observed every day. It was found that the state of the mice orally fed with heat-killed bacteria JH166 was not affected and everything was normal. The weight of the mice was weighed every 2-3 days and the weight of the mice was normal. Blood was collected from the tail every 2 weeks after feeding to prepare serum. The CD176 antibody level of the mice was detected by solid-phase enzyme-linked immunosorbent assay (ELISA) experiment. The method and reagents used were the same as those in Example 8 (1).

[0143] Table 7. CD176 antibody expression in mice after oral immunization with heat-killed bacteria JH166

[0144]

[0145] Note: Serum samples were collected from mice on days 14, 28, 42, 57, 70, 83, and 98 after they consumed a diet supplemented with heat-killed JH166 bacteria. Relative CD176 antibody levels were calculated, and the percentage change relative to the pre-immunization CD176 antibody level was determined for each time point. Increases between 10% and 30% were indicated by (+), between 30% and 50% by (++), and greater than 50% by (+++). Percent change values ​​less than 10% were considered negative (-).

[0146] Example 9 Preliminary safety evaluation experiment of JH166 bacteria injected and orally administered to mice

[0147] 1. Safety evaluation of heat-killed JH166 injected into mice

[0148] 1) Preparation of heat-killed JH166 bacteria

[0149] Bacterial strain JH166 was cultured overnight at 37°C in a shaking incubator, boiled in water for 30 minutes, and then centrifuged (5000 rpm for 15 minutes) to obtain heat-inactivated strains. Bacterial quantification was performed by turbidimetry.

[0150] 2) Safety evaluation of heat-killed JH166 injection in mice

[0151] Five female Kunming mice, 20-25 g in size, were intraperitoneally injected with heat-killed JH166 bacteria once a week for three consecutive weeks, with a dose of 5 × 10 8 CFU. The injected mice were observed daily for their mental state, diet, hair, and urination and defecation. Aside from mild inflammation at the injection site in some areas of the abdomen, the injected mice were otherwise normal. Mice were weighed every 2-3 days. A slight decrease in weight occurred after each injection, but the weights of the remaining mice remained normal (see Figure 9). Sixteen days after the final injection of inactivated JH166 bacteria, mice were dissected. Preliminary examination of various organs revealed no pathological changes. Pathological examination of sections of key mouse organs using hematoxylin-eosin staining revealed normal results. Results for selected organs are shown in Figure 13. Bacterial strain JH166 is a strain of Escherichia coli found in the human intestine. Whole-genome analysis confirmed that it is non-pathogenic. Intraperitoneal injection of heat-killed JH166 bacteria (killed bacteria) had no infectious or pathogenic effects on the experimental mice.

[0152] 2. Safety Evaluation of Oral (Gastric) Administration of Live JH166 to Mice

[0153] 1) Preparation of oral live bacteria JH166

[0154] JH166 bacteria were cultured overnight at 37°C in a shaker and then centrifuged (5000 rpm, 15 minutes) to obtain viable strains. Bacterial quantification was performed by turbidimetry.

[0155] 2) Safety evaluation of oral administration (or gavage) of live JH166 bacteria in mice

[0156] 5 female Kunming mice, size 20-25 g, oral administration (gavage) 10 8 CFU live JH166 bacteria were administered to the orally immunized mice daily for their mental state, diet, hair, and urination and defecation. Oral administration (or gavage) of live JH166 bacteria revealed no changes in the mice's condition and normal function (see Figure 10). The mice were weighed every 2-3 days and maintained normal weight. During the experiment, the mice experienced a brief weight loss due to fasting prior to gavage. However, after the start of gavage with live JH166 bacteria and the resumption of normal diet, their weight returned to normal and continued to increase. Three weeks after gavage, the mice were dissected and preliminary examination of their organs revealed no pathological changes, except for a slightly enlarged spleen. Pathological examination of HE-stained sections of key mouse organs revealed normal function. Results for selected organs are shown in Figure 13. Enterobacteriaceae are a group of Gram-negative bacteria with similar biological characteristics that primarily inhabit the intestines of humans and animals. The genus Escherichia is one such group, encompassing a variety of bacteria, with Escherichia coli being the most common in clinical practice. Escherichia coli (E. coli), commonly known as Escherichia coli, is a normal parasite in the large intestine of all mammals. It synthesizes vitamins B and K for absorption and utilization by the body. It also inhibits the excessive proliferation of putrefactive bacteria, pathogens, and fungi. However, when these bacteria leave their intestinal home and enter other parts of the body, they can cause infection. Some strains are pathogenic, causing intestinal or urinary tract infections. Molecular biological identification of bacterial strain JH166 indicates that it is safe and non-pathogenic to humans and animals. Oral administration of live JH166 bacteria is harmless to experimental mice, demonstrating its complete safety.

[0157] 3. Safety Evaluation of Oral Administration of Heat-Killed JH166 to Mice

[0158] 1) Preparation of heat-killed JH166 bacteria

[0159] Bacterial strain JH166 was cultured overnight at 37°C in a shaking incubator, boiled in water for 30 minutes, and then centrifuged (5000 rpm for 15 minutes) to obtain heat-inactivated strains. Bacterial quantification was performed by turbidimetry.

[0160] 2) Safety evaluation of oral administration of heat-killed bacteria JH166 to mice

[0161] 5 female Kunming mice, 20-25 g in size, were fed 4 g of mouse feed per adult mouse per day. 1×1010 CFU-killed bacteria were used as a special feed for 14 consecutive weeks (98 days). Daily observations were made regarding the mental state, diet, hair growth, and urination and defecation of the orally immunized mice. The mice treated with heat-killed JH166 remained healthy and normal. Mice were weighed every 2-3 days and showed normal weights (see Figure 11). Preliminary examination of the dissected organs of the mice revealed no pathological changes 98 days after the start of oral administration of the heat-killed JH166 bacteria. Pathological examination of the mouse organs using HE-stained sections of key organs also revealed normal results. Results for selected organs are shown in Figure 13. Oral administration of heat-killed JH166 bacteria (killed bacteria) was harmless to the experimental mice and is completely safe.

[0162] 4. No bacteria treatment control mice

[0163] Five female Kunming mice weighing approximately 25 grams were selected and served as blank controls without any treatment (no injection or oral administration of bacteria). Similar to the bacterial-treated mice, the orally immunized mice were observed daily for their mental state, diet, hair growth, and urination and defecation. Their growth was normal. The mice were weighed every 2-3 days and remained normal (see Figure 12). After the experiment, the mice were dissected and preliminary examination of their organs revealed no pathological changes. Pathological examination of key organs using HE staining also revealed normal results. Results for selected organs are shown in Figure 13.

[0164] Example 10

[0165] Certain bacteria have human blood group antigens on their surfaces. These bacteria can induce the production of anti-blood group antigen antibodies in the human body, which can bind to blood group antigens on the surface of human red blood cells. To test whether serum from mice immunized with JH166 bacteria can bind to blood group antigens on the surface of human red blood cells, experiments were conducted using serum from mice immunized with 5×108 cfu of JH166 bacteria intraperitoneally, along with an ABO blood typing kit (for human red blood cells) and an irregular antibody detection kit (for human red blood cells).

[0166] The ABO blood group reverse typing kit (human erythrocytes, National Medical Device Standard 20153400422) and the irregular antibody detection kit (human erythrocytes, National Medical Device Standard 20153400424) were purchased from Changchun Bode Biotechnology Co., Ltd. The ABO blood group reverse typing kit (human erythrocytes) includes A1, B, and O erythrocyte types; the irregular antibody detection kit (human erythrocytes) includes 16 erythrocyte antigens: D, C, E, c, e, M, N, s, P1, Lea, Leb, k, Fya, Fyb, Jka, and Jkb. The experimental procedures followed the test methods provided by the reagent company. Results showed that human erythrocytes (both the ABO blood group reverse typing kit and the irregular antibody detection kit) did not exhibit aggregation when treated with serum from mice immunized with JH166 bacteria.

[0167] Combined with the experimental results of Example 2, it can be concluded that the newly isolated JH166 bacterial beads do not express human blood group antigens A and B; serum after immunization with JH166 bacteria cannot bind to blood group antigens on the surface of human red blood cells, such as the classical red blood cell antigens (A, B, H) and other red blood cell antigens (D, C, E, c, e, M, N, s, P1, Lea, Leb, k, Fya, Fyb, Jka, Jkb).

[0168] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. An Escherichia coli JH166, characterized in that It can express CD176 antigen and induce the production of CD176 antibody through immunization, and its deposit number is CGMCC No. 27745.

2. The Escherichia coli JH166 according to claim 1, characterized in that Its 16S rRNA gene sequence is at least 99.86% identical to the 16S rRNA gene sequence of Escherichia coli.

3. The Escherichia coli according to claim 1, characterized in that It does not contain the pathogenic E. coli genes escV, stx2, lt, invE and aggR.

4. The Escherichia coli according to claim 1, characterized in that It does not contain the drug-resistant genes blaTEM, NDM-1, tetA, MCR-1 and blaNDM.

5. The Escherichia coli according to claim 1, characterized in that It does not express the human blood group antigens A and B.

6. Use of the Escherichia coli according to any one of claims 1 to 5 in the preparation of food, health products, food additives, bacterial components, experimental materials, engineered bacteria, anti-tumor vaccines, anti-tumor drugs, CD176 antibodies or CD176 antigens.

7. The use according to claim 6, characterized in that The bacterial component is one or more of the whole bacteria of the Escherichia coli, bacterial lysate, precipitate after lysis, and culture medium after culture.

8. The use according to claim 6, characterized in that The engineered bacteria are obtained by transforming the Escherichia coli through genetic engineering methods.

9. The use according to claim 6, characterized in that The anti-tumor vaccine comprises live or inactivated Escherichia coli bacteria.

10. The use according to claim 6, characterized in that The anti-tumor drug is a tablet, capsule, granule or injection prepared by adding an effective dose of the Escherichia coli and / or the bacterial components of the Escherichia coli according to any one of claims 1 to 5 to pharmaceutically acceptable carriers and excipients.

11. The use according to claim 6, characterized in that The CD176 antibody is induced by immunizing humans and animals with the Escherichia coli and / or bacterial components of the Escherichia coli.

12. The use according to claim 6, characterized in that The CD176 antigen is obtained by culturing the Escherichia coli and then extracting it from the Escherichia coli, the lysate of the Escherichia coli, the precipitate after lysis or the culture medium after cultivation.

13. The use according to claim 12, characterized in that The number of generations of the Escherichia coli cultured is 1 to 50.

14. Use of the Escherichia coli according to any one of claims 1 to 5 in tumor treatment.

15. The use according to claim 14, characterized in that The experimental subjects of the application are humans or mammals.

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