A non-pathogenic enterotoxigenic Escherichia coli strain expressing only F4 fimbriae adhesin and its application

Abstract

The present invention discloses a non-pathogenic Escherichia coli isolate expressing only F4 fimbriae adhesin and its application. The growth rate of the strain of the present invention is similar to that of the standard reference strain C83902 of F4ac+ enterotoxigenic Escherichia coli, and it exhibits good adhesion to the small intestinal epithelial cells of piglets in vitro experiments. Immunizing mice with the fimbriae adhesin extract extracted from the isolate or immunizing piglets with the inactivated vaccine prepared based on the isolate can induce the production of high-titer anti-FaeG antibodies, and the obtained immune piglet serum significantly inhibits the adhesion of the F4ac+ETEC domestic reference strain C83902 strain to IPEC‑J2 cells. The non-pathogenic Escherichia coli isolate is expected to become a candidate live vaccine strain for preventing diarrhea in piglets caused by infection with enterotoxigenic Escherichia coli expressing F4 fimbriae adhesin.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a non-pathogenic enterotoxigenic Escherichia coli isolate expressing only F4 pili and an application thereof. Background Art

[0002] Piglet diarrhea not only directly affects the survival rate of piglets, but also delays growth and development, reduces feed conversion rate, and increases breeding costs. Due to its high infection rate and high mortality rate, piglet diarrhea has become one of the key diseases that seriously affect the survival rate of piglets and the economic benefits of pig farming, causing continuous and significant economic losses to the global pig industry. The causes of piglet diarrhea are complex, among which pathogenic factors such as viruses and bacteria are the main causes. These pathogens infect piglets through various pathways and cause intestinal dysfunction. Common viral pathogens include porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV) and rotavirus (RV), which can damage the intestinal mucosa of piglets, causing severe watery diarrhea and dehydration. Bacterial pathogens mainly include enterotoxigenic Escherichia coli (ETEC), Salmonella spp., Clostridium perfringens, etc. These bacteria cause inflammatory reactions and diarrhea by secreting toxins and invading intestinal epithelial cells. Among them, enterotoxigenic Escherichia coli (ETEC) is one of the most important pathogens causing diarrhea in newborn piglets and weaned piglets.

[0003] Fimbrial adhesins and enterotoxins are two major virulence factors produced during the pathogenic process of ETEC. Fimbrial adhesins produced by porcine ETEC include F4 (K88), F5 (K99), F6 (987P), F18 and F41, among which F4 and F18 are the most common. Fimbrial adhesins can bind to specific receptors on the epithelial cells of the small intestine of piglets, prompting ETEC strains to adhere to the intestinal mucosa, which is one of the key links in ETEC infection. This adhesion not only helps ETEC colonize in the epithelial cells of the small intestine of piglets, but also provides conditions for the continued existence and reproduction of bacteria in the intestine. After colonization, ETEC can secrete a variety of enterotoxins, including heat-stable enterotoxin LT, heat-stable enterotoxin STa, heat-stable enterotoxin STb and enterotoxin EAST1. These toxins interfere with the water and electrolyte absorption and secretion functions of intestinal cells, leading to diarrhea and dehydration. Therefore, the expression of fimbrial adhesins and the secretion of enterotoxins are two indispensable conditions for ETEC infection. Understanding and intervening in these two key links is crucial for the development of effective prevention and treatment measures.

[0004] At present, the main measures to prevent diarrhea in piglets caused by ETEC infection include adding antibiotics to feed, feeding fimbriae adhesins or enterotoxin-specific antibodies, dietary adjustment, breeding ETEC-resistant pigs through genetic breeding methods, and adding probiotics or prebiotics to feed. Adding antibiotics to feed was once the main means of preventing ETEC infection in the pig industry. However, the long-term abuse of antibiotics has led to an increasing number of ETEC-resistant strains, and has also caused problems such as drug residues, environmental pollution and food safety, which seriously threaten public health safety. Since 2020, the infection rate of ETEC in pigs has increased. Although oral fimbriae adhesins or enterotoxin-specific antibodies show good effects in the short term, antibodies are easily degraded in the body, have a short protection period and high cost, so this may not be a sustainable solution. Breeding pigs resistant to ETEC infection through genetic breeding methods is a long-term and effective strategy, but its cycle is too long and there are many uncertainties. In addition, genetic breeding cannot cope with sudden outbreaks and the emergence of new ETEC strains. Dietary adjustment and the addition of probiotics or prebiotics to feed have shown some potential in preventing ETEC infection in piglets and improving the intestinal health of pigs, but their exact effects need further verification. Therefore, vaccination is currently considered to be the most economical and feasible measure to prevent ETEC infection. However, the existing inactivated vaccines cannot effectively induce intestinal mucosal immune response, resulting in poor immune effect, and large-dose vaccination may cause greater side effects. Live bacterial vaccines that can stimulate intestinal mucosal immune response are considered to be ideal vaccine options for preventing ETEC infection.

[0005] The attenuated or weakened strains of ETEC currently constructed using molecular biological technology have weak colonization ability in the body, short immune protection period, and safety issues. Therefore, the ideal candidate strain for ETEC live vaccine should be a naturally isolated strain that only expresses fimbriae adhesins without producing enterotoxins. This naturally isolated non-toxic ETEC strain can efficiently colonize in the small intestinal epithelial cells of piglets, induce the production of specific fimbriae adhesin antibodies, and effectively block the adhesion of bacteria to intestinal epithelial cells. In addition, this natural non-toxic live bacteria can effectively stimulate the secretion of sIgA antibodies by the small intestinal mucosa through mucosal immunization, thereby providing better immune protection. Summary of the invention

[0006] Purpose of the invention: The technical problem to be solved by the present invention is to provide a non-pathogenic enterotoxigenic Escherichia coli (ETEC) that only expresses F4 fimbriae adhesin and its application. The strain is isolated from fecal samples of healthy lactating piglets, only expresses F4 fimbriae adhesin, does not express any known enterotoxin, and has good adhesion to piglet small intestinal epithelial cells (IPEC-J2). By extracting the fimbriae adhesin of the bacteria to immunize mice or preparing the bacteria into inactivated vaccines to immunize piglets, the production of specific antibodies against FaeG can be induced, and the serum of immune piglets can effectively prevent the adhesion of ETEC strains expressing F4 fimbriae adhesin to IPEC-J2 cells in vitro, thereby showing the potential as a live vaccine to prevent F4+ETEC infection.

[0007] Technical solution: In order to solve the above technical problems, the present invention provides a strain of non-pathogenic Escherichia coli, which is classified and named Escherichia coli PEC2021-8 (Escherichia coli PEC2021-8), and the non-pathogenic Escherichia coli was deposited in the China Center for Type Culture Collection on January 26, 2024, with a preservation number of CCTCC NO: M2024224, and the preservation address is Wuhan, China.

[0008] The non-pathogenic E. coli of the present invention is a non-pathogenic E. coli strain that only expresses F4 fimbriae adhesin.

[0009] Specifically, the non-pathogenic Escherichia coli described in the present invention is the pig-derived PEC2021-8 isolate, which was isolated from a fecal sample of a healthy suckling piglet on October 5, 2021, and the collector was Duan Qiangde. Sample collection, strain isolation and identification were carried out in accordance with the "National Standard Method for Diarrhea-Causing Escherichia Coli Inspection" (GB4789.6-2016). The isolate was preserved in a 1:1 LB liquid culture medium and 50% glycerol mixture and stored at -70°C.

[0010] The porcine PEC2021-8 isolate described in the present invention has the same culture characteristics and growth characteristics as the F4ac+ETEC standard reference strain.

[0011] Among them, further, the PEC2021-8 isolate only expresses F4 fimbriae adhesin, does not contain genes encoding F5, F6, F18, F41 fimbriae adhesins and LT, STa, STb and EAST1 enterotoxins, and its culture characteristics and growth rate are consistent with the F4ac+ETEC standard reference strain C83902. The PEC2021-8 isolate can agglutinate with anti-FaeG polyclonal antibodies and has good adhesion to piglet small intestinal epithelial cells IPEC-J2.

[0012] The present invention also includes the use of the non-pathogenic Escherichia coli in the preparation of vaccines or medicines for preventing or treating piglet diarrhea.

[0013] The present invention also includes the use of the non-pathogenic Escherichia coli in the preparation of a live vaccine for preventing F4+ETEC infection.

[0014] The present invention also includes a preparation containing the Escherichia coli, which includes a bacterial suspension, fermentation broth or supernatant of Escherichia coli PEC2021-8 (Escherichia coli PEC2021-8).

[0015] The present invention also includes a fimbriae adhesin extract, which is expressed by the non-pathogenic Escherichia coli classified and named Escherichia coli PEC2021-8 (Escherichia coli PEC2021-8).

[0016] The present invention also includes the use of the fimbriae adhesin extract in the preparation of anti-FaeG IgG antibodies.

[0017] Among them, the antibody can effectively inhibit the adhesion of ETEC expressing F4 fimbriae adhesin to piglet small intestinal epithelial cells.

[0018] The present invention also includes an anti-FaeG IgG antibody, which is obtained by immunizing an animal with the non-pathogenic Escherichia coli PEC2021-8 (Escherichia coli PEC2021-8) or an inactivated vaccine prepared therefrom or the fimbriae adhesin extract.

[0019] The animals include but are not limited to mice or pigs.

[0020] Among them, immunizing mice with the fimbriae adhesin extract from the PEC2021-8 isolate can induce the production of anti-FaeG specific antibodies.

[0021] The PEC2021-8 isolate was inactivated by formaldehyde and then added with aluminum hydroxide latex particles and injected intramuscularly into the neck of 25-day-old healthy piglets with negative F4 antibodies. 9 CFU / head, immunized twice with an interval of 14 days, can induce high titer anti-FaeG antibodies, and the antibodies can effectively block the adhesion of the C83902 standard strain expressing F4 fimbriae adhesin to the small intestinal epithelial cells of piglets in vitro. Immunized piglets had no obvious clinical symptoms and pathological changes.

[0022] Furthermore, the vaccine forms of the present invention include, but are not limited to, subunit vaccines and inactivated vaccines, and also include its potential use as a live vaccine.

[0023] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: the PEC2021-8 isolate of the present invention is derived from healthy lactating piglets. The results of PCR detection and agglutination reaction show that the isolate only expresses F4 fimbriae adhesin, and does not contain genes encoding F5, F6, F18, F41 fimbriae adhesin and LT, STa, STb and EAST1 enterotoxin. The isolate has good adhesion to the small intestinal epithelial cells of piglets, and the extracted fimbriae adhesin can induce high levels of anti-FaeG antibodies after immunizing mice. After the isolate is inactivated, there are no obvious clinical symptoms and pathological changes in piglets immunized with piglets, but high titer anti-FaeG antibodies can be induced, and the antibody can effectively inhibit the adhesion of the C83902 standard strain expressing F4 fimbriae adhesin to the small intestinal epithelial cells of piglets in vitro. The isolate is expected to be used as a candidate strain for F4+ETEC vaccine (subunit, inactivated vaccine and live vaccine), and is used to prevent diarrhea in newborn piglets and weaned piglets caused by F4+ETEC infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the electrophoresis diagram of PCR identification of virulence factor genes of PEC2021-8 isolate; Lane M is 2K PlusⅡDNA Marker; Lane 1 is faeG gene; Lane 2 is fanC gene; Lane 3 is fasA gene; Lane 4 is fedF gene; Lane 5 is fim41a gene; Lane 6 is eltAB gene; Lane 7 is estA gene; Lane 8 is estB gene; Lane 9 is east1 gene;

[0025] Figure 2 These are the agglutination reaction results of the PEC2021-8 isolate; 1 is a mixture of DH5α bacterial solution and anti-FaeG polyclonal antibody, 2 is a mixture of C83902 bacterial solution and anti-FaeG polyclonal antibody, and 3 is a mixture of PEC2021-8 isolate bacterial solution and anti-FaeG polyclonal antibody.

[0026] Figure 3 The SDS-PAGE and Western blot identification diagrams of the pili adhesin extracts of the PEC2021-8 isolate. A. SDS-PAGE result diagram, 1 is the pili adhesin extract of the C83902 standard strain, 2 is the pili adhesin extract of the PEC2021-8 isolate. B. Western blot identification diagram, 1 is the pili adhesin extract of the C83902 standard strain, 2 is the pili adhesin extract of the PEC2021-8 isolate.

[0027] Figure 4 This is a graph showing the growth curve of the PEC2021-8 isolate;

[0028] Figure 5 is the adhesion ability of the PEC2021-8 isolate to IPEC-J2 cells;

[0029] Figure 6 This is a graph showing the antibody level of anti-FaeG in the serum of immunized mice detected by ELISA;

[0030] Figure 7 This is a graph showing the level of anti-FaeG antibodies in the serum of immunized piglets detected by ELISA;

[0031] Figure 8 This is a graph showing the inhibition of immune piglet serum on the adhesion of C83902 strain to IPEC-J2 cells. DETAILED DESCRIPTION

[0032] The following detailed and comprehensive description of the embodiments of the present invention is provided in conjunction with specific examples. Obviously, the described examples are only intended to illustrate a part of the embodiments of the present invention and should not be considered as limiting the scope of the present invention. In the examples, the specific conditions not particularly specified are carried out according to the conditions of normal conditions or manufacturer recommendations. The reagents or instruments used are not indicated by the manufacturer and are all commercially available conventional products.

[0033] Example 1 Isolation and identification of porcine ETEC PEC2021-8 isolate

[0034] (1) Sample collection and bacterial isolation

[0035] On October 5, 2021, 40 fresh fecal samples from suckling piglets were collected by Duan Qiangde. Sample collection, strain isolation and identification were carried out in accordance with the "National Standard Method for Diarrhea-causing Escherichia coli Detection" (GB4789.6-2016).

[0036] The specific steps are as follows:

[0037] a. Strain culture: The collected fecal samples were inoculated into intestinal bacterial enrichment medium and cultured overnight in a 37°C constant temperature shaker (220 rpm / min).

[0038] b. Use an inoculating loop to streak the enriched bacterial solution onto a MacConkey solid plate and culture in a 37°C incubator for 16-18 hours.

[0039] c. Observe the morphology of bacterial colonies, pick out red and shiny single colonies, and inoculate them into sterile LB liquid culture medium to obtain the PEC201-18 isolate.

[0040] (2) PCR identification of virulence factors of PEC201-18 isolate

[0041] The genomic DNA was extracted from the PEC2021-8 isolate by boiling lysis method and used as a PCR template. Specific primers were used to perform PCR identification on faeG gene (SEQ ID NO.1 and SEQ ID NO.2), fanC gene (SEQ ID NO.3 and SEQ ID NO.4), fasA gene (SEQ ID NO.5 and SEQ ID NO.6), fedF gene (SEQ ID NO.7 and SEQ ID NO.8), fim41a gene (SEQ ID NO.9 and SEQ ID NO.10), eltAB gene (SEQ ID NO.11 and SEQ ID NO.12), estA gene (SEQ ID NO.13 and SEQ ID NO.14), estB gene (SEQ ID NO.15 and SEQ ID NO.16) and east1 gene (SEQ ID NO.17 and SEQ ID NO.18). The specific primer sequences are shown in Table 1. The PCR reaction amplification system is shown in Table 2. The PCR reaction conditions are: 95°C pre-denaturation for 5 min, 94°C denaturation for 30 s, 50°C annealing for 30 s, 72°C extension for 1 min 30 s, 25 cycles, 72°C × 10 min, and 4°C termination. Figure 1 As shown, only the faeG gene amplified a specific band at the expected size of 849 bp, and no other primers amplified bands. This indicates that the PEC2021-8 isolate only carries the faeG gene encoding the F4 fimbriae adhesin, but not other virulence genes.

[0042] Table 1 PCR amplification primers for common virulence factors of PEC2021-8 isolates

[0043]

[0044] Table 2 PCR amplification system

[0045]

[0046]

[0047] (3) Identification of fimbriae adhesin expression in PEC2021-8 isolates

[0048] First, PEC2021-8, C83902 (purchased from the Chinese Institute of Veterinary Drug Control's bacterial strain collection center) and DH5α (Tiangen Biochemical Technology Co., Ltd.) were inoculated with fresh antibiotic-free LB liquid medium and cultured overnight at 37°C in a constant temperature shaker (220 rpm / min). The next day, the overnight culture was centrifuged at 4000 rpm for 10 min at room temperature to collect the bacteria. After the bacteria were washed three times with autoclaved PBS, the concentration of the bacteria was adjusted to 1×10 with an appropriate amount of 0.01 M PBS (PH=7.4). 10 CFU / mL. 10 μL of DH5α, C83902 and PEC2021-8 bacterial suspensions were added onto a clean glass slide, followed by 10 μL of the self-made anti-FaeG polyclonal antibody (Duan et al., 2020, A multivalent vaccine candidate targeting enterotoxigenic Escherichia coli fimbriae for broadly protecting against porcine post-weaning diarrhea), mixed well and placed at room temperature for 2 minutes to observe the formation of agglutinated particles. The results are shown in Figure 2 As shown, the C83902 standard strain and the PEC2021-8 isolate formed visible agglutinated particles after mixing with the anti-FaeG polyclonal antibody, while no agglutinated particles were observed for DH5α.

[0049] The ammonium sulfate precipitation method was used to extract the fimbriae adhesins of the F4ac standard strain C83902 and the PEC2021-8 isolate. The specific steps are as follows: first, the seed liquid of the overnight cultured F4ac standard strain C83902 and the PEC2021-8 isolate was inoculated with 500 mL of fresh non-antibiotic LB liquid at a volume ratio of 1:100, and placed in a 37°C constant temperature shaker (220 rpm / min) for shaking culture for 18 hours. The bacteria were collected by centrifugation at 4°C, 12000 rpm for 10 minutes, and the bacteria were washed twice with 0.01M PBS (PH=7.4) and then fully resuspended in 30 mL and placed at -70°C overnight. The next day, the bacterial solution was placed in a water bath at 60°C for 30 minutes, the bacterial solution was placed on ice and stirred at 100 rpm for 20 minutes on a magnetic stirrer, and centrifuged at 4°C, 10000 rpm, for 20 minutes to collect the supernatant. Add an equal volume of saturated ammonium sulfate solution to the supernatant (add slowly and stir while adding), stir on ice for 30 minutes, and place in a 4°C refrigerator overnight. The salting product was centrifuged at 10000rpm and 4°C for 15 minutes. The precipitate was collected and resuspended with 20mL PBS, and then dialyzed with 2L 0.01M PBS (PH=7.4) in a 4°C refrigerator for 48 hours to obtain the crude extract of the pili. The crude extract of the pili was analyzed by SDS-PAGE. The results are as follows: Figure 3 As shown in A, a clear band was observed at about 25 KDa for both the PEC2021-8 isolate and the C83902 reference standard strain. Western blot analysis was then performed, and the results showed that the band could be recognized by the anti-FaeG polyclonal antibody (see Figure 3 B). These results indicate that the PEC2021-8 isolate does express F4 fimbriae adhesin, confirming that it belongs to F4+ETEC. The isolate PEC2021-8 has been deposited in the China Center for Type Culture Collection (CTCCC NO: M 2024224) on January 26, 2024, in Wuhan, China, and is classified as Escherichia coli.

[0050] Example 2 Identification of biological characteristics of the pig-derived ETEC PEC2021-8 isolate

[0051] (1) Growth characteristics of ETEC PEC2021-8 isolate

[0052] The overnight culture of F4ac+ETEC standard reference strain C83902 and PEC2021-8 isolate was adjusted to OD 600nm =1.0. Then, the bacterial solution was inoculated into fresh LB liquid culture medium at a volume ratio of 1:50, and placed on a 37°C constant temperature bacterial culture shaker, and cultured at a speed of 200 rpm / min. Samples were taken every hour, and the absorbance value of the bacterial solution at a wavelength of 600 nm was measured using a spectrophotometer, and the data were recorded. The above test was repeated 3 times, and the absorbance value of the 3 OD values ​​was recorded. 600 The average value of the bacterial growth curve is plotted. Figure 4 As shown, the two strains showed the same growth rate in the lag phase, logarithmic phase and stationary phase.

[0053] (2) Adhesion ability of ETEC PEC2021-8 isolate to piglet small intestinal epithelial cells

[0054] Piglet intestinal epithelial cells IPEC-J2 were cultured in RPMI1640-F12 (1:1) medium and 10% fetal bovine serum (FBS). After the cells grew to a full monolayer, they were digested with 0.25% trypsin and inoculated into a 24-well culture plate. When the monolayer cell density reached about 3.0×10 5 Select a single colony of ETECPEC2021-8 isolated strain activated on the LB solid plate, inoculate it into LB liquid medium without antibiotics, and culture it in a constant temperature shaker at 37°C overnight. The next day, transfer the bacterial solution to fresh LB liquid medium without antibiotics at a volume ratio of 1:100. After the bacteria grow to the logarithmic growth phase, adjust the bacterial solution concentration to 1.0×108 CFU / mL. Bacteria were added at a ratio of 1:100 between the number of cells and the number of bacteria, and incubated in a 37°C constant temperature incubator for 1 hour. After incubation, the cells were washed 3 times with sterile PBS to remove non-adherent bacteria. Then, 1 mL of 0.5% Triton X-100 lysis buffer was added to each well, and the cells were placed on a shaker at room temperature and slowly shaken for 30 minutes for lysis. The lysis buffer was diluted in a gradient of 1:10, diluted with PBS, and then spread on an LB plate without antibiotics. The plates were placed in a 37°C constant temperature incubator overnight for single colony counting. Three parallel wells were set up in each group, and the experiment was repeated 3 times. The results are shown in the figure. Figure 5 As shown, the ETEC PEC2021-8 isolate had good adhesion to IPEC-J2 cells, but its adhesion ability was weaker than that of the F4ac+ ETEC standard reference strain C83902.

[0055] Example 3 Immunogenicity Detection of Porcine ETEC PEC2021-8

[0056] (1) Immunization of mice with extracted PEC2021-8 fimbriae adhesin

[0057] Twelve 6-week-old SPF BALB / c female mice were randomly divided into 2 groups, 6 mice in each group. In the first group, each mouse was immunized with 50 μg of the PEC2021-8 fimbriae adhesin extract isolated in Example 1, and 50 μL of Freund's complete adjuvant (Sigma, Catalog Number: SLCJ8308) was added; in the other group, each mouse was immunized with 50 μL of sterile PBS as a negative control. After the first immunization, booster immunizations were performed every two weeks, for a total of 3 immunizations. For the second and third immunizations, the antigen dose remained unchanged, but the adjuvant was changed to Freund's incomplete adjuvant (Sigma, Catalog Number: SLCJ3267). On the 14th day after the third immunization, the mice were killed by painless cervical dislocation, and blood was collected from the heart to collect blood for serum separation. The serum samples were stored in a refrigerator at 4°C for future use. Next, the titer of specific anti-FaeG antibodies in mouse serum was detected using an indirect ELISA method. The results are as follows. Figure 6 As shown, compared with the PBS negative control group, the pili adhesin extract of the PEC2021-8 isolate significantly induced high titer anti-FaeG antibodies. This indicates that the PEC2021-8 pili adhesins have good immunogenicity and can effectively induce antibody production in mice.

[0058] (2) PEC2021-8 inactivated vaccine for immunization of piglets

[0059] Ten 25-day-old weaned piglets were randomly divided into two groups, with 5 pigs in each group of uniform size. Group 1 was the PEC2021-8 inactivated vaccine immunization group, and Group 2 was the PBS control group. The specific immunization procedure was as follows: a total of two immunizations, the first immunization at 25 days of age, with PEC2021-8 inactivated vaccine injected intramuscularly in the neck, 2.0 mL / head (1×10 9 CFU); 14 days after the first immunization, the same method and dose were used for booster immunization. The PBS control group was injected with only 2.0 mL / head of sterile PBS each time as a control. On the 14th day after the second immunization, blood was collected from the anterior vena cava and serum was separated. The ELISA method was used to detect the level of specific anti-FaeG antibodies in the serum of immunized piglets. The results are shown in Figure 7 As shown, high levels of anti-FaeG antibodies were detected in the PEC2021-8 inactivated vaccine immunization group, while no corresponding antibodies were detected in the PBS control group, indicating that the inactivated vaccine prepared based on the PEC2021-8 isolate has good immunogenicity for immunizing piglets.

[0060] (3) Inhibition test of immune piglet serum on F4ac+ETEC adhesion

[0061] The C83902 standard strain cultured to the logarithmic growth phase was diluted to 1.0×10 7 CFU / mL. Take 150μL (about 1.5×10 6 CFU) bacterial solution was mixed with 30 μL PEC2021-8 inactivated vaccine or PBS immunized piglet serum, and supplemented with serum-free DMEM medium to a total volume of 600 μL. Then, the bacteria and serum mixture was incubated on a shaker at 30 rpm / min at room temperature for 30 min. The monolayer of IPEC-J2 cells in the 24-well cell culture plate was washed three times with sterile PBS, and then the mixture pre-incubated with mouse serum was added to the cells (bacterial number: cell number = 5:1), and placed in a 37°C constant temperature incubator for 1 hour. The total number of bacteria adhering to IPEC-J2 cells was counted using the same method as the plate counting method in the adhesion experiment.

[0062] The results are as follows Figure 8 As shown in the figure, the serum of the piglet group immunized with PEC2021-8 inactivated vaccine can significantly inhibit the adhesion of the C83902 standard strain to IPEC-J2 cells, while the serum of the PBS immunized group has no inhibitory effect on its adhesion to IPEC-J2 cells. These results show that the PEC2021-8 isolate has the potential to be used as a candidate vaccine strain for preventing diarrhea in piglets caused by F4+ETEC infection.

Claims

1. A non-pathogenic Escherichia coli, characterized in that: The non-pathogenic Escherichia coli is classified as Escherichia coli ( Escherichia coli )PEC2021-8, the non-pathogenic Escherichia coli was deposited in the China Center for Type Culture Collection on January 26, 2024, with the deposit number CCTCC NO: M2024224, and the deposit address is Wuhan, China.

2. Use of the non-pathogenic Escherichia coli according to claim 1 in the preparation of vaccines or medicines for preventing or treating piglet diarrhea.

3. Use of the non-pathogenic Escherichia coli according to claim 1 in the preparation of a live vaccine or an inactivated vaccine for preventing F4+ ETEC infection.

4. A preparation containing the Escherichia coli according to claim 1, characterized in that The preparation includes Escherichia coli ( Escherichia coli ) The bacterial suspension, fermentation broth or supernatant of PEC2021-8.

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