CN116751307A - Escherichia coli multi-epitope chimeric protein and its application - Google Patents

Abstract

The invention provides an Escherichia coli multi-epitope chimeric protein and its application. The chimeric protein uses the outer membrane protein OmpA of Escherichia coli O78 serotype as a carrier and contains B cell epitopes (OmpA-Fusion) of BamA and OmpC. ; Alternatively, the chimeric protein uses the outer membrane protein BamA of Escherichia coli O78 serotype as a carrier and contains the B cell epitopes of OmpA and OmpC (BamA-Fusion). The multi-epitope chimeric sequence of the present invention can be induced to express in Escherichia coli, and the expression product has high antigenicity. Using the multi-epitope chimeric protein and Freund's adjuvant to immunize mice together can stimulate the mouse body to produce strong immune response. The subunit vaccine provided by the invention has good immune protection effect against Escherichia coli and infection, can be used to prepare subunit vaccines against Escherichia coli, and has good application prospects.

Description

Escherichia coli multi-epitope chimeric protein and its application

Technical field

The present invention relates to the technical field of genetic engineering, specifically to an Escherichia coli multi-epitope chimeric protein and its application.

Background technique

Escherichia coli (E.coli) is an opportunistic pathogen that resides in the intestines of various animals. Under certain conditions, some Escherichia coli carrying pathogenic factors can cause a variety of local tissue and organ infections in healthy animals, such as gastrointestinal infections, urinary tract infections, and meningitis, which often bring huge pressure to the animal husbandry industry. and economic losses. At present, the main prevention and control methods for E. coli are antibiotic treatment and vaccination. However, the abuse of antibiotics has caused widespread drug resistance in E. coli, so vaccination is an ideal way. However, the development of E. coli vaccines faces severe challenges due to numerous serotypes of E. coli and the recombination of virulence genes. In addition, many commercialized E. coli vaccines have poor clinical immune effects and obvious geographical limitations, making the prevention of E. coli-related diseases still very difficult. Therefore, the development of safe and efficient new E. coli vaccines has been a research hotspot in recent years.

Multi-epitope vaccine is a new subunit vaccine designed based on the amino acid sequence of the target antigen epitope. Compared with traditional vaccines, it has the advantages of safety, non-toxic, stable and controllable, and can directly stimulate the body to produce specific immune responses, which is in line with future vaccines. The development direction has attracted increasing attention. Compared with traditional vaccines, vaccines based on epitope design have strong targeting, accurate antibody positioning, and clear immune mechanisms. However, the analysis and design of antigenic epitopes require higher requirements. Therefore, in order to successfully develop epitope vaccines, we must first screen out Suitable epitopes. Antigen epitope (epitope) includes T cell epitope and B cell epitope. It is a group in the antigen molecule that determines the antigen specificity. It can interact with T cell receptor (T cell receptor, TCR) or B cell receptor (B cell receptor). Cell receptor (BCR) specifically binds to stimulate the body to produce an immune response and form immunity to pathogenic bacteria. T cell epitopes are recognized by intracellular major histocompatibility complex (MHC) I or II molecules and presented to the cell surface, and then recognized by the TCR of CD8+ T cells and CD4+ T cells respectively. Stimulates the body to produce cellular immunity and is mainly used in the prevention and treatment of viruses and intracellular parasitic bacteria. B cell epitopes are recognized by BCR and can also be presented by MHC class II molecules. The complex after binding to MHC II molecules is recognized by BCR on the cell surface and is a soluble protein antigen conformational determinant. B cell epitopes stimulate the body to produce antibodies, which mainly exert immune functions in body fluids. Online prediction tools such as IEDB, DiscoTope, BepiPred, and ABCPred are often used to predict protein epitopes. The prediction of B cell epitopes requires the integration of physical and chemical properties, structural characteristics, statistical significance measures and other indicators of the antigen protein for epitope prediction. Such as amino acid flexibility (Flexibility), surface accessibility (Surface accessibility), local hydrophilicity (Local hydrophilicity), antigenicity, protrusion index (Protrusion index) in the primary sequence, as well as the turns and loops of the secondary structure Structure, etc., and the predicted results cannot be guaranteed to be 100% accurate and need to be further verified in experiments.

The low molecular weight of a single antigenic epitope makes it poorly immunogenic, lacks a stable three-dimensional conformation, and is easily degraded. Although multiple immunizations can achieve better immune effects, they can easily lead to immune tolerance in animals. Therefore, multiple epitopes need to be connected in series using genetic engineering technology to design a multi-epitope vaccine, and different functional regions of the same protein or proteins with different functions can be connected to enhance the immunogenicity and stability of the antigen. Currently, there are four commonly used tandem methods for designing multi-epitope vaccines: antigen epitope linear tandem, antigen epitope and carrier coupling, multi-antigen peptide and polylipopeptide multi-epitope vaccines. Antigen epitope linear concatenation is formed by directly concatenating multiple predicted antigen epitopes head to tail. This concatenation method can effectively overcome the shortcomings of a single epitope that is weak in immunogenicity and easily degraded.

Multi-epitope vaccines have unique advantages, such as high antigenicity, multivalency, good immune effect, and good safety. They have been widely used in related diseases including bacterial infections, viral diseases, parasitic infections, and anti-tumor aspects. in prevention and control research.

Contents of the invention

The object of the present invention is to provide an Escherichia coli multi-epitope chimeric protein and its application.

The present invention designs a multi-epitope chimeric sequence based on the outer membrane protein of Escherichia coli CAU0768 (O78), and uses the multi-epitope chimeric protein to construct a subunit vaccine.

In order to achieve the purpose of the present invention, in the first aspect, the present invention provides an Escherichia coli multi-epitope chimeric protein. The chimeric protein uses the outer membrane protein OmpA of Escherichia coli O78 serotype as a carrier and contains the outer membrane protein BamA. B cell epitope and B cell epitope of outer membrane protein OmpC (denoted OmpA-Fusion); or,

The chimeric protein uses the outer membrane protein BamA of Escherichia coli O78 serotype as a carrier, and contains the B cell epitope of the outer membrane protein OmpA and the B cell epitope of the outer membrane protein OmpC (denoted as BamA-Fusion).

In the present invention, the B cell epitope sequence of OmpA, the outer membrane protein of Escherichia coli O78 serotype, is shown in SEQ ID NO: 1-5;

The B cell epitope sequence of outer membrane protein BamA is shown in SEQ ID NO: 6-9;

The B cell epitope sequence of outer membrane protein OmpC is shown in SEQ ID NO: 10-12.

Further, the chimeric protein uses the outer membrane protein OmpA of Escherichia coli O78 serotype as a carrier, and the B cell epitope of the outer membrane protein BamA and the B cell epitope of the outer membrane protein OmpC are connected to the N-terminus of OmpA or through a flexible Linker (such as GGGS) is connected to the C-terminus of OmpA.

Further, the chimeric protein uses the outer membrane protein BamA of Escherichia coli O78 serotype as a carrier, and the B cell epitope of the outer membrane protein OmpA and the B cell epitope of the outer membrane protein OmpC are connected through a flexible Linker (such as GGGS). C-terminus of BamA.

In a specific embodiment of the invention, the chimeric protein OmpA-Fusion contains or consists of the following amino acid sequence:

a), the amino acid sequence shown in SEQ ID NO:13; or

b), the amino acid sequence obtained by connecting a tag to the N-terminus and/or C-terminus of a); or

A protein with the same function obtained by substituting, deleting and/or adding one or more amino acids to the amino acid sequence of c), a) or b).

In another specific embodiment of the invention, the chimeric protein BamA-Fusion contains or consists of the following amino acid sequence:

A), the amino acid sequence shown in SEQ ID NO:14; or

B), the amino acid sequence obtained by connecting a tag to the N-terminus and/or C-terminus of A); or

A protein with the same function obtained by substituting, deleting and/or adding one or more amino acids to the amino acid sequence of C), A) or B).

In a second aspect, the present invention provides nucleic acid molecules encoding the chimeric protein or biological materials containing the nucleic acid molecules; wherein the biological materials are recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineering bacteria or transgenic cell lines.

In a third aspect, the present invention provides an immunogenic composition comprising the chimeric protein.

In a fourth aspect, the present invention provides an E. coli subunit vaccine, which includes the immunogenic composition and a pharmaceutically acceptable carrier. Adjuvants are optionally included.

In a fifth aspect, the present invention provides any of the following applications of the chimeric protein:

(1) Used to prepare E. coli subunit vaccine;

(2) Preparation of reagents or kits for detecting E. coli infection;

(3) For the diagnosis of E. coli infection.

Through the above technical solutions, the present invention has at least the following advantages and beneficial effects:

(1) The present invention connects the antigenic epitopes in series to the outer membrane protein OmpA or BamA as the C-terminal or N-terminal of the carrier protein through a flexible connecting peptide Linker. The embedding of multiple B cell epitopes can enhance its antigenicity. The chimeric protein Multiple antigenic epitopes in the protein can form correct spatial structures respectively, thereby better exerting their respective biological activities and preventing mutual interference between proteins in spatial conformation.

(2) The multi-epitope chimeric sequence of the present invention can be induced and expressed in E. coli, and the expression product has high antigenicity. The multi-epitope chimeric protein of the present invention and Freund's adjuvant are used to immunize mice together, which can stimulate the mice to produce a strong immune response. After the secondary immunization, a high level of antibody titer was produced in the serum. When challenged with the lowest lethal dose concentration of E. coli CAU0768 (O78), the multi-epitope chimeric protein immunized group achieved 100% protection rate.

(3) The subunit vaccine provided by the present invention has a good immune protective effect against E. coli and infections, can be used to prepare subunit vaccines against E. coli, and has good application prospects.

Description of the drawings

Figure 1 is an SDS-PAGE diagram after purification of the multi-epitope chimeric protein in the preferred embodiment of the present invention.

Figure 2 shows the serum antibody titers of mice immunized with the multi-epitope chimeric protein in the preferred embodiment of the present invention. In the figure, **** indicates that the difference between different treatment groups is statistically significant, ****P<0.0001. ns represents the statistical comparison benchmark.

Figure 3 shows the serum antibody subtypes of mice immunized with multi-epitope chimeric proteins in a preferred embodiment of the present invention.

Figure 4 shows the cross-reaction of the serum of mice immunized with the multi-epitope chimeric protein in the preferred embodiment of the present invention.

Figure 5 shows the colony count of the infected organs of mice immunized with the multi-epitope chimeric protein in the preferred embodiment of the present invention. In the figure, *, ** and *** indicate that the differences between different treatment groups are statistically significant, * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001. ns represents the statistical comparison benchmark.

Figure 6 shows the protection rate of mice immunized with the multi-epitope chimeric protein in the preferred embodiment of the present invention. Note: The curves of the BamA-Fusion+Freund's adjuvant group and the non-immune control group overlap.

Detailed ways

The following examples are used to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products. Example 1 Construction of multi-epitope chimeric sequences

1. B cell epitope prediction

The amino acid sequences of the three outer membrane proteins OmpA, BamA and OmpC of Escherichia coli O78 were analyzed through the Bepipred, BCPred and COBEpro online prediction websites to analyze the B cell linear epitopes. The three prediction methods and immunoinformatics parameters were combined to screen the appropriate B cell epitopes. bit, using the overlapping parts of the results of different prediction methods to increase the credibility of the prediction (SEQ ID NO: 1 ~ 12). Using the antigenic regions of OmpA and BamA as the backbone, the candidate B cell epitopes of the other two proteins were connected at the C terminus through a linker peptide to construct a multi-epitope fusion protein containing multiple B cell epitopes, named OmpA-Fusion. and BamA-Fusion.

2. Synthesis of multi-epitope chimeric genes

Sangon Bioengineering (Shanghai) Co., Ltd. used a total gene synthesis method to conduct artificial chemical synthesis and cloned it into the PUC57 vector, named PUC57-OmpA-Fusion and PUC57-BamA-Fusion.

3. Secondary structure analysis of multi-epitope chimeric proteins

Use ProtParam in the ExPASy website to predict the physical and chemical properties of the amino acid sequence of the multi-epitope chimeric protein.

4. Prediction of the tertiary structure of multi-epitope chimeric proteins

The molecular biology software I-TASSER was used to predict the tertiary structure of the multi-epitope chimeric protein. The results showed that the vector can maintain a certain spatial structure after embedding multiple epitopes, and the constructed multi-epitope chimeric protein has multiple The epitope is exposed on the surface of the molecule, which meets the design requirements of multi-epitope chimerism.

5. Antigenicity analysis of multi-epitope chimeric proteins

The molecular biology software VaxiJen was used to analyze the antigenicity of the multi-epitope chimeric protein. The OmpA-Fusion score was 0.9315 and the BamA-Fusion score was 0.8394, which met the antigenicity requirements of multi-epitope proteins.

The B cell epitope sequence of OmpA, the outer membrane protein of Escherichia coli of O78 serotype, is shown in SEQ ID NO: 1-5;

The B cell epitope sequence of outer membrane protein BamA is shown in SEQ ID NO: 6-9;

The B cell epitope sequence of outer membrane protein OmpC is shown in SEQ ID NO: 10-12.

Example 2 Expression of multi-epitope genes in E. coli

1. Construction of multi-epitope chimeric sequence expression vector

The plasmids PUC57-OmpA-Fusion and PUC57-BamA-Fusion were double digested with Nco I and Xho I. After agarose gel electrophoresis, the target fragments OmpA-Fusion and BamA-Fusion were recovered and ligated with Nco I at 25°C. From plasmid pet-28a(+) double digested with Xho I, plasmid pet-28a(+)-OmpA-Fusion/BamA-Fusion was obtained.

2. Transformation of E. coli DH5α with expression vector

Add 10ul of the connected system at 25°C to 50ul of competent cells DH5α, mix gently and place in ice for 30 minutes. After 45 seconds in 42°C water bath, quickly transfer to ice and place for 2 minutes. Add 450ul of LB medium, shake at 200r/min, and incubate at 37°C for 60min. Take 200ul and 100ul of the transformation solution and spread them on the LB agar plate medium containing Kan (final concentration 50μg/ml), and culture at 37°C overnight.

3. Screening of E. coli positive clones

Use a pipette tip to pick up some colonies from a single colony, add them to 30ul of sterile water, and mix well. Take 3ul as PCR template and use universal primers for PCR identification. If the PCR result shows a specific band, select 3-5 positive bacterial liquids, take 300ul of the corresponding 1ml bacterial liquid, transfer it to LB liquid medium containing Kan, culture it to the logarithmic phase, and store the bacterial liquid in a glycerol tube at -20°C. . The remaining bacterial liquid was sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing.

4. Transformation of E. coli BL21 with expression vector

Transfer the correctly sequenced bacterial solution to 5 ml of LB medium containing Kan, and culture it at 250 r/min and 37°C for 12 hours. The Tiangen plasmid extraction kit (DP103) was used to extract the plasmid, and the recombinant plasmid was transformed into the expression strain BL21 using the same method.

5. Induced expression of E. coli positive clones

Take the positive bacteria-preserving bacterial liquid cultured overnight and add 1% of it into LB medium containing Kan (final concentration 50 μg/ml) and culture for more than 2 hours to the mid-log phase (OD ₆₀₀ = 0.6). Add IPTG to the induction tube to reach a concentration of 1 mmol/L, 250 r/min, and incubate at 37°C for about 6 hours with shaking for sampling.

6. SDS-PAGE analysis of expressed target protein

After induction, take 1 mL of bacterial solution, centrifuge at 8000 rpm for 2 minutes at 4°C to collect the cells, discard the supernatant, resuspend the cells in 100ul PBS, 100ul 2×SDS-PAGE loading buffer, boil at 100°C for 10 minutes, room temperature 12000r/min Centrifuge for 5 minutes and take the supernatant for detection by SDS-PAGE. As shown in Figure 1, the molecular weights of OmpA-Fusion (SEQ ID NO: 13) and BamA-Fusion (SEQ ID NO: 14) are both about 50 kDa, which is consistent with the theoretical value.

Example 3 Mouse immunization experiment of multi-epitope subunit vaccine

1. Mouse immunization program

Thirty female BALB/c mice aged 6-8 weeks were divided into three groups: PBS+adjuvant group, OmpA-Fusion-adjuvant group, and BamA-Fusion+adjuvant group, with 10 mice in each group. A total of three immunizations were carried out, with the interval between the first and second immunizations being 21 days, and the interval between the second and third immunizations being 14 days, with 25 μg of subunit vaccine per animal. The first dose was mixed with Freund's complete adjuvant and injected subcutaneously; the second and third doses were mixed with Freund's incomplete adjuvant and injected intraperitoneally. Blood was collected from the tail vein before immunization and 5 days after each immunization, and serum ELISA antibody analysis was performed.

2. ELISA antibody titer detection in serum of immunized mice

The indirect ELISA method was used to determine the specific IgG antibody levels in the serum of immunized mice. The specific method is as follows:

Coating: Dissolve the outer membrane protein in the protein coating solution to a final concentration of 2ug/ml, add 100ul/well to a 96-well plate, incubate at 4°C overnight, wash 4 times with 100ul PBST/well PBST, 5 minutes each time, pat dry.

Blocking: Add 200ul/well of blocking solution to the 96-well plate, block for 2 hours at 37°C, wash 4 times with PBST, 5 minutes each time, and pat dry.

Add the serum sample to be tested: 1:100 for the primary immune serum, 1:1000 for the secondary immune serum and 1:1000 for the third immune serum, 3-fold gradient dilution, add 100ul/well to a 96-well plate, and incubate at 37°C for 1.5 hours. Wash 4 times with PBST, 5 minutes each time, and pat dry.

Add secondary antibody: Dilute horseradish-labeled goat anti-mouse igG (igG-specific secondary antibody) 1:5000, add 100ul/well to a 96-well plate, incubate at 37°C for 30 minutes, wash 4 times with PBST, 5 minutes each time , pat dry.

Color development: Add 100ul/well of chromogen TMB and develop color at 37°C for 15-30 minutes. Termination: Add 50ul of stop solution/well to terminate the reaction.

Reading: Measure the OD value of each well at 450nm with 630nm as the reference wavelength, and limit the ratio (P/N) to the OD value of the negative control well to be greater than 2.1 as the critical point for determining the potency. The results are shown in Figure 2. Co-immunization with both proteins and Freund's adjuvant can stimulate a strong immune response in mice.

3. ELISA antibody subtype detection in serum of immunized mice

Except that the secondary antibodies used were horseradish-labeled goat anti-mouse igG1, igG2a, igG2b, igG3, igM and igA, the remaining methods were the same as the antibody titer detection. The results are shown in Figure 3. The antibodies detected in the serum of immunized mice were mainly igG1, igG2a, igG2b and igM, indicating that there was both humoral immune response (Th2) and cell-mediated immune response (Th1).

4. ELISA cross-reaction detection of immunized mouse serum

Fixation: Add 150ul/well of fixative to the enzyme plate and place at 37°C for 1 hour; wash the plate 4 times with 150ul/well of PBST, 5min each time, and pat dry. Add 100ul/well of bacterial antigen suspension (10 ⁸ CFU/mL) resuspended in PBS and place at 37°C until air-dried; wash 4 times with 100ul PBST washing solution to remove unbound antigen, 5 minutes each time, and pat dry. Except that the serum sample to be tested is diluted at 1:27000, the remaining steps are the same as those for antibody titer detection. The results are shown in Figure 4. The antiserum has a certain cross-protective effect on different serotypes of Escherichia coli and Shigella.

5. E. coli bacteria load in infected organs

E. coli CAU0768 (O78) was activated overnight, transferred at 1% and cultured to the logarithmic phase. On the 14th day after the third immunization, the mice in the immune group and the blank control group were intraperitoneally injected with the lowest lethal dose of log-phase E. coli. The mice were euthanized 6 hours after the challenge, and the hearts, livers, spleens, lungs, and kidneys were harvested for bacterial colony counting. The results are shown in Figure 5. Under the lowest lethal dose challenge, the bacterial load in the organs of the multi-epitope chimeric protein immunized group was significantly lower than that of the organs of unimmunized mice.

6. E. coli challenge protection rate

E. coli CAU0768 (O78) was activated overnight, transferred at 1% and cultured to the logarithmic phase. On the 14th day after the third immunization, the mice in the immune group and the blank control group were intraperitoneally injected with the lowest lethal dose of log-phase E. coli. The death of mice was monitored and recorded every day, and the mortality rate of mice was counted for a total of 7 days. As shown in Figure 6, under the lowest lethal dose challenge, unimmunized mice died within 24 hours. The survival rate of the adjuvant group was 50%, and the survival rate of the multi-epitope chimeric protein immunization group was 100%.

The above experimental results show that the novel E. coli outer membrane protein multi-epitope chimeric sequence provided by the present invention can be expressed in prokaryotic induction in E. coli, and the expression product is immunogenic and can be recognized by antibodies in the serum of immunized mice. . The vector can maintain its original spatial structure and has less secondary structure interaction between chimeric epitopes, and has high antigenicity. At the same time, multiple epitopes are exposed on the surface of the molecule, which is consistent with the design of multi-epitope. requirements and is expected to become a better immunogen. Mouse immunization experiments show that the subunit vaccine using multi-epitope chimeric protein can not only stimulate the mouse body to produce specific humoral immunity, but also produce specific cellular immunity, which has a good protection rate against E. coli infection. In vitro experiments show that the multi-epitope chimeric protein also has a certain cross-protective reaction with other Escherichia coli and Shigella.

The novel Escherichia coli multi-epitope chimeric protein designed in the present invention has high antigenicity, can be used for the preparation of Escherichia coli and Shigella subunit vaccines, and has good development prospects.

Although the present invention has been described in detail with general descriptions and specific embodiments above, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims (10)

1. Escherichia coli multi-epitope chimeric protein, characterized in that the chimeric protein uses the outer membrane protein OmpA of Escherichia coli O78 serotype as a carrier, and contains the B cell epitope of the outer membrane protein BamA and the outer membrane protein OmpC B cell epitope; or, The chimeric protein uses the outer membrane protein BamA of Escherichia coli O78 serotype as a carrier, and contains the B cell epitope of the outer membrane protein OmpA and the B cell epitope of the outer membrane protein OmpC. 2. The chimeric protein according to claim 1, characterized in that the B cell epitope sequence of outer membrane protein OmpA is shown in SEQ ID NO: 1-5; The B cell epitope sequence of outer membrane protein BamA is shown in SEQ ID NO: 6-9; The B cell epitope sequence of outer membrane protein OmpC is shown in SEQ ID NO: 10-12. 3. The chimeric protein according to claim 2, characterized in that the chimeric protein uses the outer membrane protein OmpA of O78 serotype Escherichia coli as a carrier, the B cell epitope of the outer membrane protein BamA and the outer membrane protein OmpC. The B cell epitope is connected to the N-terminus of OmpA or connected to the C-terminus of OmpA through a flexible linker. 4. The chimeric protein according to claim 2, characterized in that the chimeric protein uses the outer membrane protein BamA of Escherichia coli O78 serotype as a carrier, the B cell epitope of the outer membrane protein OmpA and the outer membrane protein OmpC. The B cell epitope is connected to the C-terminus of BamA through a flexible linker. 5. The chimeric protein according to claim 3 or 4, characterized in that the flexible Linker is GGGS. 6. The chimeric protein according to claim 5, characterized in that the chimeric protein comprises or consists of the following amino acid sequence: a), the amino acid sequence shown in SEQ ID NO:13; or b), the amino acid sequence obtained by connecting a tag to the N-terminus and/or C-terminus of a); or A protein with the same function obtained by substituting, deleting and/or adding one or more amino acids to the amino acid sequence of c), a) or b); or, A), the amino acid sequence shown in SEQ ID NO:14; or B), the amino acid sequence obtained by connecting a tag to the N-terminus and/or C-terminus of A); or A protein with the same function obtained by substituting, deleting and/or adding one or more amino acids to the amino acid sequence of C), A) or B). 7. Nucleic acid molecules encoding the chimeric protein of any one of claims 1-6 or biological materials containing the nucleic acid molecules; wherein the biological materials are recombinant DNA, expression cassettes, transposons, plasmid vectors, viruses Vectors, engineered bacteria or transgenic cell lines. 8. An immunogenic composition, characterized in that it contains the chimeric protein according to any one of claims 1-6. 9. Escherichia coli subunit vaccine, characterized in that it contains the immunogenic composition of claim 8 and a pharmaceutically acceptable carrier. 10. Any of the following applications of the chimeric protein according to any one of claims 1-6: (1) Used to prepare E. coli subunit vaccine; (2) Used to prepare reagents or kits for detecting E. coli infection.