CN116751307A - Coli multi-epitope chimeric protein and application thereof - Google Patents
Coli multi-epitope chimeric protein and application thereof Download PDFInfo
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- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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- C12N15/70—Vectors or expression systems specially adapted for E. coli
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Abstract
The invention provides an escherichia coli multi-epitope chimeric protein and application thereof, wherein the chimeric protein takes outer membrane protein OmpA of O78 serotype escherichia coli as a carrier and comprises B cell epitopes (OmpA-Fusion) of BamA and OmpC; alternatively, the chimeric protein comprises B cell epitopes of OmpA and OmpC (BamA-Fusion) using the outer membrane protein BamA of escherichia coli of O78 serotype as a vector. The multi-epitope chimeric sequence can be induced to express in escherichia coli, an expression product has higher antigenicity, and a mouse is immunized by the multi-epitope chimeric protein and Freund's adjuvant together, so that a mouse organism can be stimulated to generate strong immune response. The subunit vaccine provided by the invention has a good immune protection effect on escherichia coli and infection, can be used for preparing subunit vaccines aiming at escherichia coli, and has a good application prospect.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to an escherichia coli multi-epitope chimeric protein and application thereof.
Background
Coli (e.coli) is a conditionally pathogenic bacterium colonizing the intestinal tract of a variety of animals. Under certain conditions, some escherichia coli carrying pathogenic factors can cause a plurality of local tissue and organ infections of healthy animals, such as gastrointestinal tract infection, urinary tract infection, meningitis and the like, and huge pressure and economic loss are often brought to the livestock breeding industry. The main control method aiming at the escherichia coli at present is treatment by using antibiotics and vaccination, but the abuse of the antibiotics causes wide drug resistance of the escherichia coli, so the vaccination is a more ideal approach. However, the development of E.coli vaccines is severely challenged by the numerous E.coli serotypes, recombination of virulence genes, and the like. In addition, many commercial E.coli vaccines have poor clinical immune effects and obvious regional limitations, and the prevention of E.coli-related diseases is still very difficult. Therefore, the development of a safe and efficient novel E.coli vaccine is a recent research hotspot.
The multi-epitope vaccine is a novel subunit vaccine designed based on the amino acid sequence of the target antigen epitope, has the advantages of safety, no toxicity, stability, controllability and the like compared with the traditional vaccine, can directly stimulate an organism to generate specific immune response, accords with the development direction of the future vaccine and is increasingly focused by people. Compared with the traditional vaccine, the vaccine based on epitope design has strong targeting, accurate antibody positioning and definite immune mechanism, but has higher requirements on analysis and design of antigen epitopes, so that proper antigen epitopes are firstly screened out in order to successfully develop the epitope vaccine. An epitope (epitope) includes a T cell epitope and a B cell epitope, which are groups in an antigen molecule that determine the specificity of an antigen, and can specifically bind to a T Cell Receptor (TCR) or a B Cell Receptor (BCR), thereby stimulating an immune response in an organism and forming immunity against pathogenic bacteria. T cell epitopes are recognized by major histocompatibility complex (major histocompatibility complex, MHC) I or II molecules in cells, presented on the cell surface, then recognized by TCRs of CD8+ T cells and CD4+ T cells respectively, and stimulate the body to generate cellular immunity, and are mainly applied to 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, and complexes after the B cell epitopes are combined with the MHC class II molecules are recognized by BCR on the cell surface, and the B cell epitopes belong to soluble protein antigen conformational determinants. B cell epitopes stimulate the body to produce antibodies, which exert immune functions mainly in body fluids. IEDB, discoTope, bepiPred, ABCPred and other online prediction tools are often used for predicting antigen epitopes of proteins by people, and the prediction of B cell epitopes needs to be performed by combining indexes such as physical and chemical properties, structural characteristics, statistical significance measurement and the like of antigen proteins, such as amino acid Flexibility (Flexibility), surface accessibility (Surface accessibility), local hydrophilicity (Local hydrophilicity), antigenicity, saliency index (Protrusion index), a corner (Turn) and Loop (Loop) structure of a secondary structure and the like in a primary sequence, and the predicted result cannot be ensured to be 100% accurate and needs to be further verified in experiments.
The low molecular weight of a single epitope makes the immunogenicity poor, lacks stable stereo conformation and is easy to be degraded. Multiple immunizations can have better immune effects, but can easily lead to immune tolerance of animals. Therefore, it is necessary to design multiple epitopes into a multi-epitope vaccine by connecting different functional regions of the same protein or proteins with different functions in series using genetic engineering techniques, thereby enhancing the immunogenicity and stability of the antigen. Currently, there are mainly 4 common tandem modes for designing multi-epitope vaccines: linear tandem of epitopes, coupling of epitopes to carrier, multi-antigen peptides and multi-lipopeptide multi-epitope vaccine. The linear tandem of the antigen epitope is formed by directly tandem-connecting a plurality of predicted antigen epitopes end to end, and the tandem mode can effectively overcome the defects of weak immunogenicity and easy degradation of a single epitope.
The multi-epitope vaccine has unique superiority, such as high antigenicity, polyvalent, good immune effect, good safety and the like, and has been widely applied to prevention and treatment research of related diseases including bacterial infection, viral diseases, parasitic infection, anti-tumor aspect and the like.
Disclosure of Invention
The invention aims to provide an escherichia coli multi-epitope chimeric protein and application thereof.
The invention designs a multi-epitope chimeric sequence based on the outer membrane protein of the escherichia coli CAU0768 (O78), and utilizes the multi-epitope chimeric protein to construct subunit vaccine.
In order to achieve the object of the invention, in a first aspect, the invention provides an escherichia coli multi-epitope chimeric protein, wherein the chimeric protein takes an outer membrane protein OmpA of escherichia coli with an O78 serotype as a carrier, and comprises a B cell epitope of the outer membrane protein BamA and a B cell epitope of the outer membrane protein ompC (marked as ompA-Fusion); or alternatively, the process may be performed,
the chimeric protein takes outer membrane protein BamA of O78 serotype escherichia coli as a carrier, and comprises B cell epitopes of outer membrane protein OmpA and B cell epitopes of outer membrane protein OmpC (marked as BamA-Fusion).
In the invention, the B cell epitope sequence of O78 serotype escherichia coli outer membrane protein OmpA is shown as SEQ ID NO. 1-5;
the B cell epitope sequence of the outer membrane protein BamA is shown in SEQ ID NO. 6-9;
the B cell epitope sequence of the outer membrane protein OmpC is shown in SEQ ID NO. 10-12.
Furthermore, the chimeric protein takes the outer membrane protein OmpA of the O78 serotype escherichia coli 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 at the N end of the OmpA or connected at the C end of the OmpA through a flexible Linker (such as GGGS).
Furthermore, the chimeric protein takes the outer membrane protein BamA of O78 serotype escherichia coli 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 at the C end of the BamA through a flexible Linker (such as GGGS).
In a specific embodiment of the invention, the chimeric protein OmpA-Fusion comprises or consists of the amino acid sequence as follows:
a) An amino acid sequence shown in SEQ ID NO. 13; or (b)
b) An amino acid sequence obtained by ligating a tag to the N-terminal and/or C-terminal of a); or (b)
c) Proteins with the same function obtained by substituting, deleting and/or adding one or more amino acids to the amino acid sequence of the a) or the b).
In another embodiment of the invention, the chimeric protein BamA-Fusion comprises or consists of the amino acid sequence:
a) An amino acid sequence shown in SEQ ID NO. 14; or (b)
B) An amino acid sequence obtained by connecting a tag to the N-terminal and/or the C-terminal of A); or (b)
C) Proteins with the same function obtained by substituting, deleting and/or adding one or more amino acids to the amino acid sequence of the A) or the B).
In a second aspect, the invention provides a nucleic acid molecule encoding the chimeric protein or a biological material comprising the nucleic acid molecule; wherein the biological material is recombinant DNA, an expression cassette, a transposon, a plasmid vector, a viral vector, engineering bacteria or a transgenic cell line.
In a third aspect, the invention provides an immunogenic composition comprising the chimeric protein.
In a fourth aspect, the invention provides an E.coli subunit vaccine comprising the immunogenic composition and a pharmaceutically acceptable carrier. Optionally comprising an adjuvant.
In a fifth aspect, the invention provides any one of the following uses of the chimeric protein:
(1) Is used for preparing the subunit vaccine of the escherichia coli;
(2) Preparing a reagent or a kit for detecting the escherichia coli infection;
(3) Is used for diagnosing the escherichia coli infection.
By means of the technical scheme, the invention has at least the following advantages and beneficial effects:
according to the invention, the antigen epitope is connected in series with the C end or the N end of the carrier protein which is the outer membrane protein OmpA or BamA through the flexible connecting peptide Linker, the antigenicity of the carrier protein can be enhanced by embedding a plurality of B cell epitopes, and a plurality of antigen epitopes in the chimeric protein can respectively form a correct space structure, so that the respective biological activity is better exerted, and the mutual interference between the proteins in a space conformation is prevented.
The multi-epitope chimeric sequence can be induced to express in escherichia coli, and an expression product has higher antigenicity. The multi-epitope chimeric protein provided by the invention and Freund's adjuvant are used for jointly immunizing mice, so that the mice can be stimulated to generate strong immune response. High-level antibody titer is generated in serum after secondary immunization, and the protective rate of the multi-epitope chimeric protein immune group reaches 100% under the attack of the lowest lethal dose concentration of escherichia coli CAU0768 (O78).
The subunit vaccine provided by the invention has a good immune protection effect on escherichia coli and infection, can be used for preparing subunit vaccines aiming at escherichia coli, and has a good application prospect.
Drawings
FIG. 1 is a SDS-PAGE diagram of a multi-epitope chimeric protein purified according to a preferred embodiment of the present invention.
FIG. 2 shows the serum antibody titers of the multi-epitope chimeric proteins of the preferred embodiment of the present invention. In the figure, the differences between the different treatment groups are statistically significant, and P <0.0001.ns represents a statistical control reference.
FIG. 3 shows the immunization of mouse serum antibody subtypes with the multi-epitope chimeric proteins according to the preferred embodiment of the present invention.
FIG. 4 shows serum cross-reactions of a multi-epitope chimeric protein immunized mouse in accordance with a preferred embodiment of the invention.
FIG. 5 shows colony counts of the toxin-attacking organs of the multi-epitope chimeric protein immunized mice in the preferred embodiment of the invention. In the figures, the differences between the different treatment groups are statistically significant, P <0.05, P <0.01, and P <0.001.ns represents a statistical control reference.
FIG. 6 shows the toxicity protection rate of the multi-epitope chimeric protein immunized mice according to the preferred embodiment of the invention. And (3) injection: the curves for the BamA-Fusion +Freund's adjuvant and non-immunized control groups were coincident.
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the technical means used in the examples are conventional means well known to those skilled in the art, and all raw materials used are commercially available. EXAMPLE 1 construction of a multiple epitope chimeric sequence
1. B cell epitope prediction
The amino acid sequences of the E.coli O78 3 outer membrane proteins OmpA, bamA and OmpC were analyzed for B cell linear epitopes by means of Bepipred, BCPred and COBEpro on-line prediction websites, and appropriate B cell epitopes were selected in combination with 3 prediction methods and immunoinformatics parameters, and overlapping portions were obtained by using different prediction methods to increase the prediction reliability (SEQ ID NOS: 1 to 12). The antigenic regions of OmpA and BamA are taken as skeletons respectively, and B cell epitopes of the other two protein candidates are connected at the C end through linker peptide, so that multi-epitope Fusion proteins containing a plurality of B cell epitopes are constructed and named OmpA-Fusion and BamA-Fusion.
2. Synthesis of Multi-epitope chimeric genes
The artificial chemical synthesis is carried out by the biological engineering (Shanghai) limited company by adopting a total gene synthesis method, and the artificial chemical synthesis is cloned into a PUC57 vector, and the PUC57-OmpA-Fusion and the PUC57-BamA-Fusion are named.
3. Secondary structural analysis of polyepitope chimeric proteins
The physicochemical properties of the amino acid sequence of the multi-epitope chimeric protein were predicted using ProtParam in the ExPASy website.
4. Prediction of tertiary structure of multi-epitope chimeric proteins
The three-level structure prediction is carried out on the multi-epitope chimeric protein by utilizing molecular biology software I-TASSER, and the result shows that the carrier can maintain a certain space structure after a plurality of epitopes are embedded, and the constructed multi-epitope chimeric protein has a plurality of epitopes which are externally displayed on the surface of the molecule and meets the design requirement of multi-epitope chimeric.
5. Multi-epitope chimeric protein antigenicity analysis
The molecular biology software VaxiJen is utilized to carry out antigenicity analysis on the multi-epitope chimeric protein, the OmpA-Fusion score is 0.9315, the BamA-Fusion score is 0.8394, and the antigenicity requirement of the multi-epitope chimeric protein is met.
The B cell epitope sequence of the O78 serotype escherichia coli outer membrane protein OmpA is shown in SEQ ID NO. 1-5;
the B cell epitope sequence of the outer membrane protein BamA is shown in SEQ ID NO. 6-9;
the B cell epitope sequence of the outer membrane protein OmpC is shown in SEQ ID NO. 10-12.
EXAMPLE 2 expression of multiple epitope genes in E.coli
1. Construction of expression vector for multiple epitope chimeric sequences
The plasmids PUC57-OmpA-Fusion and PUC57-BamA-Fusion were digested with Nco I and Xho I, and after agarose gel electrophoresis, the target fragments OmpA-Fusion and BamA-Fusion were recovered, and ligated into the plasmid pet-28a (+) digested with Nco I and Xho I at 25℃to obtain the plasmid pet-28a (+) -OmpA-Fusion/BamA-Fusion.
2. Transformation of E.coli DH 5. Alpha. With expression vector
10ul of the system, which was well-connected at 25℃was added to 50ul of competent cells DH 5. Alpha. And gently mixed and placed in ice for 30min. After 45s of water bath at 42 ℃, the mixture is quickly transferred into ice and placed for 2min. 450ul of LB culture medium is added, 200r/min and the culture is carried out for 60min at 37 ℃. 200ul of 100ul of the transformation solution was spread on LB agar plate medium containing Kan (final concentration 50. Mu.g/ml), and cultured overnight at 37 ℃.
3. Screening of E.coli Positive clones
A portion of the colonies were picked from the single colonies with a gun head, added to 30ul of sterile water, and mixed well. 3ul was used as PCR template and PCR identification was performed using universal primers. If the PCR result shows the specific band, 3-5 positive bacteria solutions are selected, 300ul of LB liquid culture medium containing Kan is transferred from the corresponding 1ml of bacteria solution, and the bacteria solution is cultured to the glycerol pipe of-20 ℃ in the logarithmic phase for preservation. The remaining bacterial liquid is sent to Shanghai Biotechnology company for sequencing.
4. Expression vector transformed escherichia coli BL21
The correct bacterial liquid for sequencing is transferred into 5ml LB culture medium containing Kan, and is cultured for 12 hours at 37 ℃ at 250 r/min. Plasmid extraction was performed using the root plasmid extraction kit (DP 103), and the recombinant plasmid was transformed into the expression strain BL21 in the same manner.
5. Induction expression of E.coli positive clones
Taking overnight cultured positive bacteria-retaining bacteria solution, inoculating 1% into LB culture medium containing Kan (final concentration 50 μg/ml), culturing for more than 2 hr to mid-log (OD) 600 =0.6). IPTG was added to the induction tube to a concentration of 1mmol/L,250r/min, and the culture was performed at 37℃for about 6 hours with shaking to obtain a sample.
6. SDS-PAGE analysis of expressed proteins of interest
After the induction, 1mL of the bacterial liquid was collected by centrifugation at 8000rpm at 4℃for 2min, the supernatant was discarded, the bacterial liquid was resuspended in 100ul of PBS, 100ul of 2 XSDS-PAGE loading buffer, boiled at 100℃for 10min, centrifuged at 12000r/min for 5min, and the supernatant was detected by SDS-PAGE. As shown in FIG. 1, the molecular weights of OmpA-Fusion (SEQ ID NO: 13) and BamA-Fusion (SEQ ID NO: 14) were both about 50kDa, which corresponds to the theoretical value.
Example 3 mouse immunization experiments with Multi-epitope subunit vaccine
1. Mouse immunization program
30 female BALB/c mice of 6-8 weeks old were divided into three groups of PBS+adjuvant group, ompA-Fusion-adjuvant group, bamA-fusion+adjuvant group, 10 each. Three total immunizations were performed, one and two-and three-immune intervals 21d, and two and three-immune intervals 14d, 25 μg/each immunization subunit vaccine. Mixing Freund's complete adjuvant, and subcutaneously injecting; the second and third phases are mixed with Freund's incomplete adjuvant and injected intraperitoneally. Serum ELISA antibody analysis was performed by blood collection from the tail vein before immunization and 5d after each immunization, respectively.
2. Immune mouse serum ELISA antibody titer detection
The level of specific IgG antibodies in the serum of immunized mice was determined by indirect ELISA. The specific method comprises the following steps:
coating: the outer membrane proteins were dissolved in the protein coating solution to a final concentration of 2ug/ml,100 ul/Kong Jiazhi 96 well plates, washed 4 times at 4℃overnight, 5min each, and patted dry.
Closing: blocking solution 200 ul/Kong Jiazhi 96-well plate was blocked for 2h at 37℃and PBST was washed 4 times for 5min each time and then patted dry.
Adding a serum sample to be tested: primary serum is diluted 1:100, secondary serum and tertiary serum are diluted 3 times by gradient with 1:1000 as initial dilution concentration, 100 ul/Kong Jiazhi 96-well plates are incubated for 1.5h at 37 ℃, PBST is washed for 4 times each for 5min, and the primary serum is dried by beating.
Adding a secondary antibody: horseradish enzyme-labeled goat anti-mouse igG (igG specific secondary antibody) was diluted 1:5000 times, incubated in 100 ul/Kong Jiazhi 96-well plates at 37℃for 30min, washed with PBST for 4 times each for 5min, and then patted dry.
Color development: the color reagent TMB is added according to 100 ul/hole, and color development is carried out for 15-30min at 37 ℃. And (3) terminating: the reaction was stopped by adding 50 ul/well of stop solution.
Reading: the OD value of each well is measured at 450nm with 630nm as a reference wavelength, and the limit of the ratio (P/N) of the OD value of each well to the OD value of the negative control well is larger than 2.1 is used as a critical point for judging and determining the titer. As shown in FIG. 2, co-immunization of both proteins with Freund's adjuvant stimulated a strong immune response in mice.
3. Immune mouse serum ELISA antibody subtype detection
The procedure was followed except that the secondary antibodies used were horseradish-enzyme labeled goat anti-mice igG1, igG a, igG2b, igG3, igM and igA. As a result, as shown in FIG. 3, antibodies were detected in serum of immunized mice as mainly igG, igG, 2a, igG, 2b and igM, indicating both humoral immune response (Th 2) and cell-mediated immune response (Th 1).
4. ELISA cross-reaction detection of immune mouse serum
Fixing: placing 150 ul/Kong Jiazhi ELISA plate of the fixing solution at 37 ℃ for 1h; PBST 150 ul/well wash plate 4 times, each time5min, and drying. Cell antigen suspension resuspended in PBS was added (10 8 CFU/mL) 100 ul/well, placed at 37 ℃ until air-dried; 100ul of PBST wash was washed 4 times, unbound antigen was washed off, 5min each time, and the swatches were dried. The remaining steps are identical to the antibody titer assays except that the serum sample to be tested is diluted at a concentration of 1:27000. The results are shown in FIG. 4, where antisera have some cross-protection against E.coli of different serotypes.
5. Escherichia coli challenge visceral organ load
Coli CAU0768 (O78) was activated overnight, transferred at 1% and cultured to log phase. Mice in the immunized and blank groups were intraperitoneally injected with the lowest lethal dose of log-phase E.coli, respectively, at 14d after the three-phase immunization. Mice were euthanized 6h after challenge and colony counts were performed by heart, liver, spleen, lung, kidney. As shown in fig. 5, the visceral organ load of the multi-epitope chimeric protein immune group was significantly lower than that of the non-immunized mice at the challenge of the lowest lethal dose.
6. Escherichia coli toxicity-counteracting protecting rate
Coli CAU0768 (O78) was activated overnight, transferred at 1% and cultured to log phase. Mice in the immunized and blank groups were intraperitoneally injected with the lowest lethal dose of log-phase E.coli, respectively, at 14d after the three-phase immunization. Mice were monitored daily for mortality and recorded, and mice mortality was counted for a total of 7d. As shown in fig. 6, at the lowest lethal dose challenge, the non-immunized mice died continuously for 24h, with an adjuvant group survival rate of 50% and a multi-epitope chimeric protein immune group survival rate of 100%.
The experimental result shows that the novel escherichia coli outer membrane protein multi-epitope chimeric sequence provided by the invention can be subjected to prokaryotic induction expression in escherichia coli, and an expression product has immunogenicity and can be identified by antibodies in immune mouse serum. The carrier can maintain the original space structure of the carrier, has less mutual influence of the secondary structures among the embedded epitopes, has higher antigenicity, simultaneously has a plurality of epitopes which are externally displayed on the surface of the molecule, meets the design requirement of the multiple epitopes, and is hopeful to become a better immunogen. The mouse immunity experiment shows that the multi-epitope chimeric protein is used as subunit vaccine, which can stimulate the mouse organism to generate specific humoral immunity and generate specific cellular immunity, and has good protection rate for colibacillus infection. In vitro experiments show that the multi-epitope chimeric protein has certain cross protection reaction with other escherichia coli and shigella.
The novel escherichia coli multi-epitope chimeric protein designed by the invention has higher antigenicity, can be used for preparing escherichia coli and shigella subunit vaccines, and has good development prospect.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (10)
1. The escherichia coli multi-epitope chimeric protein is characterized in that the chimeric protein takes outer membrane protein OmpA of O78 serotype escherichia coli as a carrier and comprises B cell epitopes of outer membrane protein BamA and B cell epitopes of outer membrane protein OmpC; or alternatively, the process may be performed,
the chimeric protein takes outer membrane protein BamA of O78 serotype escherichia coli as a carrier, and comprises B cell epitopes of outer membrane protein OmpA and B cell epitopes of outer membrane protein OmpC.
2. Chimeric protein according to claim 1, characterized in that the B-cell epitope sequence of the outer membrane protein OmpA is shown in SEQ ID No. 1-5;
the B cell epitope sequence of the outer membrane protein BamA is shown in SEQ ID NO. 6-9;
the B cell epitope sequence of the outer membrane protein OmpC is shown in SEQ ID NO. 10-12.
3. Chimeric protein according to claim 2, characterized in that it uses the outer membrane protein OmpA of escherichia coli of O78 serotype as vector, the B cell epitope of the outer membrane protein BamA and the B cell epitope of the outer membrane protein OmpC are linked at the N-terminus of OmpA or at the C-terminus of OmpA by flexible Linker.
4. The chimeric protein according to claim 2, wherein the chimeric protein uses the outer membrane protein BamA of escherichia coli of O78 serotype as a carrier, and the B cell epitope of outer membrane protein OmpA and the B cell epitope of outer membrane protein OmpC are linked at the C-terminus of BamA by a flexible Linker.
5. The chimeric protein according to claim 3 or 4, wherein the flexible Linker is GGGS.
6. The chimeric protein according to claim 5, characterized in that it comprises or consists of the amino acid sequence:
a) An amino acid sequence shown in SEQ ID NO. 13; or (b)
b) An amino acid sequence obtained by ligating a tag to the N-terminal and/or C-terminal of a); or (b)
c) A protein with the same function, which is obtained by substituting, deleting and/or adding one or more amino acids in the amino acid sequence of the a) or the b); or alternatively, the process may be performed,
a) An amino acid sequence shown in SEQ ID NO. 14; or (b)
B) An amino acid sequence obtained by connecting a tag to the N-terminal and/or the C-terminal of A); or (b)
C) Proteins with the same function obtained by substituting, deleting and/or adding one or more amino acids to the amino acid sequence of the A) or the B).
7. A nucleic acid molecule encoding the chimeric protein of any one of claims 1-6 or a biological material comprising said nucleic acid molecule; wherein the biological material is recombinant DNA, an expression cassette, a transposon, a plasmid vector, a viral vector, engineering bacteria or a transgenic cell line.
8. An immunogenic composition comprising the chimeric protein of any one of claims 1-6.
9. An escherichia coli subunit vaccine comprising the immunogenic composition of claim 8 and a pharmaceutically acceptable carrier.
10. Use of any of the chimeric proteins of claims 1-6 for:
(1) Is used for preparing the subunit vaccine of the escherichia coli;
(2) The method is used for preparing a reagent or a kit for detecting the escherichia coli infection.
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