A vaccine for the prevention of Escherichia coli infection and its application

Abstract

This invention relates to a fusion protein, immunogenic composition, and recombinant vaccine for the prevention of Escherichia coli infection. The invention provides a novel target, PstS, for Escherichia coli vaccine design. Starting from the tertiary structure of PstS and YidR proteins, and combining immunoepitaxes, stable domains of PstS and YidR proteins and their variants are ultimately screened, leading to the construction of a fusion protein. Both the PstS monoantigen protein molecule and the PstS-YidR fusion protein molecule of this invention exhibit good immunogenicity, can reduce tissue lesions caused by Escherichia coli infection, and demonstrate high efficacy in preventing Escherichia coli infection, showing broad application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, particularly the field of immunotherapeutic drugs, and specifically relates to a humoral immune vaccine for the prevention of Escherichia coli infection and its application. Background Technology

[0002] Escherichia coli ( Escherichia coli Escherichia coli (E. coli), also known as pathogenic bacteria, is one of the most common commensal bacteria in the intestines of humans and animals. It is also an important pathogen that causes a variety of infectious diseases. Pathogenic E. coli can cause serious intestinal and extraintestinal infections, posing a significant threat to public health.

[0003] Pathogenic Escherichia coli can infect various tissues and cause different types of infections and diseases, including: intestinal infections, urinary tract infections, sepsis, bacteremia, meningitis, peritonitis, pneumonia, mastitis, and hemolytic uremic syndrome. Clinically, Escherichia coli infections mainly rely on antibiotic treatment; however, with the increasing antibiotic resistance of bacteria, the control of these pathogenic Escherichia coli strains faces serious challenges.

[0004] Currently, there are no commercially available preventative vaccines against Escherichia coli infection, and researchers are actively developing related vaccines. For example, existing technology patent CN106535927B (publication date 2017-03-22) discloses a polysaccharide O25B for Escherichia coli, and patent CN108430500B (publication date 2018-08-21) discloses a polysaccharide conjugate for preventing extraintestinal pathogenic Escherichia coli infection. These polysaccharide vaccines aim to develop a vaccine that targets a single type of pathogenic Escherichia coli. However, there are already more than 180 serotypes (O serotype) of pathogenic Escherichia coli, making it difficult to iterate polysaccharide vaccines, resulting in low levels of cross-immunity protection, and failing to meet the current market demand for Escherichia coli vaccines.

[0005] Phosphate-binding protein PstS is part of the phosphate-specific transport (Pst) system, located in the bacterial periplasmic space, and has a high affinity for phosphate. Previous studies have demonstrated that PstS can serve as a biosensor for phosphate contamination. For example, patent CN114965403A (publication date 2022-08-30) discloses a method for constructing a phosphate biosensor using the Pseudomonas aeruginosa PstS protein. Furthermore, in some pathogenic bacteria, PstS expression is associated with the production of virulence factors and participates in biofilm formation and host cell adhesion; however, no studies have reported that PstS can be used as a protective antigen for Escherichia coli in vaccine development.

[0006] In summary, developing a vaccine that provides broad-spectrum protection against multiple pathogenic Escherichia coli strains, is safe and effective, and easy to produce, has significant scientific and practical value. Such a vaccine could not only prevent various human infections but also potentially be applied to animal disease prevention, such as preventing mastitis in dairy cows, thus bringing significant benefits from both public health and economic perspectives. Therefore, this invention addresses the aforementioned issues by proposing a novel fusion protein design scheme to develop a highly effective, broad-spectrum vaccine for the prevention of Escherichia coli infections, potentially simultaneously combating infections from multiple pathogenic strains such as EHEC, MPEC, and UPEC. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide a novel fusion protein, immunogenic composition, recombinant vaccine, molecular architecture design, and applications for the prevention of Escherichia coli infection. This invention provides a novel target, PstS, for E. coli vaccine design. Starting from the highly conserved tertiary structures of the protein molecules of PstS and YidR, highly conserved targets in E. coli, and combining immunoepitaphs, a fusion protein is designed. Through creative screening, stable domains of PstS and YidR proteins and their variants are selected to construct the fusion protein, which is then further enhanced with Fc domains, STABILON, and other elements. This invention reveals that the novel fusion protein molecule can attenuate tissue lesions caused by E. coli infection, exhibits good immunogenicity, and provides effective prevention and immune protection, demonstrating high efficacy in preventing E. coli infection and broad application prospects. It can be used to prevent various infectious diseases caused by E. coli, such as urinary tract infections, intestinal infections, mastitis, and sepsis. Furthermore, the molecular design strategy provided by this invention can also serve as a reference for the development of vaccines against other bacterial pathogens. The present invention also provides corresponding recombinant nucleic acids, gene expression cassettes, vectors, host cells, pharmaceutical compositions, vaccines, uses, etc.

[0008] One aspect of the present invention provides a fusion protein characterized in that it comprises a phosphate-binding protein PstS antigen or a fragment thereof.

[0009] Furthermore, the fusion protein also includes the YidR antigen (DUF3748 domain-containing protein) or a fragment thereof, wherein the YidR antigen or the fragment thereof includes the full-length YidR antigen protein, the YidR antigen selection peptide, or a mutated YidR antigen selection peptide.

[0010] Furthermore, the antigen or fragment thereof is derived from bacteria.

[0011] Furthermore, the bacteria are Gram-negative.

[0012] Furthermore, the Gram-negative bacteria are Enterobacteriaceae.

[0013] Furthermore, the Enterobacteriaceae bacteria mentioned are Escherichia coli.

[0014] Furthermore, the fusion protein comprises, from the N-terminus to the C-terminus, the PstS antigen or a fragment thereof, the YidR antigen or a fragment thereof, wherein the YidR antigen or a fragment thereof is any one of the following: the full-length YidR antigen protein, the YidR antigen selection polypeptide, or a mutated YidR antigen selection polypeptide.

[0015] Further, the amino acid sequence of the PstS antigen or its fragment is shown in SEQ ID NO.1, the amino acid sequence of the full-length YidR antigen protein is shown in SEQ ID NO.11, the amino acid sequence of the YidR antigen selection peptide is shown in SEQ ID NO.2, and the amino acid sequence of the mutated YidR antigen selection peptide is shown in SEQ ID NO.3.

[0016] Furthermore, the N-terminus of the fusion protein further includes a signal peptide and / or the Fc domain of the human immunoglobulin heavy chain constant region γ1 protein (IGHG1); or, the C-terminus of the fusion protein further includes the C-terminal polypeptide fragment STABILON of the human S5a / PSMD4 proteasome subunit.

[0017] Furthermore, the signal peptide is derived from human azurocidin protein, the amino acid sequence of which is shown in SEQ ID NO: 4; the amino acid sequence of the Fc domain of IGHG1 is shown in SEQ ID NO: 5; and the amino acid sequence of STABILON is shown in SEQ ID NO. 6.

[0018] Furthermore, the different elements of the fusion protein may optionally be linked by a GGS linker sequence, a spacer sequence, and / or a GS linker sequence.

[0019] Further, the amino acid sequence of the spacer sequence is shown in SEQ ID NO: 7, the amino acid sequence of the GGS linker sequence is shown in SEQ ID NO: 8, and the amino acid sequence of the GS linker sequence is shown in SEQ ID NO: 9.

[0020] Furthermore, it includes any one of the following four fusion proteins:

[0021] (1) Fusion protein A: From N-terminus to C-terminus, it contains: signal peptide, Fc domain, GGS linker, PstS antigen or fragment thereof, GS linker, STABILON;

[0022] (2) Fusion protein B: From the N-terminus to the C-terminus, it contains the following components in sequence: signal peptide, Fc domain, GGS linker, YidR antigen-selecting peptide, spacer sequence, YidR antigen-selecting peptide, GS linker, and STABILON.

[0023] (3) Fusion protein C: From N-terminus to C-terminus, it includes: signal peptide, Fc domain, GGS linker, PstS antigen or fragment thereof, spacer sequence, full-length YidR antigen protein, GS linker, STABILON;

[0024] (4) Fusion protein D: From N-terminus to C-terminus, it contains: signal peptide, Fc domain, GGS linker, PstS antigen or fragment thereof, spacer sequence, mutated YidR antigen-selective peptide, GS linker, STABILON;

[0025] Further, the amino acid sequence of the PstS antigen or its fragment is shown in SEQ ID NO.1, the amino acid sequence of the full-length YidR antigen protein is shown in SEQ ID NO.11, the amino acid sequence of the YidR antigen selection peptide is shown in SEQ ID NO.2, the amino acid sequence of the mutated YidR antigen selection peptide is shown in SEQ ID NO.3, the amino acid sequence of the signal peptide is shown in SEQ ID NO:4, the amino acid sequence of the Fc domain is shown in SEQ ID NO:5, the amino acid sequence of STABILON is shown in SEQ ID NO.6, the amino acid sequence of the spacer sequence is shown in SEQ ID NO:7, the amino acid sequence of the GGS linker sequence is shown in SEQ ID NO:8, and the amino acid sequence of the GS linker sequence is shown in SEQ ID NO:9.

[0026] Furthermore, the amino acid sequence of the fusion protein is shown in SEQ ID NO.10.

[0027] Another aspect of the present invention provides a recombinant nucleic acid molecule, characterized in that it comprises a nucleic acid encoding a fusion protein according to any one of the present invention.

[0028] Another aspect of the present invention provides a recombinant gene expression cassette, characterized in that it comprises the recombinant nucleic acid molecule described in the present invention.

[0029] Furthermore, the recombinant gene expression cassette also includes one or more of a promoter, a terminator, and a regulatory sequence.

[0030] Another aspect of the present invention provides a recombinant vector, characterized in that it comprises the recombinant nucleic acid molecule described in the present invention or the recombinant gene expression cassette described in the present invention.

[0031] Furthermore, the recombinant vector comprises a prokaryotic vector or a eukaryotic vector.

[0032] Furthermore, the prokaryotic vector includes, but is not limited to, Escherichia coli vectors.

[0033] Furthermore, the Escherichia coli vector includes, but is not limited to, pET vector, pGEX vector, pMAL vector, pBAD vector, pUC vector, and pBR vector.

[0034] Furthermore, the eukaryotic vector includes, but is not limited to, yeast expression vectors, insect expression vectors, and mammalian cell expression vectors.

[0035] Furthermore, the yeast expression vector includes, but is not limited to, pPICZ vector, pGAPZ vector, pYES vector, pGAP vector, pAO815 vector, and pPIC9 vector.

[0036] Another aspect of the present invention provides a recombinant host cell, characterized in that it comprises the recombinant nucleic acid molecule described in the present invention, or the recombinant gene expression cassette described in the present invention, or the recombinant vector described in the present invention.

[0037] Furthermore, the recombinant host cell comprises a eukaryotic cell or a prokaryotic cell.

[0038] Furthermore, the eukaryotic cells include mammalian cells, insect cells, and yeast cells.

[0039] Furthermore, the yeast cells include, but are not limited to, Saccharomyces cerevisiae, Pichia pastoris, and Hansenula polymorpha.

[0040] Furthermore, the prokaryotic cells include, but are not limited to, Escherichia coli cells, Bacillus subtilis cells, and Pseudomonas cells.

[0041] Furthermore, the *E. coli* cells include, but are not limited to, BL21(DE3), DH5α, TOP10, and Rosetta.

[0042] Another aspect of the present invention provides an immunogenic composition or pharmaceutical composition, characterized in that it comprises one or more fusion proteins according to any one of the present invention, and / or one or more recombinant nucleic acid molecules according to the present invention, and / or one or more recombinant gene expression cassettes according to the present invention, and / or one or more recombinant vectors according to the present invention, and / or one or more recombinant host cells according to the present invention.

[0043] Furthermore, the immunogenic composition or pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0044] Furthermore, the immunogenic composition or pharmaceutical composition may also contain other drugs for the prevention of diseases caused by Escherichia coli.

[0045] Furthermore, the other medications used to prevent diseases caused by Escherichia coli include antibiotics.

[0046] Another aspect of the present invention provides a recombinant vaccine, characterized in that it comprises one or more fusion proteins according to any one of the present invention, and / or one or more recombinant nucleic acid molecules according to the present invention, and / or one or more recombinant gene expression cassettes according to the present invention, and / or one or more recombinant vectors according to the present invention, and / or one or more recombinant host cells according to the present invention, and / or one or more immunogenic compositions or pharmaceutical compositions according to the present invention.

[0047] Furthermore, the recombinant vaccine is a nucleic acid vaccine or a subunit vaccine.

[0048] Furthermore, the recombinant vaccine is a nucleic acid vaccine.

[0049] Another aspect of the present invention provides the use of one or more fusion proteins according to any one of the present invention, and / or one or more recombinant nucleic acid molecules according to the present invention, and / or one or more recombinant gene expression cassettes according to the present invention, and / or one or more recombinant vectors according to the present invention, and / or one or more recombinant host cells according to the present invention, and / or one or more immunogenic compositions or pharmaceutical compositions according to the present invention, and / or one or more recombinant vaccines according to the present invention in the preparation of medicaments for the prevention of diseases and / or secondary infections caused by Escherichia coli.

[0050] Another aspect of the present invention provides a method for preventing diseases and / or secondary infections caused by Escherichia coli, characterized in that it includes administering to a subject one or more fusion proteins of any one of the present invention, and / or one or more recombinant nucleic acid molecules of the present invention, and / or one or more recombinant gene expression cassettes of the present invention, and / or one or more recombinant vectors of the present invention, and / or one or more recombinant host cells of the present invention, and / or one or more immunogenic compositions or pharmaceutical compositions of the present invention, and / or one or more recombinant vaccines of the present invention.

[0051] Furthermore, the diseases and / or secondary infections caused by the Escherichia coli include intestinal infections, urinary tract infections, sepsis, bacteremia, meningitis, peritonitis, pneumonia, mastitis, and hemolytic uremic syndrome.

[0052] Furthermore, the intestinal infection includes enteritis and / or diarrhea.

[0053] Furthermore, the urinary tract infection includes cystitis and / or pyelonephritis.

[0054] The fusion protein, immunogenic composition, recombinant vaccine, etc. of the present invention have the following beneficial technical effects:

[0055] 1. This invention discovers that novel fusion molecules possess excellent immunogenicity and can provide immune protection, making them suitable for the development of nucleic acid vaccines or subunit vaccines. The immunogenic composition of this invention can provide effective immune protection against Escherichia coli infection.

[0056] 2. This invention can serve as a universal antigenic framework for nucleic acid vaccines or subunit vaccines, exhibiting strong immunogenicity. Compared to traditional vaccines, it is not limited by serotype and can induce broad-spectrum immune protection.

[0057] 3. This invention employs a reverse vaccinology strategy to screen potential Escherichia coli antigens, ultimately identifying multiple candidate antigens, including PstS and YidR. Based on immunoepitope prediction analysis, PstS and YidR were evaluated and determined to be the optimal candidate antigens. Furthermore, to improve the expression of candidate antigens and enhance their immunogenicity, N-terminal truncation or amino acid mutations at specific sites were performed on known PstS and YidR antigens.

[0058] 4. This invention starts from the tertiary structure of PstS and YidR proteins and combines them with immunoepitaxes to design a fusion protein that can be stably expressed. Through creative screening, stable domains of PstS and YidR proteins and their variants were finally selected to construct the fusion protein, and the selected polypeptide fragment has a complete and stable 3D structure.

[0059] 5. Example 6 of the present invention shows that the fusion molecule based on the present invention can effectively promote the high abundance expression and secretion of bacterial proteins in eukaryotic cells, and the expressed protein structure is correct and stable, which is conducive to the presentation of immune epitopes, thereby further improving the immune protection effect and providing a new technology for the design of immunotherapeutic drugs for Escherichia coli multivalent vaccines.

[0060] 6. The mouse gavage infection model experiment in Example 7 of this invention showed that, compared with the PBS control group, the bacterial load in the ileum and colon tissues of mice vaccinated with the vaccine of this invention was significantly reduced. Statistical analysis showed that this reduction was statistically significant (P<0.001). This result demonstrates that the vaccine based on this invention can effectively reduce Escherichia coli colonization in the intestine and provide effective local immune protection.

[0061] 7. Example 7 of this invention uses tissue sections and HE staining for in-depth analysis, and the results further confirm the protective effect of the vaccine of this invention. Specifically, the results are as follows: a) In colon tissue, the intestinal villi structure of mice in the normal control group (uninfected and untreated) was intact, with no obvious lesions or inflammatory infiltration. This provides a benchmark for assessing pathological changes in other groups. b) The intestines of mice in the PBS control group (i.e., those challenged by bacteria but not vaccinated) showed severe pathological changes, with a large number of inflammatory cells infiltrating and accumulating in the submucosa. This reflects the significant damage to intestinal tissue caused by Escherichia coli infection. c) Although the mice vaccinated with the vaccine of this invention showed mild pathological changes (minor dissolution of intestinal villi and inflammatory cell infiltration and accumulation in the mucosa), the degree of lesions was significantly milder compared to the PBS control group. This indicates that the vaccine of this invention can significantly alleviate tissue lesions caused by Escherichia coli infection.

[0062] 8. Based on the combined bacterial load and histopathological results of Example 7 of this invention, the vaccine of this invention exhibits a dual protective effect: on the one hand, it significantly reduces the number of bacteria in the intestine, and on the other hand, it effectively alleviates tissue lesions caused by infection. This comprehensive protective effect lays a solid foundation for the potential clinical application of the vaccine of this invention.

[0063] 9. The fusion protein vaccine provided by this invention exhibits excellent immunogenicity and protective efficacy, showing great potential in preventing Escherichia coli infection. This novel vaccine design strategy provides a reference for the development of vaccines against other bacterial pathogens. Attached Figure Description

[0064] Figures 1A-1B This is a schematic diagram of the 3D structure of the two PstS fusion proteins of the present invention; wherein, Figure 1A This is a schematic diagram of the 3D structure of the PstS-YidR full-length protein fusion protein. Figure 1BA schematic diagram of the 3D structure of the fusion protein containing the PstS-YidR-tagged peptide.

[0065] Figure 2 This is a schematic diagram of the molecular structure of vaccines A, B, C, and D used in the comparative experiments of this invention.

[0066] Figure 3 This is a schematic diagram of a circular plasmid containing the molecular structure of the fusion protein of the present invention.

[0067] Figure 4A -D represents the quality control peak diagram and purity test results of the nucleic acid vaccine used in the comparative experiment in this invention embodiment; wherein... Figure 4A The image shows the quality control peaks and purity test results for vaccine A. Figure 4B The results show the quality control peaks and purity test results for vaccine B. Figure 4C The results show the quality control peaks and purity test results for vaccine C. Figure 4D This is a peak diagram of the quality control of vaccine D and the results of purity testing.

[0068] Figures 5A-5C The results of in vitro expression analysis (WB) of vaccines A, B, C, and D transfected into HEK293 cells according to this invention are shown below. Figure 5A The results of Western blot analysis of in vitro expression of vaccine A in HEK293 cells. Figure 5B The results of Western blot analysis of vaccine B expression in HEK293 cells transfected in vitro. Figure 5C The results of Western blot analysis of the in vitro expression of vaccines C and D in HEK293 cells.

[0069] Figure 6 This is a schematic diagram of the immunization, challenge, and sampling process for the mouse gavage infection experiment of this invention.

[0070] Figures 7A-7B These are the bacterial load test results of the ileum and colon tissues of mice in each experimental group after immunization and challenge according to this invention; among them, Figure 7A This is a comparative analysis of bacterial load (number of colonies on agar plates) in the ileum tissue of mice after immunization and challenge. Figure 7B This is a comparative analysis of bacterial load (number of colonies on a plate) in the colon tissue of mice after immunization and challenge.

[0071] Figure 8 The results of staining of pathological tissue sections of colon tissue from mice after immunization and challenge. Detailed Implementation

[0072] Terms and Definitions

[0073] The term "Escherichia coli" refers to Escherichia coliEscherichia coli, also known as coliform bacteria, is a Gram-negative, rod-shaped, facultative anaerobic bacterium belonging to the Enterobacteriaceae family.

[0074] The term "Escherichia coli infection" refers to various diseases caused by Escherichia coli, including urinary tract infections, diarrhea, sepsis, meningitis, etc.

[0075] The term "PstS" refers to a phosphate-specific transport system substrate-binding protein, which is an important component of the phosphate transport system in Escherichia coli. Preferably, the amino acid sequence of PstS is shown in SEQ ID NO: 1.

[0076] The term "YidR" refers to a conserved protein in Escherichia coli containing the DUF3748 domain-containing protein. Its function is not fully elucidated, but it may be involved in bacterial stress responses and biofilm formation. Preferably, the amino acid sequence of YidR or a variant thereof is as shown in SEQ ID NO: 11, 2, or 3.

[0077] The term "immune response" refers to a humoral response, a cellular response, or both in an organism. Immune responses can be measured by assays, including but not limited to assays measuring the presence or amount of antibodies that specifically recognize proteins or cell surface proteins, assays measuring T cell activation or proliferation, and / or assays measuring the regulation of the activity or expression of one or more cytokines.

[0078] The terms "administration" or "vaccination" refer to the administration of the nucleic acid vaccine or vaccine composition based on the present invention, preferably via intramuscular or subcutaneous route, although other routes of administration may also be used, such as oral, intranasal (e.g., aerosol or other non-injection), intralymphatic, intradermal, intraperitoneal, rectal or vaginal administration, or by combination of routes.

[0079] The term "expression" includes any step involved in polypeptide production, including but not limited to: transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0080] The term "recombinant nucleic acid molecule" refers to a polynucleotide having a sequence that is not linked together in nature. Recombinant polynucleotides can be contained in a suitable vector, which can then be used to transform into a suitable host cell.

[0081] The term "recombinant expression vector" refers to a DNA structure containing a polynucleotide that is used to express, for example, a desired polypeptide. A recombinant expression vector may include, for example, a collection of genetic elements that regulate gene expression, such as promoters and enhancers; (2) a structural or coding sequence that is transcribed into mRNA and translated into a protein; and (3) a transcriptional subunit containing appropriate transcription and translation initiation and termination sequences.

[0082] The term "mRNA" refers to messenger RNA, also known as "messenger RNA," which is a type of single-stranded ribonucleic acid transcribed from one strand of DNA as a template. It carries genetic information and can guide protein synthesis.

[0083] The term "5'-UTR" refers to the "5' untranslated region" or "5'UTR," which is a portion of a gene transcribed into a primary RNA transcript (precursor mRNA) located upstream of the coding sequence.

[0084] The term "3'-UTR" refers to the "3'-untranslated region" or "3'UTR," which refers to the region located at the 3' end of a gene, downstream of the stop codon in a protein-coding region, and which is transcribed but not translated into an amino acid sequence, or the corresponding region in an RNA molecule.

[0085] The term "host cell" refers to a cell into which exogenous polynucleotides have been introduced, including the progeny of such cells.

[0086] The terms "individual," "patient," or "subject" include mammals. Mammals include, but are not limited to, domesticated animals (e.g., pigs, cattle, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), and rodents (e.g., rabbits, mice, and rats).

[0087] The terms "transformation," "transfection," and "transduction" have the meanings commonly understood by those skilled in the art: the process of introducing exogenous DNA or RNA into a host.

[0088] The term "drug combination" or "pharmaceutical composition" refers to excipients widely used in the pharmaceutical manufacturing industry. The primary purpose of using a carrier is to provide a pharmaceutical composition that is safe to use, stable in nature, and / or has specific functionalities, as well as to provide a method for its effective absorption in a subject.

[0089] The term "prevention" refers to the reduction of symptoms after contracting a disease by exposing (e.g., administering medication) a subject to a recombinant vaccine, composition, etc. based on the present invention before contracting the disease, compared to the absence of exposure, and does not imply the necessity of completely suppressing the disease.

[0090] Unless otherwise defined or clearly indicated by the context, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art described herein.

[0091] This invention discloses a novel method for preparing an Escherichia coli vaccine and its application. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included within the scope of this invention. The method and application of this invention have been described through preferred embodiments, and those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0092] The fusion protein and encoding nucleic acid and their elements provided by this invention, as well as the preparation method and application, all utilize commercially available raw materials and reagents. Based on conventional knowledge in molecular cloning, expression construction, vaccine preparation, and immunization, those skilled in the art can implement the methods and embodiments of this invention.

[0093] The present invention will be further illustrated below with reference to the embodiments. Preferably, a nucleic acid vaccine architecture is selected for the preparation of the recombinant vaccine.

[0094] Example 1: PstS antigen screening

[0095] Because Escherichia coli antigens are serotype-dependent, this invention employs a reverse vaccinology strategy to screen for potential Escherichia coli antigens. The primary goal of the screening is to identify antigens that are highly expressed in the transcriptome and are highly conserved across different Escherichia coli subtypes.

[0096] The specific screening steps are as follows:

[0097] (1) Collect transcriptome data of different subtypes of Escherichia coli strains.

[0098] (2) Bioinformatics analysis of transcriptome data to identify genes with high abundance expression.

[0099] (3) The conservation of these highly expressed genes among different subtypes was analyzed by comparative genomics.

[0100] Through the aforementioned systematic screening, this invention ultimately identified several candidate antigens, including PstS and YidR. Subsequently, based on immunoepitaxy prediction analysis, PstS and YidR were evaluated and determined to be the optimal candidate antigens.

[0101] In addition, in order to improve the expression of candidate antigens and enhance their immunogenicity, N-terminal truncation or amino acid mutations at individual sites were performed on known PstS and YidR antigens.

[0102] Example 2: Design of a fusion protein based on PstS-YidR protein

[0103] Based on the screening results in Example 1, a fusion protein design for PstS and YidR was developed and compared with a PstS monoantigen vaccine in order to obtain a vaccine with better immune protection.

[0104] Based on the design requirements of multi-antigen vaccines, the PstS-YidR fusion protein needs to possess a stable three-dimensional conformation to better express and secrete it, and to provide more epitope information, thereby improving the immunogenicity of the antigen. Therefore, this invention, starting from the tertiary structure of the PstS-YidR fusion protein and combining it with immunoepitope screening, constructs two fusion protein molecules:

[0105] (1) The PstS antigen protein is fused with the full-length YidR antigen protein;

[0106] (2) The PstS antigen protein is fused with the YidR antigen selection stable domain peptide.

[0107] like Figure 1A (Schematic diagram of the 3D structure of the PstS-YidR full-length protein fusion protein) and Figure 1B (A schematic diagram of the 3D structure of the fusion protein of the PstS-YidR-tipped peptide is shown.) Both fusion proteins constructed using these methods possess stable and complete spatial conformations, and the structures of the two antigens do not interfere with each other. Theoretically, both meet the requirements for fusion molecule expression. Therefore, the two fusion proteins were used as antigen sequences for vaccine design and comparative experiments in subsequent embodiments of this invention.

[0108] Example 3: Construction of Recombinant Nucleic Acid Vaccine

[0109] To prepare a recombinant nucleic acid vaccine containing the antigen of the present invention, and to compare whether the vaccine based on the present invention has good in vitro expression effects, exemplary schematic diagrams of the vaccine molecular structures involved in the embodiments are shown below. Figure 2 As shown, from the N-terminus to the C-terminus, the following elements are sequentially included: signal peptide, Fc domain, target antigen region, and STABILON. These elements can be directly linked or linked through different linkers, such as GGS linker, spacer sequence, or GS linker. Figure 2The four vaccines illustrated (A, B, C, and D) differ in their target antigen regions. The target antigens are, respectively, the full-length PstS antigen and the full-length YidR antigen protein (linked by a spacer sequence), the selected peptides of the PstS antigen and the YidR antigen (linked by a spacer sequence), and the selected peptides of the PstS antigen and the mutated YidR antigen (linked by a spacer sequence). To prepare vaccines capable of producing... Figure 2 The recombinant nucleic acid vaccine for the protein shown first constructs a gene expression cassette for expressing the antigen sequence described in this invention. The expression cassette, from the 5' end to the 3' end, sequentially includes: a 5' UTR, a CDS region, a 3' UTR, and a PolyA region, wherein the CDS region contains the fusion molecular structure described in this invention. Subsequently, the complete gene expression cassette sequence is optimized based on codon degeneracy, and the DNA sequence is directly obtained through gene synthesis (commissioned by GenScript). Finally, the synthesized gene expression cassette DNA sequence is inserted into an expression vector suitable for in vitro RNA transcription, such as... Figure 3 As shown, a vector plasmid for preparing a recombinant nucleic acid vaccine was obtained.

[0110] According to the above method, a carrier for use in subsequent embodiments is prepared:

[0111] (1) Preparation vector of recombinant nucleic acid vaccine A based on the present invention

[0112] Step a: Synthesize the fusion gene fragment “signal peptide-human IGHG Fc domain-PstS antigen-STABILON”. The human IGHG Fc domain and PstS antigen are linked by the GGS linker sequence shown in SEQ ID NO: 8, and the PstS antigen and STABILON are linked by the GS linker sequence shown in SEQ ID NO: 9. The amino acid sequence of the signal peptide is shown in SEQ ID NO: 4, the amino acid sequence of the human IGHG Fc domain is shown in SEQ ID NO: 5, the amino acid sequence of the PstS antigen is shown in SEQ ID NO: 1, and the amino acid sequence of STABILON is shown in SEQ ID NO: 6.

[0113] Step b: Construct a nucleic acid vaccine architecture carrier.

[0114] The nucleic acid vaccine architecture vector includes 5'-UTR and 3'-UTR, and can be a vector for producing any form of RNA vaccine or a vector for producing DNA vaccines.

[0115] Step c: Prepare recombinant plasmids.

[0116] The gene synthesized in step a is inserted into the vector architecture in step b to obtain the recombinant nucleic acid vaccine A preparation vector based on the present invention. Figure 3 (As shown).

[0117] (2) Preparation vector of recombinant nucleic acid vaccine B based on the present invention

[0118] Step a: Synthesize the fusion gene fragment “signal peptide-human IGHG Fc domain-YidR antigen-selective peptide-STABILON”. The human IGHG Fc domain and the YidR antigen-selective peptide are linked by the GGS linker sequence shown in SEQ ID NO: 8, and the YidR antigen-selective peptide and STABILON are linked by the GS linker sequence shown in SEQ ID NO: 9. The amino acid sequence of the signal peptide is shown in SEQ ID NO: 4, the amino acid sequence of the human IGHG Fc domain is shown in SEQ ID NO: 5, the amino acid sequence of the YidR antigen-selective peptide is shown in SEQ ID NO: 2, and the amino acid sequence of STABILON is shown in SEQ ID NO: 6.

[0119] Step b: Construct a nucleic acid vaccine architecture carrier.

[0120] The nucleic acid vaccine architecture vector includes 5'-UTR and 3'-UTR, and can be a vector for producing any form of RNA vaccine or a vector for producing DNA vaccines.

[0121] Step c: Prepare recombinant plasmids.

[0122] The gene synthesized in step a is inserted into the vector architecture in step b to obtain the recombinant nucleic acid vaccine B preparation vector based on the present invention. Figure 3 (As shown).

[0123] (3) Preparation vector of recombinant nucleic acid vaccine C based on the present invention

[0124] Step a: Synthesize the fusion gene fragment “signal peptide-human IGHG Fc domain-PstS antigen-YidR full-length protein-STABILON”. The human IGHG Fc domain and PstS antigen are linked by the GGSlinker sequence shown in SEQ ID NO: 8. The PstS antigen and the full-length YidR protein are linked by the spacer sequence shown in SEQ ID NO: 7. The full-length YidR protein and STABILON are linked by the GSlinker sequence shown in SEQ ID NO: 9. The amino acid sequence of the signal peptide is shown in SEQ ID NO: 4. The amino acid sequence of the human IGHG Fc domain is shown in SEQ ID NO: 5. The amino acid sequence of the PstS antigen is shown in SEQ ID NO: 1. The amino acid sequence of the full-length YidR protein is shown in SEQ ID NO: 11. The amino acid sequence of STABILON is shown in SEQ ID NO: 6.

[0125] Step b: Construct a nucleic acid vaccine architecture carrier.

[0126] The nucleic acid vaccine architecture vector includes 5'-UTR and 3'-UTR, and can be a vector for producing any form of RNA vaccine or a vector for producing DNA vaccines.

[0127] Step c: Prepare recombinant plasmids.

[0128] The gene synthesized in step a is inserted into the vector architecture in step b to obtain the recombinant nucleic acid vaccine C preparation vector based on the present invention. Figure 3 (As shown).

[0129] (4) Preparation vector of recombinant nucleic acid vaccine D based on the present invention

[0130] Step a: Synthesize the fusion gene fragment “signal peptide-human IGHG Fc domain-PstS antigen-mutated YidR antigen selection peptide-STABILON”. The human IGHG Fc domain and PstS antigen are linked by the GGS linker sequence shown in SEQ ID NO:8. The PstS antigen and the mutated YidR antigen selection peptide are linked by the spacer sequence shown in SEQ ID NO:7. The mutated YidR antigen selection peptide and STABILON are linked by the GS linker sequence shown in SEQ ID NO:9. The amino acid sequence of the signal peptide is shown in SEQ ID NO:4. The amino acid sequence of the human IGHG Fc domain is shown in SEQ ID NO:5. The amino acid sequence of the PstS antigen is shown in SEQ ID NO:1. The amino acid sequence of the mutated YidR antigen selection peptide is shown in SEQ ID NO:3. The amino acid sequence of STABILON is shown in SEQ ID NO:6.

[0131] Step b: Construct a nucleic acid vaccine architecture carrier.

[0132] The nucleic acid vaccine architecture vector includes 5'-UTR and 3'-UTR, and can be a vector for producing any form of RNA vaccine or a vector for producing DNA vaccines.

[0133] Step c: Prepare recombinant plasmids.

[0134] The gene synthesized in step a is inserted into the vector architecture in step b to obtain the recombinant nucleic acid vaccine preparation vector D based on the present invention. Figure 3 (As shown).

[0135] Table 1. Protein amino acid sequences involved in this invention.

[0136] amino acid sequence and sequence number PstS antigen EASLTGAGATFPAPVYAKWADTYQKETGNKVNYQGIGSSGGVKQIIANTVDFGASDAPLSDEKLAQEGLFQFPTVIGGVVLAVNIPGLKSGELVLDGKTLGDIYLGKIKKWDDEAIAKLNPGLKLPSQNIAVVRRADGSGTSFVFTSYLAKVNEEWKNNVGT GSTVKWPIGLGGKGNDGIAAFVQRLPGAIGYVEYAYAKQNNLAYTKLISADGKPVSPTEENFANAAKGADWSKTFAQDLTNQKGEDAWPITSTTFILIHKDQKKPEQGTEVLKFFDWAYKTGAKQANDLDYASLPDSVVEQVRAAWKTNIKDSSGKPLY (SEQ IDNO: 1) YidR antigen-selected peptide fragment RAMKQITFAPRNHLTNTTWTPDSQWLVFDVRPSGASFTGETIERVNIHTGEVEVIYRASQGAHVGVVTVHPKSEKYVFIHGPENPEDETWHYDFHHRRGVIAEGGKVSNLDAMDITAPYTPGALRGGSHVHVFSPNGERVSFTYNDHVMHELDPALDLRNVGVAAPFGPVNVQKQHPREYSGSHWCVLVSKTTPTPQPGSDEIN RAYEEGWVGNHALAFIGDTLSPKGEKVPELFIVELPQDEAGWKAAGDAPLSGTETTLPAPPRGVVQRRLTFTHHRAYPGLVNVPRHWVRCNPQGTQIAFLMR DDNGIMQLWLISPQGGEPRQLTHQKTDIQSAFNWHPSGEWLGFVLDNRIACAHAQSGEVEYLTENHANPPSADAVVFSPDGQWLAWMEGGQLWITETDR (SEQ ID NO: 2) Mutated YidR antigen-selective peptide RAMKQITFAPRNHLLTNTNTWTPDSQWLVFDVRPSGASFTGETIERVNIHTGEVEVIYRASQGAHVGVVTVHPKSEKYVFIHGPENPDETWHYDFHHRRGVIAEGGKVSNLDAMDITAPYTPGALRGGSHVHVFSPNGERVSFTYNDHVMHELDPALDLRNVGVAAPFGPVNVQKQHPREYSGSHWCVLVSKATPAPQPGSDEINRAYEEGWVGNHALAFIGDTLSPKGEKVPELFIVELPQDEAGWKAAGDAPLSGTEATLPAPPRGVVQRRLTFAHHRAYPGLVNVPRHWVRCNPQGTQIAFLMRDDNGIMQLWLISPQGGEPRQLTHNKTDIQSAFNWHPSGEWLGFVLDNRIACAHAQSGEVEYLTENHANPPSADAVVFSPDGQWLAWMEGGQLWITETDR (SEQ ID NO: 3) Signal peptide MTRLTVLALLAGLLASSRA (SEQ ID NO: 4) Fc domain of human IGHG EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 5) STABILON KDGKKDKKEEDKK (SEQ ID NO: 6) Spacer sequence GGSGGGGSGG (SEQ ID NO: 7) GGS linker GGSGGSGGSG (SEQ ID NO: 8) GS linker GSGSGSG (SEQ ID NO: 9) PstS-truncated YidR fusion protein EASLTGAGATFPAPVYAKWADTYQKETGNKVNYQGIGSSGGVKQIIANTVDFGASDAPLSDEKLAQEGLFQFPTVIGGVVLAVNIPGLKSGELVLDGKTLGDIYLGKIKKWDDEAIAKLNPGLKLPSQNIAVVRRADGSGTSFVFTSYLAKVNEEWKNNVGTGSTVKWPIGLGGKGNDGIAAFVQRLPGAIGYVEYAYAKQNNLAYTKLISADGKPVSPTEENFANAAKGADWSKTFAQDLTNQKGEDAWPITSTTFILIHKDQKKPEQGTEVLKFFDWAYKTGAKQANDLDYASLPDSVVEQVRAAWKTNIKDSSGKPLYGGSGGGGSGGRAMKQITFAPRNHLLTNTNTWTPDSQWLVFDVRPSGASFTGETIERVNIHTGEVEVIYRASQGAHVGVVTVHPKSEKYVFIHGPENPDETWHYDFHHRRGVIAEGGKVSNLDAMDITAPYTPGALRGGSHVHVFSPNGERVSFTYNDHVMHELDPALDLRNVGVAAPFGPVNVQKQHPREYSGSHWCVLVSKTTPTPQPGSDEINRAYEEGWVGNHALAFIGDTLSPKGEKVPELFIVELPQDEAGWKAAGDAPLSGTETTLPAPPRGVVQRRLTFTHHRAYPGLVNVPRHWVRCNPQGTQIAFLMRDDNGIMQLWLISPQGGEPRQLTHQKTDIQSAFNWHPSGEWLGFVLDNRIACAHAQSGEVEYLTENHANPPSADAVVFSPDGQWLAWMEGGQLWITETDR (SEQ ID NO: 10) Full-length YidR antigen protein MMAGPVLYQDRAMKQITFAPRNHLLTNTNTWTPDSQWLVFDVRPSGASFTGETIERVNIHTGEVEVIYRASQGAHVGVVTVHPKSEKYVFIHGPENPDETWHYDF HHRRGVIAEGGKVSNLDAMDITAPYTPGALRGGSHVHVFSPNGERVSFTYNDHVMHELDPALDLRNVGVAAPFGPVNVQKQHPREYSGSHWCVLVSKTTPTPQPG SDEINRAYEEGWVGNHALAFIGDTLSPKGEKVPELFIVELPQDEAGWKAAGDAPLSGTETTLPAPPRGVVQRRLTFTHHRAYPGLVNVPRHWVRCNPQGTQIAFL MRDDNGIMQLWLISPQGGEPRQLTHQKTDIQSAFNWHPSGEWLGFVLDNRIACAHAQSGEVEYLTENHANPPSADAVVFSPDGQWLAWMEGGQLWITETDR (SEQ ID NO:11)

[0137] Example 4: Preparation of the recombinant nucleic acid vaccine of the present invention

[0138] (1) Preparation of capped mRNA vaccines

[0139] Step a: Linearize the vector plasmid used in Example 3 for producing capped mRNA vaccines by enzyme digestion to obtain a linearized plasmid for in vitro transcription.

[0140] Step b: The linearized plasmid was subjected to an in vitro co-transcriptional capping reaction to add a 7-methylguanylate cap structure to the 5' end of the transcribed mRNA and the template DNA was degraded.

[0141] (2) Preparation of uncapped mRNA vaccines

[0142] Step a: Linearize the vector plasmid used in Example 3 for producing uncapped mRNA vaccines by enzyme digestion to obtain a linearized plasmid for in vitro transcription.

[0143] Step b: Perform an in vitro uncapped transcription reaction on the linearized plasmid and degrade the template DNA.

[0144] (3) DNA vaccine preparation

[0145] Step a: Amplify the vector plasmid used in Example 3 for producing DNA vaccines to obtain a large number of target plasmids for purification.

[0146] Step b: Extract and purify the target plasmid using an endotoxin-free plasmid extraction and purification kit.

[0147] Example 5: Quality control of recombinant nucleic acid in vitro transcription and vaccine preparation according to the present invention

[0148] Vaccine A (recombinant nucleic acid vaccine A based on the present invention), vaccine B (recombinant nucleic acid vaccine B based on the present invention), vaccine C (recombinant nucleic acid vaccine C based on the present invention), and vaccine D (recombinant nucleic acid vaccine D based on the present invention) were prepared using the capped mRNA vaccine preparation method described in Example 4. The purity of the produced recombinant nucleic acids was tested, and the purity of all recombinant nucleic acids used in the experiments was greater than or equal to 85%. The quality control peak diagram of the recombinant nucleic acids based on the present invention is shown below. Figure 4A , Figure 4B , Figure 4C , Figure 4D As shown. Specifically, it is described as follows: (1) The recombinant nucleic acid vaccine A based on the present invention has a purity of 90.3%, and the quality control peak diagram and purity test results are as follows. Figure 4A (2) The recombinant nucleic acid vaccine B based on the present invention has a purity of 88.5%, and the quality control peak diagram and purity test results are as follows: Figure 4B (3) The recombinant nucleic acid vaccine C based on the present invention has a purity of 88.5%, and the quality control peak diagram and purity test results are as follows: Figure 4C (4) The recombinant nucleic acid vaccine D based on the present invention has a purity of 85%, and the quality control peak diagram and purity test results are as follows: Figure 4D The purity levels described above all meet the quality requirements for cell transfection experiments and vaccine production.

[0149] Example 6: In vitro expression effect of the recombinant nucleic acid of the present invention

[0150] The vaccines from Example 5 were transfected into HEK293T cells using cell transfection reagents. After 48 hours of in vitro culture, the proteins were collected and subjected to Western blot analysis. The molecular weights of the proteins from vaccines A, B, C, and D were calculated, as shown in Table 2.

[0151] Table 2. Protein molecular weight of vaccines A, B, C, and D

[0152]

[0153] Figures 5A-5C The results of in vitro expression analysis (WB) on HEK293 cells transfected with vaccines A, B, C, and D are presented. Figure 5A The results of Western blot analysis of in vitro expression of vaccine A in HEK293 cells. Figure 5B The results of Western blot analysis of vaccine B expression in HEK293 cells transfected in vitro. Figure 5C The results show the in vitro expression of antigens in HEK293 cells transfected with vaccines C and D (Western Blot analysis). The results indicate that the expressed antigens are all humoral immune antigens, which theoretically should be significantly expressed in the supernatant.

[0154] like Figures 5A-5C As shown, vaccines A, B, C, and D all showed detectable target protein signals in the supernatant, and their molecular weights were all as expected. This demonstrates that the fusion proteins based on this invention can not only be successfully translated and correctly folded in eukaryotic cells, but also have a stable structure and can be secreted extracellularly.

[0155] contrast Figure 5A , Figure 5B The results showed that the expression level of vaccine A in the supernatant was significantly higher than that of vaccine B, proving that the secretion effect of PstS antigen alone was superior to that of YidR antigen; according to Figure 5C The results showed that the expression level of vaccine D in the supernatant was significantly higher than that of vaccine C, proving that the design of vaccine D is superior among the two PstS-YidR fusion protein designs. Choosing the mutated YidR antigen-selected peptide of this invention, compared with the full-length YidR antigen protein, can significantly improve the secretion efficiency of PstS-YidR fusion protein.

[0156] The comparative analysis above shows that vaccine A, expressing the PstS antigen protein, and vaccine D, expressing the "PstS antigen-mutated YidR antigen selection multiple" fusion protein, are better choices for vaccine design, as they are more conducive to antigen presentation and activation of humoral immunity. Therefore, subsequent animal experiments will be conducted using vaccines A and D to verify their immunoprotective effects. Simultaneously, the immunoprotective effects of single-antigen and multi-antigen fusion proteins will be compared to determine if there are differences.

[0157] Example 7: Preventive effect of the recombinant nucleic acid vaccine of the present invention in a mouse gavage infection model.

[0158] To verify whether the nucleic acid vaccine based on the present invention has an immune protective effect, this embodiment conducted immunization and challenge comparison experiments on mice immunized with vaccine A (experimental group), mice immunized with vaccine D (experimental group), and mice that were not immunized (blank control group, i.e., treated with PBS).

[0159] Fifteen male BALB / c mice aged 6-8 weeks, weighing 18-25g, were selected for the experiment. All mice were housed in individual cages with constant temperature and humidity, and were acclimatized to the environment for 7 days prior. The housing temperature was 20-26℃, and the humidity was 40-70%. A day-night cycle was implemented, with light from 8:00 AM to 8:00 PM and darkness from 8:00 PM to 8:00 AM the following day. Sufficient feed was continuously provided, with unlimited access to sterile water via a continuous water bottle. After acclimatization, the mice were randomly divided into three groups of five, each mouse tagged with an ear tag. Details are shown in Table 3. Dosages in this table and below refer to the amount of active ingredient.

[0160] Table 3. Grouping and Immunization Procedure for Mouse Immunization Experiment in Example 5

[0161]

[0162] Mice in each group were immunized twice according to the immunization protocol in Table 3. On Day 32, mice were administered 100 μl of a mixture of 50 mg / ml penicillin and streptomycin antibiotics by gavage once daily for 3 days. On Day 35, mice were fasted overnight before bacterial challenge and were given 100 μl of Escherichia coli bacterial suspension (Enterohemorrhagic Escherichia coli EHEC, 1×10⁻⁶). 9 CFU was administered via gavage. Mice were fed normally after gavage, and their clinical manifestations were observed and recorded daily. Mice were sacrificed on Day 40 for sampling. The immunization, challenge, and sampling procedures are as follows: Figure 6 As shown in the figure. Ileal and colonic tissues of mice were collected, and the bacterial load and histopathological sections were tested to comprehensively evaluate the immunoprotective effect of the vaccine.

[0163] Ileal and colon tissues were collected from each mouse. 10 mg of ileal and colon tissue was aseptically excised from each tissue and ground on ice with sterile PBS buffer until no significant precipitation occurred. Blood was diluted 1:1000 with sterile PBS buffer, and 100 μl of the diluted solution was evenly spread onto LB agar plates. The plates were then incubated at 37°C. After 24 hours of bacterial culture, the bacterial clones were counted and photographed to observe the protective effect of the vaccine on mice. The results are as follows: Figures 7A-7B As shown. Figure 7AThe image shows a comparative analysis of bacterial load (number of colonies on agar plates) in the ileum tissue of mice after immunization. The results show that, compared with the blank control group mice (immunized with PBS), the Escherichia coli load in the vaccine-immunized group mice (immunized with vaccine A and vaccine D) was significantly reduced. The bacterial load in the ileum tissue of the blank control group mice was about 5 times that of the vaccine-immunized group mice. Figure 7B The image shows a comparative analysis of bacterial load (number of colonies on agar plates) in the colon tissue of mice after immunization. The results show that, compared with the blank control group mice (immunized with PBS), the Escherichia coli load in the vaccine-immunized group mice (immunized with vaccine A and vaccine D) was significantly reduced. The bacterial load in the colon tissue of the blank control group mice was about 4 times that of the vaccine-immunized group mice.

[0164] Colon tissue was collected from each mouse, fixed with paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Pathological changes in the colon, kidney, and spleen tissues were observed under a microscope. The results are as follows: Figure 8 As shown. Figure 8 Comparative photographs of colon tissue sections (HE staining) from mice after immunization with the virus show that the intestinal villi structure of normal mice remained intact, without obvious lesions or inflammatory infiltration. In the blank control group (immunized with PBS), a large number of inflammatory cells infiltrated and accumulated in the submucosa of the intestine, exhibiting symptoms of Escherichia coli infection. In the vaccine-immunized mice (immunized with vaccines A and D), a small number of intestinal villi were broken and dissolved, and some inflammatory cells infiltrated and accumulated in the mucosa. Analysis of these results indicates that mice immunized with vaccines A and D based on this invention all showed resistance to Escherichia coli infection, demonstrating that the vaccine based on this invention can induce immune protection against Escherichia coli in mice and prevent Escherichia coli infection.

[0165] In summary, both the novel antigen PstS and the PstS-YidR fusion protein provided by this invention can induce effective protective immunity in mouse model animals, demonstrating good preventive effects against Escherichia coli infection. Therefore, this invention can be applied to the production and development of immunotherapeutic drugs, filling a gap in the current field of Escherichia coli vaccine development, and possesses extremely high commercial value and broad application prospects.

[0166] The embodiments described above are merely examples for clearly illustrating the present disclosure and are not intended to limit the implementation of the present disclosure. Those skilled in the art can make other variations or modifications based on the above description. It is neither necessary nor possible to exhaustively describe all possible implementations. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this disclosure should be included within the scope of protection of the claims of this disclosure.

Claims

1. A fusion protein for preventing Escherichia coli infection, characterized in that, Selected from either of the following two fusion proteins: Fusion protein A: From N-terminus to C-terminus, the sequence is: signal peptide, Fc domain, PstS antigen, STABILON; Fusion protein D: From N-terminus to C-terminus, it consists of: signal peptide, Fc domain, PstS antigen, mutated YidR antigen-selective peptide, and STABILON. The amino acid sequence of the PstS antigen is shown in SEQ ID NO.1; the amino acid sequence of the mutated YidR antigen-selective polypeptide is shown in SEQ ID NO.3; the amino acid sequence of the Fc domain is shown in SEQ ID NO.5; and the amino acid sequence of the STABILON is shown in SEQ ID NO.

6. The different elements of the fusion protein may optionally be linked by a GGS linker sequence, a spacer sequence, and / or a GSlinker sequence.

2. The fusion protein according to claim 1, characterized in that, The signal peptide is derived from human azurocidin protein, and its amino acid sequence is shown in SEQ ID NO:

4.

3. The fusion protein according to claim 1 or 2, characterized in that, The amino acid sequence of the spacer sequence is shown in SEQ ID NO: 7, the amino acid sequence of the GGS linker sequence is shown in SEQ ID NO: 8, and the amino acid sequence of the GS linker sequence is shown in SEQ ID NO:

9.

4. A recombinant nucleic acid molecule, characterized in that, Encodes the fusion protein according to any one of claims 1-3.

5. A recombinant gene expression cassette, characterized in that, It includes the recombinant nucleic acid molecule of claim 4.

6. A recombinant vector, characterized in that, It comprises the recombinant nucleic acid molecule of claim 4 or the recombinant gene expression cassette of claim 5.

7. A recombinant host cell, characterized in that, It comprises the recombinant nucleic acid molecule of claim 4, or the recombinant gene expression cassette of claim 5, or the recombinant vector of claim 6.

8. An immunogenic composition or pharmaceutical composition, characterized in that, It comprises one or more of the fusion proteins of any one of claims 1-3, and / or one or more of the recombinant nucleic acid molecules of claim 4, and / or one or more of the recombinant gene expression cassettes of claim 5, and / or one or more of the recombinant vectors of claim 6, and / or one or more of the recombinant host cells of claim 7.

9. The immunogenic composition or pharmaceutical composition according to claim 8, characterized in that, The immunogenic composition or pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

10. The immunogenic composition or pharmaceutical composition according to claim 8 or 9, characterized in that, The immunogenic composition or pharmaceutical composition may also contain other drugs for the prevention of diseases caused by Escherichia coli.

11. A recombinant vaccine, characterized in that, The composition comprises one or more of the fusion proteins of any one of claims 1-3, and / or one or more of the recombinant nucleic acid molecules of claim 4, and / or one or more of the recombinant gene expression cassettes of claim 5, and / or one or more of the recombinant vectors of claim 6, and / or one or more of the recombinant host cells of claim 7, and / or one or more of the immunogenic compositions or pharmaceutical compositions of any one of claims 8-10.

12. The recombinant vaccine according to claim 11, characterized in that, The recombinant vaccine is a nucleic acid vaccine or a subunit vaccine.

13. Use of one or more fusion proteins according to any one of claims 1-3, and / or one or more recombinant nucleic acid molecules according to claim 4, and / or one or more recombinant gene expression cassettes according to claim 5, and / or one or more recombinant vectors according to claim 6, and / or one or more recombinant host cells according to claim 7, and / or one or more immunogenic compositions or pharmaceutical compositions according to any one of claims 8-10, and / or one or more recombinant vaccines according to claim 11 or 12 in the preparation of medicaments for the prevention of diseases and / or secondary infections caused by Escherichia coli.

14. The use according to claim 13, characterized in that, The diseases and / or secondary infections caused by Escherichia coli include intestinal infections, urinary tract infections, sepsis, bacteremia, meningitis, peritonitis, pneumonia, mastitis, and hemolytic uremic syndrome.

15. The use according to claim 14, characterized in that, The intestinal infection is enteritis and / or diarrhea.

16. The use according to claim 14, characterized in that, The urinary tract infection is cystitis and / or pyelonephritis.

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