Vaccine for preventing Escherichia coli infection and application thereof

Abstract

The invention relates to a fusion protein, an immunogenic composition, a recombinant vaccine and the like for preventing Escherichia coli infection. The invention provides a new target PstS for Escherichia coli vaccine design, starting from a protein molecule tertiary structure of PstS and YidR, combining with immune epitopes and the like, finally screening to obtain a PstS protein, a YidR protein and stable structural domains of variants of the PstS protein and the YidR protein, and constructing to obtain a fusion protein. The PstS single antigen protein molecule or the PstS-YidR fusion protein molecule provided by the invention has good immunogenicity, can weaken tissue lesions caused by Escherichia coli infection, has good immunogenicity, can efficiently prevent Escherichia coli infection, and has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biological medicine technology, especially the field of immunopharmaceutical technology, and specifically relates to a humoral immune vaccine for preventing Escherichia coli infection and its applications, etc. Background Art

[0002] Escherichia coli ( Escherichia coli ), also known as E. coli, is one of the most common symbiotic bacteria in the intestines of humans and animals, and is also an important pathogen causing various infectious diseases. Pathogenic Escherichia coli can cause severe intestinal and extra-intestinal infections, posing a major threat to public health.

[0003] Pathogenic Escherichia coli can infect a variety of tissues and cause different types of infections and diseases, including: intestinal infections, urinary tract infections, septicemia, bacteremia, meningitis, peritonitis, pneumonia, mastitis, hemolytic uremic syndrome, etc. Clinically, Escherichia coli infections mainly rely on antibiotic treatment. However, with the continuous increase in bacterial antibiotic resistance, the prevention and control of these pathogenic Escherichia coli face severe challenges.

[0004] Currently, there is no preventive vaccine for Escherichia coli infection on the market, and researchers are actively developing related vaccines. For example, the prior art patent CN106535927B (publication date: 2022-08-30) discloses a polysaccharide O25B of Escherichia coli, and the patent CN108430500B (publication date: 2018-08-21) discloses a polysaccharide conjugate for preventing extra-intestinal pathogenic Escherichia coli infection. These polysaccharide vaccines aim to develop a vaccine that can target a single type of pathogenic Escherichia coli. However, the serotypes (O serotypes) of pathogenic Escherichia coli have exceeded 180. The iteration of polysaccharide vaccines is difficult, and the degree of cross-immune protection is low, which cannot meet the current market demand for Escherichia coli vaccines.

[0005] The phosphate-binding protein PstS (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. Existing studies have proven that PstS can be used as a biosensor for phosphate pollution. For example, the patent CN114965403A (publication date: 2022-08-30) discloses a method for constructing a phosphate biosensor using the PstS protein of Pseudomonas aeruginosa. In addition, in some pathogenic bacteria, the expression of PstS is related to the production of virulence factors and is involved in biofilm formation and host cell adhesion. However, there has been no study reporting that PstS can be used as a protective antigen of Escherichia coli for vaccine development.

[0006] In summary, developing a vaccine that can provide broad-spectrum protection against a variety of pathogenic Escherichia coli, is safe, effective, and easy to produce has important scientific significance and application value. Such a vaccine can not only prevent various infections in humans, but may also be applied to the prevention of animal diseases, such as preventing mastitis in dairy cows, thus bringing significant benefits at the public health and economic levels. Therefore, in view of the above problems, the present invention proposes a new fusion protein design scheme, aiming to develop an efficient and broad-spectrum vaccine for preventing Escherichia coli infection, which is expected to cope with the infections of various pathogenic strains such as EHEC, MPEC, and UPEC at the same time. Summary of the Invention

[0007] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a new fusion protein for preventing Escherichia coli infection, an immunogenic composition, a recombinant vaccine, and molecular architecture design and applications, etc. The present invention provides a new target PstS that can be used for the design of Escherichia coli vaccines, and starting from the protein molecular tertiary structures of the highly conserved targets PstS and YidR of Escherichia coli, combined with immunogenic epitopes, etc., a fusion protein is designed. Through creative screening, the stable domains of the PstS protein and the YidR protein and their variants are finally selected to construct a fusion protein, and further elements such as the Fc domain and STABILON are added. The present invention discovers that the new fusion protein molecule can weaken the tissue lesions caused by Escherichia coli infection, has good immunogenicity, plays an effective preventive and immunoprotective role, can efficiently prevent Escherichia coli infection, has broad application prospects, and can be used to prevent various infectious diseases caused by Escherichia coli, such as urinary tract infection, intestinal infection, mastitis, sepsis, etc. In addition, the molecular design strategy provided by the present invention can also provide 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 (Phosphate-binding protein PstS) antigen or a fragment thereof.

[0009] Furthermore, the fusion protein further comprises a YidR antigen (DUF3748 domain-containing protein) or a fragment thereof, and the YidR antigen or a fragment thereof comprises the full-length YidR antigen protein, a truncated polypeptide of the YidR antigen, or a truncated polypeptide of the mutated YidR antigen.

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

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

[0012] Furthermore, the Gram-negative bacterium is a bacterium of the Enterobacteriaceae family.

[0013] Furthermore, the bacterium of the Enterobacteriaceae family is Escherichia coli.

[0014] Furthermore, the fusion protein sequentially includes 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 full-length YidR antigen protein, a truncated YidR polypeptide, or a truncated YidR polypeptide after mutation.

[0015] Furthermore, the amino acid sequence of the PstS antigen or a fragment thereof is as shown in SEQ ID NO.1, the amino acid sequence of the full-length YidR antigen protein is as shown in SEQ ID NO.11, the amino acid sequence of the truncated YidR polypeptide is as shown in SEQ ID NO.2, and the amino acid sequence of the truncated YidR polypeptide after mutation is as 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 the human Azurocidin protein, and its amino acid sequence is as shown in SEQ ID NO: 4; the amino acid sequence of the Fc domain of IGHG1 is as shown in SEQ ID NO: 5; the amino acid sequence of STABILON is as shown in SEQ ID NO.6.

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

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

[0020] Furthermore, it includes any one of the following 4 fusion proteins: (1) Fusion protein A: sequentially includes from the N-terminus to the C-terminus: a signal peptide, an Fc domain, a GGS linker, the PstS antigen or a fragment thereof, a GS linker, and STABILON; (2)Fusion protein B: sequentially includes from the N-terminus to the C-terminus: a signal peptide, an Fc domain, a GGS linker, a truncated YidR antigen polypeptide, a spacer sequence, a truncated YidR antigen polypeptide, a GS linker, and STABILON; (3)Fusion protein C: sequentially includes from the N-terminus to the C-terminus: a signal peptide, an Fc domain, a GGS linker, the PstS antigen or a fragment thereof, a spacer sequence, the full-length YidR antigen protein, a GS linker, and STABILON; (4)Fusion protein D: sequentially includes from the N-terminus to the C-terminus: a signal peptide, an Fc domain, a GGS linker, the PstS antigen or a fragment thereof, a spacer sequence, a truncated YidR antigen polypeptide after mutation, a GS linker, and STABILON; Furthermore, the amino acid sequence of the PstS antigen or a fragment thereof is as shown in SEQ ID NO.1, the amino acid sequence of the full-length YidR antigen protein is as shown in SEQ ID NO.11, the amino acid sequence of the truncated YidR antigen polypeptide is as shown in SEQ ID NO.2, the amino acid sequence of the truncated YidR antigen polypeptide after mutation is as shown in SEQ ID NO.3, the amino acid sequence of the signal peptide is as shown in SEQ ID NO: 4, the amino acid sequence of the Fc domain is as shown in SEQ ID NO: 5, the amino acid sequence of STABILON is as shown in SEQ ID NO.6, the amino acid sequence of the spacer sequence is as shown in SEQID NO: 7, the amino acid sequence of the GGS linker sequence is as shown in SEQID NO: 8, and the amino acid sequence of the GS linker sequence is as shown in SEQ ID NO: 9; Furthermore, the amino acid sequence of the fusion protein is as shown in SEQ ID NO.10.

[0021] Another aspect of the present invention provides a recombinant nucleic acid molecule, characterized in that it contains a nucleic acid encoding the fusion protein according to any one of the present invention.

[0022] Another aspect of the present invention provides a recombinant gene expression cassette, characterized in that it contains the recombinant nucleic acid molecule of the present invention.

[0023] Furthermore, the recombinant gene expression cassette further includes one or more of a promoter, a terminator, and regulatory sequences.

[0024] Another aspect of the present invention provides a recombinant vector, characterized in that it contains the recombinant nucleic acid molecule of the present invention or the recombinant gene expression cassette of the present invention.

[0025] Furthermore, the recombinant vector comprises a prokaryotic vector or a eukaryotic vector.

[0026] Furthermore, the prokaryotic vector includes but is not limited to an Escherichia coli vector.

[0027] Furthermore, the Escherichia coli vector includes but is not limited to pET vector, pGEX vector, pMAL vector, pBAD vector, pUC vector, pBR vector.

[0028] Furthermore, the eukaryotic vector includes but is not limited to a yeast expression vector, an insect expression vector, a mammalian cell expression vector.

[0029] Furthermore, the yeast expression vector includes but is not limited to pPICZ vector, pGAPZ vector, pYES vector, pGAP vector, pAO815 vector, pPIC9 vector.

[0030] Another aspect of the present invention provides a recombinant host cell, characterized by comprising the recombinant nucleic acid molecule of the present invention, or the recombinant gene expression cassette of the present invention, or the recombinant vector of the present invention.

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

[0032] Furthermore, the eukaryotic cell comprises a mammalian cell, an insect cell, a yeast cell.

[0033] Furthermore, the yeast cell includes but is not limited to Saccharomyces cerevisiae, Pichia pastoris, Hansenula yeast.

[0034] Furthermore, the prokaryotic cell includes but is not limited to Escherichia coli cell, Bacillus subtilis cell, Pseudomonas cell.

[0035] Furthermore, the Escherichia coli cell includes but is not limited to BL21(DE3), DH5α, TOP10, Rosetta.

[0036] Another aspect of the present invention provides an immunogenic composition or a pharmaceutical composition, characterized by comprising one or more of the fusion proteins of any one of the present invention, and / or one or more of the recombinant nucleic acid molecules of the present invention, and / or one or more of the recombinant gene expression cassettes of the present invention, and / or one or more of the recombinant vectors of the present invention, and / or one or more of the recombinant host cells of the present invention.

[0037] Furthermore, the immunogenic composition or the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0038] Furthermore, the immunogenic composition or the pharmaceutical composition further comprises other drugs for preventing diseases caused by Escherichia coli.

[0039] Furthermore, the other drugs for preventing diseases caused by Escherichia coli contain antibiotics.

[0040] Another aspect of the present invention provides a recombinant vaccine, which is characterized by comprising 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.

[0041] Furthermore, the recombinant vaccine is a nucleic acid vaccine or a subunit vaccine.

[0042] Furthermore, the recombinant vaccine is a nucleic acid vaccine.

[0043] Another aspect of the present invention provides the use of 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 in the preparation of drugs for preventing diseases caused by Escherichia coli and / or secondary infections.

[0044] Another aspect of the present invention provides a method for preventing diseases caused by Escherichia coli and / or secondary infections, which is characterized by comprising 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.

[0045] Furthermore, the diseases and / or secondary infections caused by Escherichia coli include intestinal infections, urinary tract infections, septicemia, bacteremia, meningitis, peritonitis, pneumonia, mastitis, hemolytic uremic syndrome.

[0046] Furthermore, the intestinal infection includes enteritis and / or diarrhea.

[0047] Furthermore, the urinary tract infection includes cystitis and / or pyelonephritis.

[0048] The fusion protein, immunogenic composition, recombinant vaccine, etc. of the present invention have the following beneficial technical effects: 1. The present invention discovers that the new fusion molecule has good immunogenicity and can provide an immune protection effect, and can be used for the research and development of nucleic acid vaccines or subunit vaccines. The immunogenic composition of the present invention can provide effective immune protection against Escherichia coli infection.

[0049] 2. The present invention can be used as a general antigen framework for nucleic acid vaccines or subunit vaccines, has strong immunogenicity, and can induce broad-spectrum immune protection without being restricted by serotypes compared with traditional vaccines.

[0050] 3. The present invention adopts the reverse vaccinology strategy to screen potential antigens of Escherichia coli, and finally identifies multiple candidate antigens including PstS and YidR. Based on immunogenic epitope prediction analysis, PstS and YidR are evaluated and determined to be the best candidate antigens. In addition, in order to improve the expression of candidate antigens and enhance the immunogenicity and other characteristics of antigens, N-terminal truncation or amino acid mutation at individual sites of the known PstS and YidR antigens is carried out.

[0051] 4. Starting from the tertiary structure of the protein molecules of PstS and YidR and combining with immunogenic epitopes, the present invention designs a fusion protein that can be stably expressed. Through creative screening, the stable domains of PstS protein and YidR protein and their variants are finally selected to construct the fusion protein, and the truncated polypeptide fragment has a complete and stable 3D structure.

[0052] 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 has a correct and stable structure, which is beneficial to the presentation of immunogenic epitopes, thereby further improving the immune protection effect and providing a new technology for the field of immunopharmaceutical design of Escherichia coli multivalent vaccines.

[0053] 6. The mouse gavage infection model experiment in Example 7 of the present invention shows that in the ileum and colon tissues, the bacterial load of the mouse group inoculated with the vaccine of the present invention is significantly reduced compared with the PBS control group. Statistical analysis shows that this reduction has a significant difference (P < 0.001). This result proves that the vaccine based on the present invention can effectively reduce the colonization of Escherichia coli in the intestine and provide effective local immune protection.

[0054] 7. In Example 7 of the present invention, in-depth detection was carried out by means of tissue section and HE staining, and the results further confirmed the protective effect of the vaccine of the present invention. The specific manifestations are as follows: a) In the colon tissue, the intestinal villi structure of the mice in the normal control group (without infection and immunization treatment) was intact, and no obvious lesions and inflammatory infiltration were observed. This provided a benchmark for evaluating the pathological changes in other groups. b) The intestines of the mice in the PBS control group (i.e., the group that received bacterial challenge but did not receive the vaccine) showed severe pathological changes, and a large number of inflammatory cells infiltrated and aggregated in the submucosa of the intestinal mucosa. This reflected the significant damage of Escherichia coli infection to the intestinal tissue. c) Although slight pathological changes occurred in the group of mice inoculated with the vaccine of the present invention (a small amount of dissolution of intestinal villi and inflammatory cell infiltration and aggregation in the mucosal layer), compared with the PBS control group, the degree of lesions was significantly lighter. This indicates that the vaccine of the present invention can significantly reduce the tissue lesions caused by Escherichia coli infection.

[0055] 8. Based on the comprehensive results of bacterial load and histopathology in Example 7 of the present invention, the vaccine of the present 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 the tissue lesions caused by infection. This comprehensive protective effect lays a solid foundation for the potential clinical application of the vaccine of the present invention.

[0056] 9. The fusion protein vaccine provided by the present invention exhibits excellent immunogenicity and protective effect, and has great potential in preventing Escherichia coli infection. This new vaccine design strategy provides a reference for the development of vaccines against other bacterial pathogens. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figures 1A - 1B It is a schematic diagram of the 3D structure of two fusion proteins of PstS of the present invention; among them, Figure 1A is a schematic diagram of the 3D structure of the fusion protein of the full-length protein of PstS-YidR, Figure 1B is a schematic diagram of the 3D structure of the fusion protein of the truncated polypeptide of PstS-YidR.

[0058] Figure 2 It is a schematic diagram of the molecular structures of vaccines A, B, C, and D used in the comparative experiment in the embodiment of the present invention.

[0059] Figure 3 It is a schematic diagram of a circular plasmid containing the molecular architecture of the fusion protein of the present invention.

[0060] Figure 4A -D is the quality control peak map and purity detection result of the nucleic acid vaccine used in the comparative experiment in the embodiment of the present invention; among them, Figure 4A is the quality control peak map and purity detection result of vaccine A, Figure 4B is the quality control peak map and purity detection result of vaccine B,Figure 4C The quality control peak map and purity test results of Vaccine C Figure 4D The quality control peak map and purity test results of Vaccine D

[0061] Figures 5A - 5C The in vitro expression WB (Western blot) test results of Vaccines A, B, C, and D of the present invention transfected into HEK293 cells, where Figure 5A The in vitro expression WB test results of Vaccine A transfected into HEK293 cells Figure 5B The in vitro expression WB test results of Vaccine B transfected into HEK293 cells Figure 5C The in vitro expression WB test results of Vaccines C and D transfected into HEK293 cells..

[0062] Figure 6 The schematic diagram of the immunization, challenge, and sampling processes of the mouse gavage infection experiment of the present invention

[0063] Figures 7A - 7B The bacterial load test results of the ileum and colon tissues of mice in each experimental group after immunization and challenge of the present invention; among them, Figure 7A The comparative analysis chart of the bacterial load (colony number on the plate) in the ileum tissue of mice after immunization and challenge Figure 7B The comparative analysis chart of the bacterial load (colony number on the plate) in the colon tissue of mice after immunization and challenge

[0064] Figure 8 The pathological tissue section staining results of the colon tissue of mice after immunization and challenge Detailed implementation manners

[0065] Terms and definitions The term "Escherichia coli" refers to Escherichia coli , also known as E. coli, is a Gram-negative, rod-shaped, facultative anaerobic bacterium belonging to the Enterobacteriaceae family

[0066] The term "Escherichia coli infection" refers to various diseases caused by Escherichia coli, including urinary tract infections, diarrhea, sepsis, meningitis, etc

[0067] The term "PstS" refers to the phosphate-specific transport system substrate-binding protein (Phosphate-specific transport system substrate-binding protein), which is an important component of the Escherichia coli phosphate transport system. Preferably, the amino acid sequence of PstS is as shown in SEQ ID NO: 1

[0068] The term "YidR" refers to a conserved protein in Escherichia coli that contains a DUF3748 domain (DUF3748 domain-containing protein). Its function has not been fully elucidated, but it may be involved in bacterial stress responses and biofilm formation. Preferably, the amino acid sequence of YidR or its variant is as shown in SEQ ID NO: 11, 2, or 3.

[0069] The term "immune response" refers to a humoral response, a cellular response, or both a humoral and cellular response in an organism. The immune response can be measured by assays that include, but are not limited to, assays for measuring the presence or amount of antibodies that specifically recognize a protein or cell surface protein, assays for measuring T cell activation or proliferation, and / or assays for measuring the regulation of the activity or expression of one or more cytokines.

[0070] The term "administration" or "inoculation" refers to administration, preferably via the intramuscular or subcutaneous route, of the nucleic acid vaccine or vaccine composition of the present invention, although other routes of administration can also be used, such as oral, intranasal (e.g., aerosol or other non-injection administration), intralymphatic, intradermal, intraperitoneal, rectal, or vaginal administration, or by combined routes.

[0071] 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.

[0072] The term "recombinant nucleic acid molecule" refers to a polynucleotide having sequences that are not linked together in nature. The recombinant polynucleotide can be included in a suitable vector, and the vector can be used to transform a suitable host cell.

[0073] The term "recombinant expression vector" refers to a DNA construct used for expressing, for example, a polynucleotide encoding a desired polypeptide. The recombinant expression vector can include, for example, a transcriptional subunit that contains (1) a collection of genetic elements that regulate gene expression, such as promoters and enhancers; (2) a structure or coding sequence that is transcribed into mRNA and translated into a protein; and (3) appropriate transcriptional and translational start and stop sequences.

[0074] The term "mRNA" refers to messenger RNA, Messenger RNA, and its Chinese translation is "messenger ribonucleic acid", which is a single-stranded ribonucleic acid transcribed from one strand of DNA as a template and carrying genetic information that can direct protein synthesis.

[0075] The term "5'-UTR" refers to the "5' untranslated region" or "5'UTR", which is a part of a gene that is transcribed into a primary RNA transcript (precursor mRNA) and is located upstream of the coding sequence.

[0076] The term "3'-UTR" refers to the "3'-untranslated region" or "3'UTR", and relates to the region located at the 3'-end of a gene, downstream of the stop codon in the protein-coding region, and which is transcribed but not translated into an amino acid sequence, or relates to the corresponding region in an RNA molecule.

[0077] The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including progeny of such cells.

[0078] The terms "individual", "patient" or "subject" include mammals. Mammals include, but are not limited to, domestic animals (e.g., pigs, cows, sheep, cats, dogs, horses, etc.), primates (e.g., humans and non-human primates such as monkeys), and rodents (e.g., rabbits, mice, rats, etc.).

[0079] The terms "transformation, transfection, transduction" have the meanings generally understood by those skilled in the art, which refer to the process of introducing exogenous DNA or RNA into a host.

[0080] The term "drug combination" or "pharmaceutical composition" refers to an auxiliary material widely used in the field of drug production. The main purpose of using a carrier is to provide a drug composition that is safe to use, has stable properties and / or has specific functionality, and also to provide a method for effective absorption in a subject's body.

[0081] The term "prevention" means that before suffering from a disease, by exposing (e.g., administering) a subject to a recombinant vaccine, composition, etc. based on the present invention, the symptoms after suffering from the disease are alleviated compared to not being exposed, and it does not necessarily mean completely suppressing the onset of the disease.

[0082] Unless otherwise defined or clearly indicated by the context, all technical and scientific terms in this disclosure have the same meaning as commonly understood by those of ordinary skill in the art to which this disclosure pertains.

[0083] The present invention discloses a method for preparing a new Escherichia coli vaccine and its application. Those skilled in the art can draw on the content of this article and appropriately modify the process parameters to achieve it. 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 the present invention. The method and application of the present invention have been described through preferred embodiments, and those skilled in the art can obviously make changes or appropriate modifications and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0084] In the fusion protein, coding nucleic acid and its elements, preparation method and application provided by the present invention, the raw materials and reagents used can all be obtained commercially. Based on the basic knowledge of conventional molecular cloning, expression construction, vaccine preparation, immunology, etc. in the art, those skilled in the art can all implement the methods of the examples of the present invention.

[0085] The following further illustrates the present invention in conjunction with examples. Among them, as a preference, a nucleic acid vaccine framework is selected for the preparation of recombinant vaccines.

[0086] Example 1 Screening of PstS antigen Due to the limitations of serotypes of Escherichia coli antigens, the present invention adopts a reverse vaccinology strategy to screen potential antigens of Escherichia coli. The main goal of the screening is to find antigens that are highly expressed in the transcriptome and highly conserved among different subtypes of Escherichia coli.

[0087] The specific screening steps are as follows: (1) Collect transcriptome data of different subtypes of Escherichia coli strains.

[0088] (2) Perform bioinformatics analysis on the transcriptome data to identify genes with high abundance expression.

[0089] (3) Analyze the conservation of these highly expressed genes among different subtypes by comparative genomics methods.

[0090] After the above systematic screening, the present invention finally identified multiple candidate antigens including PstS and YidR. Subsequently, based on immunogenic epitope prediction analysis, PstS and YidR were evaluated and determined to be the best candidate antigens.

[0091] In addition, in order to improve the expression of candidate antigens, enhance the immunogenicity and other characteristics of antigens, N-terminal truncation or amino acid mutation at individual sites was performed on the known PstS and YidR antigens.

[0092] Example 2 Design of PstS-YidR fusion protein Based on the screening results in Example 1, a fusion protein design of PstS and YidR was made, and a comparison was made with the PstS single antigen vaccine, in order to obtain a vaccine with better immune protection effect.

[0093] Based on the design requirements of multi-antigen vaccines, the PstS-YidR fusion protein needs to have a stable three-dimensional conformation in order to better express, secrete, and provide more epitope information to enhance the immunogenicity of the antigen. Therefore, starting from the tertiary structure of the PstS-YidR fusion protein and combining immunogenic epitope screening, two fusion protein molecules were constructed: (1) Fusion of the PstS antigen protein with the full-length YidR antigen protein; (2) The PstS antigen protein is fused with a truncated and stable domain polypeptide of the YidR antigen.

[0094] Such as Figure 1A (Schematic diagram of the 3D structure of the fusion protein of the full-length PstS-YidR protein) and Figure 1B (Schematic diagram of the 3D structure of the fusion protein of the truncated polypeptide of PstS-YidR) shown, the fusion proteins constructed by both methods have stable and complete spatial conformations, and the structures of the two antigens do not interfere with each other. Theoretically, both meet the expression requirements of the fusion molecule. Therefore, the two fusion proteins are used as antigen sequences for the vaccine design and comparative experiments in the subsequent examples of the present invention.

[0095] Example 3 Construction of Recombinant Nucleic Acid Vaccines In order to prepare recombinant nucleic acid vaccines containing the antigens of the present invention and to compare whether the vaccines based on the present invention have good in vitro expression effects, exemplarily, the schematic diagram of the vaccine molecular architecture involved in the examples is as Figure 2 shown, and successively includes the following elements from the N-terminus to the C-terminus: signal peptide, Fc domain, target antigen region, STABILON. Each element can be directly connected or connected through different linkers, such as GGS linker, spacer sequence or GS linker. Figure 2 The four vaccines, namely vaccine A, vaccine B, vaccine C, and vaccine D, are different in the target antigen region. The target antigens are PstS antigen, full-length proteins of PstS antigen and YidR antigen (which can be connected by a spacer sequence between them), truncated polypeptides of PstS antigen and YidR antigen (which can be connected by a spacer sequence between them), and truncated polypeptides of PstS antigen and mutated YidR antigen (which can be connected by a spacer sequence between them). In order to prepare recombinant nucleic acid vaccines capable of producing the proteins as Figure 2 shown, first, a gene expression cassette is constructed to express the antigen sequence of the present invention. The expression cassette successively includes from the 5'-end to the 3'-end: 5'UTR, CDS region, 3'UTR, PolyA. Among them, the CDS region contains the fusion molecular architecture of the present invention. Subsequently, based on codon degeneracy, the complete gene expression cassette sequence is optimized, and the DNA sequence is directly obtained by gene synthesis (entrusted to GenScript Corporation for synthesis). Finally, the synthesized gene expression cassette DNA sequence is inserted into an expression vector available for in vitro RNA transcription, as Figure 3 shown, to obtain the vector plasmid for preparing recombinant nucleic acid vaccines.

[0096] According to the above method, vectors for subsequent examples are prepared: (1) Preparation of the vector based on the recombinant nucleic acid vaccine A of the present invention Step a: Synthesize the fusion gene fragment of "signal peptide - Fc domain of human IGHG - PstS antigen - STABILON". The Fc domain of human IGHG and the PstS antigen are connected by the GGS linker sequence shown in SEQ ID NO: 8 in the amino acid sequence, and the PstS antigen and STABILON are connected by the GS linker sequence shown in SEQ ID NO: 9 in the amino acid sequence. The amino acid sequence of the signal peptide is shown in SEQ ID NO: 4, the amino acid sequence of the Fc domain of human IGHG 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.

[0097] Step b: Construct a nucleic acid vaccine framework vector.

[0098] The nucleic acid vaccine framework vector contains 5'-UTR and 3'-UTR, and can be a vector for producing any form of RNA vaccine or a vector for producing DNA vaccine.

[0099] Step c: Prepare a recombinant plasmid.

[0100] Insert the gene synthesized in step a into the vector framework of step b to obtain the preparation vector of recombinant nucleic acid vaccine A based on the present invention ( Figure 3 as shown).

[0101] (2) Preparation vector of recombinant nucleic acid vaccine B based on the present invention Step a: Synthesize the fusion gene fragment of "signal peptide - Fc domain of human IGHG - truncated polypeptide of YidR antigen - STABILON". The Fc domain of human IGHG and the truncated polypeptide of YidR antigen are connected by the GGS linker sequence shown in SEQ ID NO: 8 in the amino acid sequence, and the truncated polypeptide of YidR antigen and STABILON are connected by the GS linker sequence shown in SEQ ID NO: 9 in the amino acid sequence. The amino acid sequence of the signal peptide is shown in SEQ ID NO: 4, the amino acid sequence of the Fc domain of human IGHG is shown in SEQ ID NO: 5, the amino acid sequence of the truncated polypeptide of YidR antigen is shown in SEQID NO.2, and the amino acid sequence of STABILON is shown in SEQ ID NO: 6.

[0102] Step b: Construct a nucleic acid vaccine framework vector.

[0103] The nucleic acid vaccine framework vector contains 5'-UTR and 3'-UTR, and can be a vector for producing any form of RNA vaccine or a vector for producing DNA vaccine.

[0104] Step c: Prepare the recombinant plasmid.

[0105] Insert the gene synthesized in step a into the vector framework of step b to obtain the vector for preparing the recombinant nucleic acid vaccine B based on the present invention ( Figure 3 as shown).

[0106] (3) Vector for preparing the recombinant nucleic acid vaccine C based on the present invention Step a: Synthesize the "signal peptide - Fc domain of human IGHG - PstS antigen - full - length YidR protein - STABILON" fusion gene fragment. The Fc domain of human IGHG and the PstS antigen are linked by the GGSlinker sequence shown in the amino acid sequence of SEQ ID NO: 8. The PstS antigen and the full - length YidR protein are linked by the spacer sequence shown in the amino acid sequence of SEQ ID NO: 7. The full - length YidR protein and STABILON are linked by the GSlinker sequence shown in the amino acid sequence of 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 Fc domain of human IGHG 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, and the amino acid sequence of STABILON is shown in SEQ ID NO: 6.

[0107] Step b: Construct the nucleic acid vaccine framework vector.

[0108] The nucleic acid vaccine framework vector contains 5'-UTR and 3'-UTR, and can be a vector for producing any form of RNA vaccine or a vector for producing DNA vaccine.

[0109] Step c: Prepare the recombinant plasmid.

[0110] Insert the gene synthesized in step a into the vector framework of step b to obtain the vector for preparing the recombinant nucleic acid vaccine C based on the present invention ( Figure 3 as shown).

[0111] (4) Vector for preparing the recombinant nucleic acid vaccine D based on the present invention Step a: Synthesize the "signal peptide - Fc domain of human IGHG - PstS antigen - truncated polypeptide of mutated YidR antigen - STABILON" fusion gene fragment. The Fc domain of human IGHG and the PstS antigen are linked by the GGS linker sequence shown in SEQ ID NO:8. The PstS antigen and the truncated polypeptide of the mutated YidR antigen are linked by the spacer sequence shown in SEQ ID NO:7. The truncated polypeptide of the mutated YidR 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 Fc domain of human IGHG 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 truncated polypeptide of the mutated YidR antigen is shown in SEQ ID NO:3, and the amino acid sequence of STABILON is shown in SEQ ID NO:6.

[0112] Step b: Construct a nucleic acid vaccine framework vector.

[0113] The nucleic acid vaccine framework vector contains 5'-UTR and 3'-UTR, and can be a vector for producing any form of RNA vaccine or a vector for producing DNA vaccine.

[0114] Step c: Prepare a recombinant plasmid.

[0115] Insert the gene synthesized in step a into the vector framework of step b to obtain the preparation vector of the recombinant nucleic acid vaccine D based on the present invention ( Figure 3 as shown).

[0116] Table 1 Protein amino acid sequences involved in the present invention Amino acid sequence and sequence number PstS antigen EASLTGAGATFPAPVYAKWADTYQKETGNKVNYQGIGSSGGVKQIIANTVDFGASDAPLSDEKLAQEGLFQFPTVIGGVVLAVNIPGLKSGELVLDGKTLGDIYLGKIKKWDDEAIAKLNPGLKLPSQNIAVVRRADGSGTSFVFTSYLAKVNEEWKNNVGTGSTVKWPIGLGGKGNDGIAAFVQRLPGAIGYVEYAYAKQNNLAYTKLISADGKPVSPTEENFANAAKGADWSKTFAQDLTNQKGEDAWPITSTTFILIHKDQKKPEQGTEVLKFFDWAYKTGAKQANDLDYASLPDSVVEQVRAAWKTNIKDSSGKPLY (SEQ ID NO: 1) Truncated polypeptide fragment of YidR antigen RAMKQITFAPRNHLLTNTNTWTPDSQWLVFDVRPSGASFTGETIERVNIHTGEVEVIYRASQGAHVGVVTVHPKSEKYVFIHGPENPDETWHYDFHHRRGVIAEGGKVSNLDAMDITAPYTPGALRGGSHVHVFSPNGERVSFTYNDHVMHELDPALDLRNVGVAAPFGPVNVQKQHPREYSGSHWCVLVSKTTPTPQPGSDEINRAYEEGWVGNHALAFIGDTLSPKGEKVPELFIVELPQDEAGWKAAGDAPLSGTETTLPAPPRGVVQRRLTFTHHRAYPGLVNVPRHWVRCNPQGTQIAFLMRDDNGIMQLWLISPQGGEPRQLTHQKTDIQSAFNWHPSGEWLGFVLDNRIACAHAQSGEVEYLTENHANPPSADAVVFSPDGQWLAWMEGGQLWITETDR (SEQ ID NO: 2) Mutated truncated polypeptide of YidR antigen 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 MMAGPVLYQDRAMKQITFAPRNHLLTNTNTWTPDSQWLVFDVRPSGASFTGETIERVNIHTGEVEVIYRASQGAHVGVVTVHPKSEKYVFIHGPENPDETWHYDFHHRRGVIAEGGKVSNLDAMDITAPYTPGALRGGSHVHVFSPNGERVSFTYNDHVMHELDPALDLRNVGVAAPFGPVNVQKQHPREYSGSHWCVLVSKTTPTPQPGSDEINRAYEEGWVGNHALAFIGDTLSPKGEKVPELFIVELPQDEAGWKAAGDAPLSGTETTLPAPPRGVVQRRLTFTHHRAYPGLVNVPRHWVRCNPQGTQIAFLMRDDNGIMQLWLISPQGGEPRQLTHQKTDIQSAFNWHPSGEWLGFVLDNRIACAHAQSGEVEYLTENHANPPSADAVVFSPDGQWLAWMEGGQLWITETDR (SEQ ID NO:11) Example 4 Preparation of the recombinant nucleic acid vaccine of the present invention (1) Preparation of capped mRNA vaccine Step a: Digest the vector plasmid used for producing the capped mRNA vaccine in Example 3 to linearize it, and obtain the linearized plasmid for in vitro transcription.

[0117] Step b: Perform an in vitro co-transcription capping reaction on the linearized plasmid, add the 7-methylguanylate cap structure to the 5' end of the transcribed mRNA, and degrade the template DNA.

[0118] (2) Preparation of uncapped mRNA vaccine Step a: Digest and linearize the vector plasmid used for producing the uncapped mRNA vaccine in Example 3 to obtain the linearized plasmid for in vitro transcription.

[0119] Step b: Conduct an in vitro uncapped transcription reaction on the linearized plasmid and degrade the template DNA.

[0120] (3) Preparation of DNA vaccine Step a: Amplify the vector plasmid used for producing the DNA vaccine in Example 3 to obtain a large amount of target plasmid for purification.

[0121] Step b: Extract and purify the target plasmid using an endotoxin-removing plasmid extraction and purification kit.

[0122] Example 5 Quality control of in vitro transcription of the recombinant nucleic acid of the present invention and vaccine preparation Prepare vaccines A (recombinant nucleic acid vaccine A based on the present invention), B (recombinant nucleic acid vaccine B based on the present invention), C (recombinant nucleic acid vaccine C based on the present invention), and D (recombinant nucleic acid vaccine D based on the present invention) by the method for preparing the capped mRNA vaccine in Example 4. Detect the purity of the produced recombinant nucleic acid. The purity of the recombinant nucleic acid used in the experiment is greater than or equal to 85%. The quality control peak diagrams of the recombinant nucleic acid based on the present invention are as shown in Figure 4A 、 Figure 4B 、 Figure 4C 、 Figure 4D . Specifically described as: (1) Recombinant nucleic acid vaccine A based on the present invention, with a purity of 90.3%. The quality control peak diagram and the purity detection results are as shown in Figure 4A ; (2) Recombinant nucleic acid vaccine B based on the present invention, with a purity of 88.5%. The quality control peak diagram and the purity detection results are as shown in Figure 4B ; (3) Recombinant nucleic acid vaccine C based on the present invention, with a purity of 88.5%. The quality control peak diagram and the purity detection results are as shown in Figure 4C ; (4) Recombinant nucleic acid vaccine D based on the present invention, with a purity of 85%. The quality control peak diagram and the purity detection results are as shown in Figure 4D . The above purities all meet the quality requirements for cell transfection experiments and vaccine production.

[0123] Example 6 In vitro expression effect of the recombinant nucleic acid of the present invention Transfect the vaccines in Example 5 into HEK293T cells through a cell transfection reagent. After culturing in vitro for 48 hours, collect the protein, conduct Western blot detection, and calculate the protein molecular weights of vaccines A, B, C, and D, as shown in Table 2.

[0124] Table 2 Protein molecular weights of vaccines A, B, C, and D

[0125] Figures 5A - 5C Shows the in vitro expression WB (Western blot) test results of vaccines A, B, C, and D transfected into HEK293 cells, where Figure 5A is the in vitro expression WB test result of vaccine A transfected into HEK293 cells, Figure 5B is the in vitro expression WB test result of vaccine B transfected into HEK293 cells, Figure 5C is the in vitro expression WB test result of vaccines C and D transfected into HEK293 cells. The results show that the expressed antigens are all humoral immune antigens and theoretically significant expression can be detected in the supernatant.

[0126] As Figures 5A - 5C shown, signals of the target proteins were detected in the supernatants of vaccines A, B, C, and D, and the molecular weights were all in line with expectations. This proves that the fusion protein based on the present invention can not only be successfully translated and correctly folded in eukaryotic cells, but also has a stable structure and can be secreted extracellularly.

[0127] Comparing Figure 5A , Figure 5B results, the expression level of vaccine A in the supernatant is significantly higher than that of vaccine B, proving that when the PstS antigen is expressed alone, the secretion effect is better than that of the YidR antigen; according to Figure 5C the results shown, the expression level of vaccine D in the supernatant is significantly higher than that of vaccine C, proving that among the two PstS-YidR fusion protein designs, the design method of vaccine D is better, and choosing the truncated polypeptide of the mutated YidR antigen of the present invention instead of the full-length YidR antigen protein can significantly improve the secretion efficiency of the PstS-YidR fusion protein.

[0128] From the above comparative analysis, it can be seen that vaccine A expressing the PstS antigen protein and vaccine D expressing the "PstS antigen - truncated polypeptide of the mutated YidR antigen" fusion protein are better choices for vaccine design, which is more conducive to antigen presentation and the activation of humoral immunity. Therefore, vaccines A and D are subsequently used for animal experiments to verify the immune protection effect. At the same time, compare whether there are differences in the immune protection effects between single antigen and multi-antigen fusion proteins.

[0129] Example 7 Preventive effect of the recombinant nucleic acid vaccine based on the present invention in a mouse gavage infection model To verify whether the nucleic acid vaccine based on the present invention has an immune protection effect, in this example, mice immunized with vaccine A (experimental group), mice immunized with vaccine D (experimental group) and non-immunized mice (blank control group, i.e., treated with PBS) were respectively subjected to an immunization and challenge comparison experiment.

[0130] Fifteen male BALB / C mice, 6 - 8 weeks old and weighing 18 - 25 g, were selected for the experiment. They were all housed in independent cages with constant temperature and humidity, and were allowed to adapt to the environment for 7 days in advance. The temperature in the breeding room was 20 - 26 °C, the humidity was 40 - 70%, with a light - dark cycle. There was light from 8 am to 8 pm and darkness from 8 pm to 8 am the next day. Sufficient feed was continuously provided, and the mice could freely ingest it without limit. They drank sterile water, and the water bottles supplied water continuously for free ingestion. After the mice were adaptively bred, they were randomly divided into 3 groups, with 5 mice in each group, and each mouse was marked with an ear tag. Specifically as shown in Table 3, the dose in this table and in the following text refers to the amount of the active ingredient.

[0131] Table 3 Grouping and immunization process of mouse immunization experiment in Example 5

[0132] The mice in each group were immunized twice according to the immunization process in Table 3. On Day 32, the mice were gavaged with 100 μl of a penicillin - streptomycin antibiotic mixture at a concentration of 50 mg / ml once a day for 3 consecutive days. On Day 35, the mice were fasted overnight before bacterial challenge. The mice were gavaged with 100 μl of Escherichia coli bacterial solution (enterohemorrhagic Escherichia coli EHEC, 1×10 9 CFU), and after gavaging, they were normally bred. The clinical manifestations of the mice were observed, monitored, and recorded every day. On Day 40, the mice were sacrificed for sampling. The immunization, challenge, and sampling processes were as Figure 6 shown. The ileum and colon tissues of the mice were collected to detect the bacterial load in the tissues and histological sections, and the immune protection effect of the vaccine was comprehensively evaluated.

[0133] For each mouse, 10 mg of ileum and colon tissues were aseptically excised. The ileum and colon tissues were added to sterile PBS buffer and ground on ice until there was no obvious precipitate. The blood was diluted 1:1000 with sterile PBS buffer, and after mixing, 100 μl of the diluted solution was evenly spread on LB solid medium. The culture dish was placed in an incubator at 37 °C for culture. After 24 h of bacterial culture, the culture dish was taken out, and the bacterial colonies were counted and photographed to observe the protective effect of the vaccine on the mice. The results were as Figures 7A - 7B shown. Figure 7A This is a comparative analysis chart of the bacterial load (colony number on the plate) in the ileum tissue of mice after immune challenge. The results showed that compared with the blank control group of mice (immunized with PBS), the Escherichia coli load in the vaccine - immunized groups of mice (immunized with Vaccine A and Vaccine D) decreased significantly. The bacterial load in the ileum tissue of the blank control group of mice was about 5 times that of the vaccine - immunized groups of mice. Figure 7BIt is a comparative analysis chart of the bacterial load (colony forming units on plates) in the colon tissues of mice after immune challenge. The results show that compared with the blank control group of mice (immunized with PBS), the Escherichia coli load in the vaccine-immunized groups of mice (immunized with Vaccine A and Vaccine D) decreased significantly, and the bacterial load in the colon tissues of the blank control group of mice was approximately 4 times that of the vaccine-immunized groups of mice.

[0134] The colon tissues of each mouse were taken, fixed with paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin respectively, and the pathological changes in the colon, kidney, and spleen tissues were observed under a microscope. The results are as Figure 8 shown. Figure 8 It is a comparative photo of the colon tissue sections (HE staining) of mice after immune challenge. The results show that the intestinal villi structure of normal mice is intact, without obvious lesions and inflammatory infiltration; a large number of inflammatory cells infiltrated and aggregated in the submucosa of the intestinal mucosa of the blank control group of mice (immunized with PBS), showing symptoms after Escherichia coli infection; a small number of intestinal villi in the vaccine-immunized groups of mice (immunized with Vaccine A and Vaccine D) were broken and dissolved, and partial inflammatory cell infiltration and aggregation appeared in the mucosal layer. Analyzing the above results, the mice immunized with Vaccine A and Vaccine D based on the present invention all showed resistance to Escherichia coli infection, proving that the vaccines based on the present invention can induce mice to produce immune protection against Escherichia coli and prevent Escherichia coli infection.

[0135] In summary, both the novel antigen PstS and the PstS-YidR fusion protein provided by the present invention can induce effective protective immunity in the model animal mice and have a good preventive effect against Escherichia coli infection. Therefore, the present invention can be applied to the production and research and development of immune drugs, filling the gap in the current research and development field of Escherichia coli vaccines, and having extremely high commercial value and broad application prospects.

[0136] The above embodiments of the present disclosure are only examples for clearly illustrating the present disclosure and are not limitations on the implementation manners of the present disclosure. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present disclosure shall be included within the protection scope of the claims of the present disclosure.

Claims

1. A fusion protein for preventing Escherichia coli infection, characterized in that: It comprises a phosphate-binding protein PstS (Phosphate-binding protein PstS) antigen or a fragment thereof, and the amino acid sequence of the PstS antigen protein is shown in SEQ ID NO.

1.

2. The fusion protein according to claim 1, characterized in that It also contains YidR antigen (DUF3748 domain-containing protein) or a fragment thereof, wherein the YidR antigen or the fragment thereof contains a full-length YidR antigen protein, a truncated YidR antigen polypeptide, or a mutated YidR antigen truncated polypeptide.

3. According to the fusion protein of claims 1 and 2, the antigen or fragment thereof is derived from bacteria; preferably, the bacteria is a Gram-negative bacterium; preferably, the Gram-negative bacterium is an Enterobacteriaceae bacterium; preferably, the Enterobacteriaceae bacterium is Escherichia coli.

4. The fusion protein according to claims 2 and 3, characterized in that The fusion protein comprises, from N-terminus to 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 YidR antigen full-length protein, the YidR antigen truncated polypeptide, or the mutated YidR antigen truncated polypeptide; preferably, the amino acid sequence of the PstS antigen or a fragment thereof is as shown in SEQ ID NO.1, the amino acid sequence of the YidR antigen full-length protein is as shown in SEQ ID NO.11, the amino acid sequence of the YidR antigen truncated polypeptide is as shown in SEQ ID NO.2, and the amino acid sequence of the mutated YidR antigen truncated polypeptide is as shown in SEQ ID NO.

3.

5. The fusion protein according to claims 1-4, characterized in that The N-terminus of the fusion protein further comprises a signal peptide and / or the Fc domain of human immunoglobulin heavy chain constant region γ1 protein (IGHG1); or, the C-terminus of the fusion protein further comprises the C-terminal polypeptide fragment STABILON of the human S5a / PSMD4 proteasome subunit.

6. The fusion protein according to claim 5, characterized in that The signal peptide is derived from human Azurocidin protein, and its amino acid sequence 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.

7. The fusion protein according to claim 5 or 6, characterized in that The different elements of the fusion protein may be optionally connected via a GGS linker sequence, a spacer sequence, and / or a GS linker sequence; preferably, the amino acid sequence of the spacer sequence is as shown in SEQ ID NO: 7, the amino acid sequence of the GGS linker sequence is as shown in SEQ ID NO: 8, and the amino acid sequence of the GS linker sequence is as shown in SEQ ID NO:

9.

8. The fusion protein according to any one of claims 1 to 7, characterized in that Contains any one of the following 4 fusion proteins: Fusion protein A: from N-terminus to C-terminus, it includes: signal peptide, Fc domain, GGS linker, PstS antigen or its fragment, GS linker, STABILON; Fusion protein B: from N-terminus to C-terminus, it includes: signal peptide, Fc domain, GGS linker, YidR antigen truncated polypeptide, spacer sequence, YidR antigen truncated polypeptide, GS linker, STABILON; Fusion protein C: from N-terminus to C-terminus, it includes: signal peptide, Fc domain, GGS linker, PstS antigen or its fragment, spacer sequence, YidR antigen full-length protein, GS linker, STABILON; Fusion protein D: from N-terminus to C-terminus, it includes: signal peptide, Fc domain, GGS linker, PstS antigen or its fragment, spacer sequence, mutated YidR antigen truncated polypeptide, GS linker, STABILON; Preferably, the amino acid sequence of the PstS antigen or its fragment is as shown in SEQ ID NO.1, the amino acid sequence of the full-length protein of the YidR antigen is as shown in SEQ ID NO.11, the amino acid sequence of the YidR antigen truncated polypeptide is as shown in SEQ ID NO.2, the amino acid sequence of the mutated YidR antigen truncated polypeptide is as shown in SEQ ID NO.3, the amino acid sequence of the signal peptide is as shown in SEQ ID NO: 4, the amino acid sequence of the Fc domain is as shown in SEQ ID NO: 5, the amino acid sequence of STABILON is as shown in SEQ ID NO.6, the amino acid sequence of the spacer sequence is as shown in SEQ ID NO: 7, the amino acid sequence of the GGS linker sequence is as shown in SEQ ID NO: 8, and the amino acid sequence of the GS linker sequence is as shown in SEQ ID NO: 9; More preferably, the amino acid sequence of the fusion protein is as shown in SEQ ID NO.

10.

9. A recombinant nucleic acid molecule, characterized in that Comprising a nucleic acid encoding the fusion protein according to any one of claims 1 to 8.

10. A recombinant gene expression cassette, characterized in that: Comprising the recombinant nucleic acid molecule of claim 9.

11. A recombinant vector, characterized in that: Comprising the recombinant nucleic acid molecule according to claim 9, or the recombinant gene expression cassette according to claim 10.

12. A recombinant host cell, characterized in that Comprising the recombinant nucleic acid molecule of claim 9, or the recombinant gene expression cassette of claim 10, or the recombinant vector of claim 11.

13. An immunogenic composition or a pharmaceutical composition, characterized in that: Comprising one or more fusion proteins of any one of claims 1 to 8, and / or one or more recombinant nucleic acid molecules of claim 9, and / or one or more recombinant gene expression cassettes of claim 10, and / or one or more recombinant vectors of claim 11, and / or one or more recombinant host cells of claim 12; preferably, the immunogenic composition or pharmaceutical composition further comprises a pharmaceutically acceptable carrier; more preferably, the immunogenic composition or pharmaceutical composition further comprises other drugs for preventing diseases caused by Escherichia coli; most preferably, the other drugs for preventing diseases caused by Escherichia coli comprise antibiotics.

14. A recombinant vaccine, characterized in that: Comprising one or more fusion proteins of any one of claims 1 to 8, and / or one or more recombinant nucleic acid molecules of claim 9, and / or one or more recombinant gene expression cassettes of claim 10, and / or one or more recombinant vectors of claim 11, and / or one or more recombinant host cells of claim 12, and / or one or more immunogenic compositions or pharmaceutical compositions of claim 13; preferably, the recombinant vaccine is a nucleic acid vaccine or a subunit vaccine; more preferably, the recombinant vaccine is a nucleic acid vaccine.

15. Use of one or more fusion proteins according to any one of claims 1 to 8, and / or one or more recombinant nucleic acid molecules according to claim 9, and / or one or more recombinant gene expression cassettes according to claim 10, and / or one or more recombinant vectors according to claim 11, and / or one or more recombinant host cells according to claim 12, and / or one or more immunogenic compositions or pharmaceutical compositions according to claim 13, and / or one or more recombinant vaccines according to claim 14 in the preparation of a medicament for preventing diseases and / or secondary infections caused by Escherichia coli.

16. The use according to claim 15, characterized in that The diseases and / or secondary infections caused by the Escherichia coli include intestinal infection, urinary tract infection, sepsis, bacteremia, meningitis, peritonitis, pneumonia, mastitis, and hemolytic uremic syndrome; preferably, the intestinal infection includes enteritis and / or diarrhea; preferably, the urinary tract infection includes cystitis and / or pyelonephritis.

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