Immune composition comprising fatty acid-modified recombinant mrka protein and use thereof

Abstract

Provided are a fatty acid-modified recombinant MrkA protein, an immune composition comprising the recombinant MrkA protein, and a use thereof. Further provided is an immune composition in which polysaccharides are displayed on the fatty acid-modified recombinant MrkA protein by means of a SpyTag / SpyCatcher linker, to enhance immunogenicity.

Description

An immunoassay containing fatty acid-modified recombinant MrkA protein and its application. Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an immune composition containing recombinant MrkA protein with fatty acid modification and its application. Background Technology

[0002] Klebsiella pneumoniae (Kp) is a capsule-bound Gram-negative bacterium widely distributed in the natural environment and the digestive tract of mammals. Kp is an opportunistic pathogen, typically causing infection through wounds, invasive medical device catheters, or nosocomial infections, leading to pneumonia, bacteremia, and urinary tract infections. ICU patients and the elderly, among others with weakened immune systems, are the most susceptible to infection. Literature reports that Kp infection accounts for 83% of deaths in ventilator-associated pneumonia (VAP) cases. Klebsiella pneumoniae infection is also a leading cause of neonatal death in developing countries. Klebsiella pneumoniae naturally carries the β-lactamase gene SHV-1 and continuously acquires antibiotic resistance genes through plasmids and other transferable genetic material, such as aminoglycoside resistance genes, quinolone resistance genes, polymyxin resistance genes, and tigecycline resistance genes. Therefore, Klebsiella pneumoniae is one of the most difficult pathogens to treat. Klebsiella pneumoniae is also a vector for resistance genes, transferring drug-resistant genes from the environment to clinical pathogens. It is currently considered a major source and transmission tool of antibiotic resistance worldwide.

[0003] Among numerous strategies for preventing and controlling bacterial infections, vaccination is considered the most economical and effective method. Compared to viruses, bacteria have larger genes, more virulence factors, and more complex pathogenic mechanisms, immune evasion mechanisms, and drug resistance mechanisms. Therefore, the selection and design of antigens, delivery vectors, adjuvants, and their compatibility in bacterial vaccines require more careful consideration. The safety, accessibility, conservation, and abundance of antigens are the most important factors in vaccine antigen selection. Type 3 fimbriae are distributed throughout the surface of bacteria and are ubiquitous in Klebsiella pneumoniae, mediating its adhesion to the respiratory tract or other tissues. MrkA protein is a major component of type 3 fimbriae filaments, is associated with biofilm formation, and is highly conserved in Klebsiella pneumoniae and even Enterobacteriaceae, making it an ideal candidate antigen. Studies have shown that directly extracting type 3 fimbriae complexes from Klebsiella pneumoniae or immunizing mice with recombinantly expressed MrkA protein can induce protective antibodies in mice. Positively isolated MrkA-specific monoclonal antibodies from antiserum also exhibit protective activity. However, the directly extracted type 3 fimbriae complex is highly heterogeneous, and the recombinant expression of MrkA protein exists in the form of inclusion bodies. Subsequent problems include protein refolding, structural confirmation, stability, and relatively weak immunogenicity, which prevents MrkA protein from becoming a candidate protective antigen.

[0004] Bacteria possess a class of proteins whose N-terminal cysteine ​​(Cys) is modified with fatty acid and anchored to the cell surface via fatty acid chains; these are called bacterial lipoproteins. The in vivo synthesis of bacterial lipoproteins is guided by a signal peptide. Bioinformatics and statistical analysis of the signal peptide sequences of naturally occurring and non-fatty acid modified bacterial proteins revealed that the signal peptide of fatty acid modified proteins contains nonpolar amino acids near the signal peptidase cleavage site, exhibiting a degree of conservation. The conserved sequence of the last four amino acids in this region was summarized as [L / V / I][A / S / T / V / I][G / A / S][C], where the C-terminal cysteine ​​is the lipid modification site. Guided by the signal peptide, nascent peptide chains are localized to the extracellular space via the Sec or TAT secretion pathway. Lgt enzymes, using phosphatidylglycerol (PG) as a substrate, attach diacylglycerol to the sulfhydryl group of Cys. LspA enzymes cleave the portion of the signal peptide excluding the C-terminal Cys group. Lnt enzymes, using phosphatidylethanolamine (PE) as a substrate, transfer a fatty acid chain to the alpha-amino group of Cys. Bacterial fatty acid modification is heterogeneous, mainly manifested in the number and length of fatty acid chains. The acyl carbon chain length of bacterial lipoproteins is usually between C14 and C18, but the vast majority are C16.

[0005] Displaying antigens on the surface of protein nanocarriers is an effective way to improve antigen immunogenicity. A modular preparation scheme, which combines nanoparticles and loaded antigens through a universal, efficient, and controllable linking method, effectively solves problems such as low expression efficiency, particle assembly failure, structural instability, and loss of immunogenicity caused by carrier-antigen fusion expression. The SpyTag / SpyCatcher linking system is one of the most thoroughly studied protein linking systems. Due to its rapid reaction, specificity, broad tolerance to reaction conditions, and irreversibility, this system is widely used in vaccine development and research. By fusing it with pathogen antigens and suitable carrier proteins, precise covalent linking of antigens and carrier proteins can be achieved. In particular, it allows for repeated display of antigens on the surface of self-polymerizing nanocarrier proteins, improving antigen presentation efficiency and immunogenicity. This linkage system originates from the CnaB2 domain of the fibronectin-binding protein of Streptococcus pyogenes. Lys31 and Asp117 of the CnaB2 domain can form intramolecular isopeptide bonds. After the CnaB2 domain is split into an N-terminal domain containing Lys31 (SpyCatcher, 116aa) and a C-terminal polypeptide containing Asp117 (SpyTag, 13aa), the two fragments can still form specific isopeptide bonds. To further optimize the SpyTag / SpyCatcher ligation system, Mark Howarth's team analyzed the relationship between its structure and function, demonstrating the minimum SpyCatcher required to form isopeptide bonds. To improve the ligation reaction speed and efficiency, the team artificially designed the SpyTag(13aa) / SpyCatcher(116aa) ligation system, developing the SpyTag002 / SpyCatcher002 and SpyTag003 / SpyCatcher003 ligation systems.

[0006] Currently, among the subunit vaccine technologies for Klebsiella pneumoniae, protein subunit vaccines and polysaccharide conjugate vaccines are the main candidate vaccines. Antigen selection is limited to polysaccharide antigens (OPS or CPS) or protein antigens, resulting in weak immunogenicity and the induction of immune memory only against either polysaccharide or protein antigens. To address the characteristics of susceptible populations and improve antigen immunogenicity while simultaneously inducing a strong immune response and immune memory against both Klebsiella pneumoniae polysaccharides and proteins, this invention selects the conserved protein MrkA, distributed throughout the surface of Klebsiella pneumoniae, as the protein antigen. Through fatty acid modification, it self-polymerizes into nanoparticles. Compared to unmodified MrkA protein, this fatty acid-modified recombinant MrkA protein can induce faster and stronger humoral immunity through injection immunization, and can also induce mucosal and humoral immunity through respiratory nebulization. Furthermore, by employing a pluggable modular preparation technology to display the polysaccharide antigen on the surface of the fatty acid-modified recombinant MrkA protein nanoparticles, a dual-antigen immunoassay for Klebsiella pneumoniae is provided. Compared with existing Klebsiella pneumoniae subunit vaccine regimens, the immune composition provided by this invention can induce immune memory not only against Klebsiella pneumoniae polysaccharides, but also against Klebsiella pneumoniae surface proteins. In particular, it can induce mucosal immunity through respiratory nebulization. Summary of the Invention

[0007] In view of the problems existing in the prior art, the present invention provides an immune composition containing fatty acid modified recombinant MrkA protein and its application, specifically a fatty acid modified recombinant MrkA protein and an immune composition containing such fatty acid modified recombinant MrkA protein and its application.

[0008] According to a first aspect of the present invention, a recombinant MrkA protein modified with fatty acid esterification is provided, which contains an N-terminal cysteine ​​(Cys) modified with fatty acid esterification; wherein the N-terminal cysteine ​​(Cys) modified with fatty acid esterification has a thiol group modified with diacylglycerol, or the thiol group of Cys is modified with diacylglycerol and the α-amino group is modified with an acyl group; wherein the carbon chain length of the acyl group is C16 to C18.

[0009] Preferably, the recombinant MrkA protein comprises a polypeptide with the amino acid sequence shown in SEQ ID NO.1.

[0010] According to a second aspect of the present invention, an immune composition containing fatty acid-modified recombinant MrkA protein is provided, the immune composition containing fatty acid-modified recombinant MrkA protein and a polysaccharide coupled thereto.

[0011] The polysaccharide is covalently linked to the fatty acid-modified recombinant MrkA protein via a linker.

[0012] The linker is an isopeptide linker, preferably a SpyCatcher / SpyTag linker.

[0013] The polysaccharide forms a polysaccharide-protein conjugate containing SpyCatcher protein with the recombinant fusion protein containing SpyCatcher protein, and the polysaccharide-protein conjugate containing SpyCatcher protein is covalently linked to the fatty acid-modified MrkA protein containing SpyTag polypeptide through the formation of isopeptide bonds.

[0014] The SpyCatcher protein is a polypeptide containing any of the amino acid sequences shown in SEQ ID NO. 11-13, and the SpyTag polypeptide is any of the amino acid sequences shown in SEQ ID NO. 2-4.

[0015] When the recombinant fusion protein containing SpyCatcher protein contains the amino acid sequence shown in SEQ ID NO.11, the SpyTag polypeptide contained in the corresponding fatty acid-modified recombinant MrkA protein preferably uses the amino acid sequence shown in SEQ ID NO.2.

[0016] When the recombinant fusion protein containing SpyCatcher protein contains the amino acid sequence shown in SEQ ID NO.12, the SpyTag polypeptide contained in the corresponding fatty acid-modified recombinant MrkA protein preferably uses the amino acid sequence shown in SEQ ID NO.3.

[0017] When the recombinant fusion protein containing SpyCatcher protein contains the amino acid sequence shown in SEQ ID NO.13, the SpyTag polypeptide contained in the corresponding fatty acid-modified recombinant MrkA protein preferably uses the amino acid sequence shown in SEQ ID NO.4.

[0018] Taking SpyCatcher protein containing the amino acid sequence shown in SEQ ID NO. 11 and SpyTag polypeptide containing the amino acid sequence shown in SEQ ID NO. 2 as examples, the amino acid sequence of the fatty acid-modified recombinant MrkA protein containing SpyTag polypeptide is shown in SEQ ID NO. 21. The amino acid sequence of the polysaccharide-protein conjugate containing SpyCatcher protein is shown in SEQ ID NO. 22. To facilitate protein purification, a purification tag can also be added. Taking the commonly used His tag as an example, the amino acid sequence of the fatty acid-modified recombinant MrkA protein containing SpyTag polypeptide and His tag is shown in SEQ ID NO. 18, and the amino acid sequence of the polysaccharide-protein conjugate containing SpyCatcher protein and His tag is shown in SEQ ID NO. 16.

[0019] The polysaccharide-protein conjugate containing SpyCatcher protein can be prepared by chemical coupling or by biological coupling. The polysaccharide includes Klebsiella pneumoniae polysaccharide, specifically Klebsiella pneumoniae O polysaccharide (OPS) and / or Klebsiella pneumoniae capsular polysaccharide (CPS).

[0020] Taking Klebsiella pneumoniae O-polysaccharide as an example, a Klebsiella pneumoniae O-polysaccharide protein conjugate containing SpyCatcher protein was prepared using a bio-conjugation method. The preparation method is as follows: a DNA fragment containing the coding sequence of Neisseria meningitidis O-oligosaccharide transferase PglL, a DNA fragment containing the coding sequence of signal peptide, SpyCatcher protein, and O glycosylation motif, were introduced into Klebsiella pneumoniae with an O antigen transferase gene deficiency. The recombinant bacteria were cultured to obtain a conjugate of Klebsiella pneumoniae O-polysaccharide and recombinant SpyCatcher protein containing O glycosylation motif. The amino acid sequence of Neisseria meningitidis O-oligosaccharide transferase PglL is shown in SEQ ID NO.15, and the amino acid sequence of recombinant SpyCatcher protein containing O glycosylation motif is shown in SEQ ID NO.16.

[0021] Alternatively, a DNA fragment containing the coding sequence of Neisseria meningitidis O-oligosaccharide transferase PglL, a DNA fragment containing the coding sequence of signal peptide, SpyCatcher protein and O glycosylation motif, and a DNA sequence containing the O polysaccharide synthesis locus of Klebsiella pneumoniae can be introduced into Escherichia coli with an O antigen transferase gene deficiency. The recombinant bacteria can then be cultured to obtain a conjugate of Klebsiella pneumoniae O polysaccharide and recombinant SpyCatcher protein containing the O glycosylation motif.

[0022] According to a third aspect of the present invention, a method for preparing recombinant MrkA protein modified with fatty acid as described above is provided, the method comprising the following steps:

[0023] 1) DNA fragments containing signal peptide coding sequences, MrkA protein coding sequences, and SpyTag polypeptide coding sequences, or with or without such sequences, were introduced into E. coli, and recombinant E. coli were cultured to express recombinant MrkA protein with N-terminal fatty acid modification.

[0024] 2) Cultivate the recombinant Escherichia coli described in step 1) to express the fatty acid-modified recombinant MrkA protein;

[0025] 3) The recombinant Escherichia coli was lysed, a surfactant was added, and the fatty acid-modified recombinant MrkA fusion protein was extracted and purified from the lysate.

[0026] The signal peptide is characterized by the following amino acid sequence at the last four C-terminal positions: [L / V / I][A / S / T / V / I][G / A / S][C]; the amino acid sequence of the signal peptide is shown in any one of SEQ ID NO.5-SEQ ID NO.7. The DNA fragment containing the signal peptide coding sequence, the MrkA protein coding sequence, or the SpyTag polypeptide coding sequence, may or may not contain the SpyTag polypeptide coding sequence, can be introduced into *E. coli* via a recombinant expression vector or integrated into the *E. coli* genome.

[0027] According to a fourth aspect of the present invention, a method for preparing a composition containing fatty acid-modified recombinant MrkA protein as described above is provided. The method comprises mixing fatty acid-modified recombinant MrkA protein containing SpyTag polypeptide and a bacterial polysaccharide protein conjugate containing SpyCatcher protein in a certain proportion, and reacting fully under suitable conditions to obtain an immune composition.

[0028] According to the fifth aspect of the technical solution of the present invention, an application is provided for the fatty acid-modified recombinant MrkA protein as described above in the preparation of a product that can induce the production of antigen-specific antibodies in animals. The fatty acid-modified recombinant MrkA protein is administered via intramuscular injection, subcutaneous injection, nasal instillation, or nebulized inhalation.

[0029] According to a sixth aspect of the present invention, a composition containing fatty acid-modified recombinant MrkA protein as described above is provided for use in the preparation of a product that can induce the production of antigen-specific antibodies in animals. The immunization composition is administered via intramuscular injection, subcutaneous injection, nasal drops, or nebulized inhalation.

[0030] According to a seventh aspect of the present invention, an immunization vaccine is provided, the vaccine comprising the above-described fatty acid-modified recombinant MrkA protein, or the above-described composition containing fatty acid-modified recombinant MrkA protein.

[0031] According to an eighth aspect of the present invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising the above-described fatty acid-modified recombinant MrkA protein, or the above-described composition containing fatty acid-modified recombinant MrkA protein.

[0032] According to a ninth aspect of the present invention, a biomaterial is provided, said biomaterial being any of the following:

[0033] C1) A nucleic acid molecule encoding the fatty acid-modified recombinant MrkA protein as described in any one of claims 1-14;

[0034] C2) An expression cassette containing the nucleic acid molecule described in C1);

[0035] C3) A recombinant vector containing the nucleic acid molecule described in C1), or a recombinant vector containing the expression cassette described in C2);

[0036] C4) Recombinant microorganisms containing the nucleic acid molecules described in C1), or recombinant microorganisms containing the expression cassette described in C2), or recombinant microorganisms containing the recombinant vector described in C3);

[0037] C5) Recombinant cells containing the nucleic acid molecule described in C1), or recombinant cells containing the expression cassette described in C2), or recombinant cells containing the recombinant vector described in C3).

[0038] Compared with the prior art, the present invention has at least the following beneficial effects:

[0039] This invention discloses a fatty acid-modified recombinant Klebsiella pneumoniae MrkA protein, which can be polymerized into nanoparticles. A single injection immunization of mice induces high titers of MrkA-specific IgG antibodies, and three injections protect mice against lethal challenges from clinically resistant strains of Klebsiella pneumoniae. Respiratory nebulization of mice, after three immunizations, yields detectable high titers of MrkA-specific serum IgG and IgA antibodies. Compared to the unmodified recombinant MrkA protein, this antigen induces faster and stronger humoral immunity via injection and mucosal immunity via respiratory nebulization. Furthermore, this invention utilizes a SpyTag / SpyCatcher linker to display Klebsiella pneumoniae polysaccharide antigens on the fatty acid-modified recombinant MrkA protein, enhancing the immunogenicity of the polysaccharide and thus providing a Klebsiella pneumoniae multi-antigen immunoassay composition. Attached Figure Description

[0040] Figure 1 shows the SDS-PAGE identification of recombinant MrkAST protein expression modified with fatty acid esterification containing SpyTag peptide; the left image in Figure 1 shows the Coomassie Brilliant Blue staining results; the right image in Figure 1 shows the Anti-His tag monoclonal antibody detection results.

[0041] Figure 2 shows the SDS-PAGE identification of the fatty acid-modified recombinant MrA protein Ni affinity chromatography purified sample containing SpyTag peptide.

[0042] Figure 3 shows the size exclusion chromatography of recombinant MrkA protein modified with fatty acid esterification containing SpyTag peptide.

[0043] Figure 4 shows the SDS-PAGE identification of the fatty acid-modified recombinant MrkA protein molecule containing SpyTag peptide after being purified by size exclusion chromatography.

[0044] Figure 5 shows the relative molecular weight (Q-Tof) of fatty acid-modified recombinant MrkA protein containing SpyTag peptide;

[0045] Figure 6 shows the transmission electron microscopy (TEM) image of the fatty acid-modified recombinant MrkA protein containing SpyTag peptide.

[0046] Figure 7 shows the SDS-PAGE identification of the unfatty acid modified recombinant MrA protein containing SpyTag peptide;

[0047] Figures 8A, 8B, and 8C show the detection of IgG antibody titers after intramuscular immunization of fatty acid-modified recombinant MrkA protein; Figure 8A shows the results after one immunization lasting two weeks; Figure 8B shows the results after two immunizations lasting two weeks; and Figure 8C shows the results after three immunizations lasting two weeks.

[0048] Figure 9 shows the protective effect of intramuscular immunization with fatty acid-modified recombinant MrkA protein in a lethal bacteremia model.

[0049] Figure 10 shows the detection of IgG and IgA antibody titers in mucosal immune serum of fatty acid-modified recombinant MrkA protein;

[0050] Figure 11. SDS-PAGE and WB identification of Klebsiella pneumoniae O2 type O polysaccharide-protein conjugate;

[0051] Figure 12 shows the SDS-PAGE and WB identification of Klebsiella pneumoniae O1 type O polysaccharide-protein conjugate;

[0052] Figure 13 shows the linking and electrophoretic identification of fatty acid-modified recombinant MrkA protein with pneumonia Kleb O2 type O polysaccharide protein conjugate.

[0053] Figure 14 shows the fatty acid-modified Klebsiella pneumoniae double antigen immunoassay composition LipoMrkA-OPS. KpO2 Size exclusion chromatography and electrophoretic identification diagrams;

[0054] Figure 15 shows the fatty acid-modified Klebsiella pneumoniae double antigen immunoassay composition LipoMrkA-OPS. KpO2 Dynamic light scattering (DLS) identification diagram;

[0055] Figure 16 shows the fatty acid-modified Klebsiella pneumoniae double antigen immunoassay composition LipoMrkA-OPS. KpO2 Transmission electron microscopy (TEM) images;

[0056] Figure 17 shows the linker and electrophoretic identification of the non-fatty acid modified recombinant MrkA protein and the pneumonia Kleb O2 type O polysaccharide protein conjugate.

[0057] Figure 18. Connection and electrophoretic identification of fatty acid-modified recombinant MrA protein with pneumonia Kleb O1 type O polysaccharide protein conjugate;

[0058] Figure 19. Fatty acid-modified Klebsiella pneumoniae double antigen immunoassay composition LipoMrkA-OPS KpO1 Size exclusion chromatography and electrophoretic identification diagrams. Detailed Implementation

[0059] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0060] The present invention will be further illustrated below with reference to specific embodiments.

[0061] pET30a(+) was purchased from Novagen.

[0062] Escherichia coli BL21DE3 was purchased from Thermo Fisher Scientific, product number EC0114.

[0063] Escherichia coli C43DEDE3 was purchased from Sigma, catalog number CMC0019.

[0064] Escherichia coli W3110 was purchased from ATCC, catalog number ATCC27325.

[0065] Strain 355 of Klebsiella pneumoniae, with accession number CGMCC No.19879, can be obtained from the Academy of Military Medical Sciences.

[0066] Anti-His tag mouse monoclonal antibody was purchased from Sigma, catalog number A7058.

[0067] The chromatographic medium, Sepharose FF, is from Cytiva, catalog number 17057502.

[0068] Sephadex G25 fine chromatography packing material was purchased from Cytiva, product catalog number 17003202.

[0069] SOURCE30Q chromatography packing material was purchased from Cytiva, product catalog number 17127503.

[0070] The SOURCE 30S chromatography packing material was purchased from Cytiva, catalog number 17127302.

[0071] Superdex TM The 75 Increase 10 / 300 GL pre-loaded column was purchased from Cytiva, catalog number 29148721.

[0072] Superdex TM The 200 Increase 10 / 300 GL pre-loaded column was purchased from Cytiva, catalog number 28990944.

[0073] BALB / c mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0074] High-resolution mass spectrometer: Xevo G2-XS QTof (Waters).

[0075] SEQ ID NO.1: Partial sequence of MrkA protein:

[0076] SEQ ID NO.2: SpyTag peptide:

[0077] SEQ ID NO.3: SpyTag002 peptide:

[0078] SEQ ID NO.4: SpyTag003 peptide:

[0079] SEQ ID NO.5:

[0080] ML: MKATKLVLGAVILGSTLLAGC;

[0081] SEQ ID NO.6:

[0082] rAg:MKKLLIAAMMAAALAAC;

[0083] SEQ ID NO.7:

[0084] P4: MKTTLKMTALAALSAFVLAGC;

[0085] SEQ ID NO.8: MLMrkAST:

[0086] SEQ ID NO.9: rAgMrkAST:

[0087] SEQ ID NO.10: P4-MrkAST:

[0088] SEQ ID NO.11: miniSC:

[0089] SEQ ID NO.12: (SpyCatcher002 amino acid sequence):

[0090] SEQ ID NO.13: (SpyCatcher003 amino acid sequence):

[0091] SEQ ID NO.14: pelB-MrkAST:

[0092] SEQ ID NO.15: Amino acid sequence of PglL:

[0093] SEQ ID NO.16: Amino acid sequence of recombinant SpyCatcher protein with O-glycosylation (4573SC amino acid sequence):

[0094] SEQ ID NO.17: Nucleotide sequence of recombinant SpyCatcher protein with O-glycosylation:

[0095] SEQ ID NO.18 Fatty acid-modified recombinant MrkAST protein amino acid sequence:

[0096] SEQ ID NO.19 Recombinant MrkAST amino acid sequence:

[0097] SEQ ID NO.20: MLMrkA:

[0098] SEQ ID NO.21: MLMrkAST (without His tag):

[0099] SEQ ID NO.22: Amino acid sequence of recombinant SpyCatcher protein with O-glycosylation (4573SC amino acid sequence, no His tag):

[0100] Example 1: Preparation of recombinant MrkA protein modified with fatty acid esterification containing SpyTag peptide

[0101] (1.1) Construction of a recombinant MrkA protein expression vector containing fatty acid modification of SpyTag peptide

[0102] Based on the Klebsiella pneumoniae type 3 fimbriae major subunit (GenBank: EU682505.2) sequence published in GenBank, recombinant MrkA proteins were designed using SWISS-MODEL or other artificial intelligence tools. The Escherichia coli Murein-lipoprotein signal peptide sequence (EU900370.1), Neisseria meningitidis Ag473 signal peptide sequence (AY566590.1), and Haemophilus influenzae P4 signal peptide sequence (M68502.1) published in GenBank were fused to the N-terminus of the structurally optimized recombinant MrkA protein. At the same time, a SpyTag polypeptide (SEQ ID NO.2) and a His tag were added, and they were named MLMrkAST, rAgMrkAST, and P4MrkAST, respectively. The DNA sequences were optimized and synthesized by Sangon Biotech (Shanghai) Co., Ltd. based on the codons preferred by Escherichia coli.

[0103] The optimized MLMrkAST nucleotide sequence is shown in SEQ ID NO.8, the optimized rAgMrkAST nucleotide sequence is shown in SEQ ID NO.9, and the optimized P4SC nucleotide sequence is shown in SEQ ID NO.10.

[0104] In SEQ ID NO.8, positions 1 to 6 are NdeⅠ restriction sites, positions 4 to 66 are the signal peptide coding sequence shown in SEQ ID NO.5, positions 67 to 93 are the linker coding sequence, positions 94 to 657 are the recombinant MrkA protein coding sequence, positions 658 to 681 are the linker coding sequence, positions 682 to 720 are the SpyTag polypeptide tag coding sequence, positions 721 to 750 are the linker and His tag coding sequences, and positions 754 to 761 are NotⅠ restriction sites;

[0105] In SEQ ID NO.9, positions 1 to 6 are NdeⅠ restriction sites, positions 4 to 54 are the signal peptide coding sequence shown in SEQ ID NO.6, positions 55 to 81 are the linker coding sequence, positions 82 to 645 are the recombinant MrkA protein coding sequence, positions 646 to 669 are the linker coding sequence, positions 670 to 708 are the SpyTag polypeptide tag coding sequence, positions 709 to 738 are the linker and His tag coding sequences, and positions 742 to 749 are NotⅠ restriction sites;

[0106] In SEQ ID NO.10, positions 1 to 6 are NdeⅠ restriction sites, positions 4 to 66 are the signal peptide coding sequence shown in SEQ ID NO.7, positions 67 to 93 are the linker coding sequence, positions 94 to 657 are the recombinant MrkA protein coding sequence, positions 658 to 681 are the linker coding sequence, positions 682 to 720 are the SpyTag polypeptide tag coding sequence, positions 721 to 750 are the linker and His tag coding sequences, and positions 754 to 761 are NotⅠ restriction sites;

[0107] The optimized sequences were cloned into expression vectors. Taking the pET30a vector as an example, the obtained DNA fragments were inserted between the NdeⅠ and NotⅠ restriction sites of pET30a to construct the pET30-MLMrkAST, pET30-rAgMrkAST, and pET30-P4MrkAST expression vectors.

[0108] (1.2) Construction of recombinant MrkA protein expression strain containing SpyTag polypeptide with fatty acid modification

[0109] Taking SEQ ID NO.8 as an example, the constructed expression vector pET30-MLMrkAST was introduced into Escherichia coli C43DE3 (purchased from Sigma, catalog number CMC0019) or BL21DE3 (Thermo Fisher Scientific, catalog number EC0114) host cells, and spread on LB solid medium containing a final concentration of 50 μg / mL kanamycin. The positive clones are the expression strains C43DE3 / pET30-MLMrkAST or BL21DE3 / pET30-MLMrkAST.

[0110] (1.3) Expression and purification of recombinant bacterial MrkA protein containing SpyTag polypeptide and fatty acid modification

[0111] Single clones of recombinant bacteria C43DE3 / pET30-MLMrkAST or BL21DE3 / pET30-MLMrkAST were inoculated into LB medium containing kanamycin at a final concentration of 50 μg / mL and cultured at 37°C until the OD600 was approximately 0.6. Then, IPTG at a final concentration of 0.5 mM was added, and the temperature was lowered to 25°C for induction for 6 to 20 hours.

[0112] The following day, the bacterial cultures induced at 25℃ were collected, centrifuged to obtain bacterial cells, and suspended in distilled water at a ratio of 1:30 (w / v). The cells were then sonicated, centrifuged to separate the precipitate and supernatant, and the precipitate was resuspended in an equal volume of water. The sample was prepared with 5X reducing buffer (250 mM pH 6.8 Tris-HCl, 10% SDS, 0.5% bromophenol blue, 50% glycerol, 500 mM DTT), boiled for 10 min, and subjected to 15% SDS-PAGE electrophoresis. After electrophoresis, the sample was transferred to a PVDF membrane and transferred at a constant voltage of 20 V for 1 h. The results were detected using an Anti-His tag monoclonal antibody (Sigma, A7058), as shown in Figure 1. The left image in Figure 1 shows the Coomassie brilliant blue staining results; the right image shows the Anti-His tag monoclonal antibody (Sigma, A7058) detection results.

[0113] SDS-PAGE and WB identification showed that both strains could express recombinant MrkAST protein. Taking C43DE3 / pET30-MLMrkAST as an example, the culture volume was expanded and the recombinant MrkA protein was purified.

[0114] The sample was purified using a Chelating affinity chromatography column (Φ1.6cm×15cm).

[0115] After harvesting the bacterial cells, the cells were suspended in Ni-A1 buffer (20mM pH7.5 Tris-HCl + 0.3M NaCl + 5mM imidazole + 1% Triton X100) at a ratio of 1:20 (W / V), sonicated, and the supernatant was collected by centrifugation and purified by Chelating affinity chromatography.

[0116] First, wash the column bed with at least 3 column volumes of 0.5M NaOH aqueous solution. Then, equilibrate to pH neutral with deionized water. Next, equilibrate with at least 3 column volumes of 0.2M NiSO4 aqueous solution. Then, equilibrate with 1 column volume of Ni-B buffer (20mM pH 7.5 Tris-HCl, 0.3M NaCl, 500mM imidazole + Triton X100). Finally, equilibrate with at least 1 column volume of Ni-A1 buffer (20mM pH 7.5 Tris-HCl + 0.3M NaCl + 5mM imidazole + 1% Triton X100). Samples containing MLMrkAST were purified using a Chelating affinity chromatography column. Unbound proteins were washed with Ni-A1 buffer (20 mM pH 7.5 Tris-HCl + 0.3 M NaCl + 5 mM imidazole + 1% Triton X100) to equilibrate for at least 5 column volumes. Then, the samples were eluted with 10%, 30%, and 100% Ni-B buffer (20 mM pH 7.5 Tris-HCl, 0.3 M NaCl, 500 mM imidazole + 1% Triton X100). Samples eluted with 30% Ni-B or 100% Ni-B buffer were collected to obtain preliminarily purified samples (Figure 2).

[0117] The sample was purified using Superdex 200 Increase.

[0118] The Superdex 200 Increase column was equilibrated with SEC buffer (5 mM pH 7.4 PB + 0.9% NaCl + 0.01% Tween 80). Cytiva (28990944) was used to purify 1 mL of the 100% eluted sample purified by Ni affinity chromatography. The results showed that the target protein was eluted in the range of 8 mL to 16 mL (Figure 3). The SDS-PAGE electrophoresis diagram (Figure 4) showed that the protein with a retention volume in the range of 10 mL to 16 mL had higher purity and was suitable for subsequent experiments.

[0119] (1.4) Characterization of recombinant MrkA protein containing SpyTag peptide fatty acid modification

[0120] To characterize the MLMrkAST protein, Shanghai Zhongke New Life Biotechnology Co., Ltd. was commissioned to perform N-terminal sequencing on the purified sample using the Edman degradation method. The results showed sequencing failure and did not match the theoretical sequence, suggesting that the N-terminus of the recombinant protein was blocked. Further mass spectrometry (XevoG2-XS Q-Tof (Waters)) was used to determine the molecular weight of MLMrkAST (Figure 5). The theoretical molecular weight of the unmodified recombinant MrkA protein (without N-terminal Cys) was 22939.96 Da (SEQ ID NO.19). The results showed that the molecular weights of MLMrkAST were mainly 23855.25 Da, 23841.25 Da, and 23827.00 Da, consistent with the molecular weights of recombinant MrkAST containing thiol groups modified with diacylglycerol and α-amino groups modified with acyl groups. The corresponding three fatty acid chains had slightly different lengths: 23827.00 Da corresponded to (C16, C16, C16), 23841.25 Da to (C17, C16, C16), and 23855.25 Da to (C18, C16, C16 or C17, C17, C16). There were also a few peaks at 23867.25 Da and 23878.75 Da, corresponding to fatty acid chain forms such as (C18, C17, C16) and (C18, C17, C17). MLMrkAST contains an N-terminal Cys group with a thiol group modified by diacylglycerol and an α-amino group modified by an acyl group, wherein the fatty acid chain length of the three acyl groups is C16-C18.

[0121] Based on the above calculations and the technical solution of this invention, the N-terminus of the recombinant MrkA protein prepared by this invention is modified with fatty acid modification, and the recombinant MrkA protein prepared by this method is named LipoMrkAST.

[0122] The appearance of the purified LipoMrkAST lipoprotein was observed using transmission electron microscopy (TEM), and the results showed that the obtained LipoMrkAST was an irregular particle.

[0123] (1.5) Preparation of unfatty acid modified recombinant MrA protein (MrkAST) containing SpyTag peptide

[0124] The signal peptide of the recombinant MrkA protein was replaced with the bacterial pelB signal peptide. The amino acid sequence of the recombinant mature MrkAST peptide is shown in SEQ ID NO.19, and its nucleotide sequence is shown in SEQ ID NO.14.

[0125] In SEQ ID NO.14, positions 1 to 6 are NdeⅠ restriction sites, positions 4 to 69 are the coding sequence for the pelB signal peptide, positions 70 to 633 are the coding sequence for the MrkA protein, positions 634 to 696 are the coding sequence for the SpyTag polypeptide, positions 697 to 726 are the coding sequences for the linker and His tag, and positions 730 to 737 are the NotⅠ restriction sites;

[0126] Unfatty acid-modified recombinant MrkA protein (MrkAST) was prepared according to the expression and purification methods described in this embodiment, and the purification results are shown in Figure 7.

[0127] Example 2: Preparation of recombinant MrkA protein without SpyTag peptide fatty acid modification

[0128] (2.1) Construction of the fatty acid-modified recombinant MrkA protein (MLMrkA) expression vector

[0129] Based on the Klebsiella pneumoniae type 3 fimbriae major subunit (GenBank: EU682505.2) protein sequence published in GenBank, recombinant MrkA protein was designed using SWISS-MODEL or other artificial intelligence tools. The Escherichia coli Murein-lipoprotein signal peptide sequence (EU900370.1) published in GenBank was fused to the N-terminus of the structurally optimized recombinant MrkA protein, and a His tag was added. It was named MLMrkA, and the DNA sequence was optimized and synthesized by Sangon Biotech (Shanghai) Co., Ltd. according to the codons preferred by Escherichia coli.

[0130] The optimized MLMrkA nucleotide sequence is shown in SEQ ID NO.20; in SEQ ID NO.20, positions 1 to 6 are NdeⅠ restriction sites, positions 4 to 66 are the signal peptide coding sequence shown in SEQ ID NO.5, positions 67 to 93 are the linker coding sequence, positions 94 to 657 are the recombinant MrkA protein coding sequence, positions 658 to 687 are the linker and His tag coding sequences, and positions 691 to 698 are NotⅠ restriction sites;

[0131] The optimized sequence was cloned between the NdeⅠ and NotⅠ restriction sites of the pET30a vector to construct the pET30-MLMrkA expression vector.

[0132] (2.2) Construction of a fatty acid-modified recombinant MrkA protein (MLMrkA) expression strain

[0133] The constructed expression vector pET30-MLMrkA was introduced into Escherichia coli C43DE3 (purchased from Sigma, catalog number CMC0019) host cells and plated on LB solid medium containing a final concentration of 50 μg / mL kanamycin. The positive clones were the expression strains C43DE3 / pET30-MLMrkA.

[0134] (2.3) Expression and purification of fatty acid-modified recombinant Klebsiella pneumoniae MrkA protein (MLMrkA)

[0135] Single clones of recombinant bacteria C43DE3 / pET30-MLMrkA were picked and inoculated into LB medium containing kanamycin at a final concentration of 50 μg / mL. The medium was cultured at 37°C until the OD600 was about 0.6. Then, IPTG at a final concentration of 0.5 mM was added, and the temperature was lowered to 25°C for induction for 6-20 h.

[0136] The sample was purified using a Chelating affinity chromatography column (Φ1.6cm×15cm).

[0137] After harvesting the bacterial cells, the cells were suspended in Ni-A1 buffer (20mM pH7.5 Tris-HCl + 0.3M NaCl + 5mM imidazole + 1% Triton X100) at a ratio of 1:20 (W / V), sonicated, and the supernatant was collected by centrifugation and purified by Chelating affinity chromatography.

[0138] First, wash the column bed with at least 3 column volumes of 0.5M NaOH aqueous solution. Then, equilibrate to pH neutral with deionized water. Next, equilibrate with at least 3 column volumes of 0.2M NiSO4 aqueous solution. Then, equilibrate with one column volume of Ni-B buffer (20mM pH 7.5 Tris-HCl, 0.3M NaCl, 500mM imidazole + Triton X100). Finally, equilibrate with at least one column volume of Ni-A1 buffer (20mM pH 7.5 Tris-HCl + 0.3M NaCl + 5mM imidazole + 1% Triton X100). Samples containing MLMrkA were purified using a Chelating affinity chromatography column. Unbound proteins were washed with Ni-A1 buffer (20 mM pH 7.5 Tris-HCl + 0.3 M NaCl + 5 mM imidazole + 1% Triton X100) to equilibrate for at least 5 column volumes. The samples were then eluted with 10%, 30%, and 100% Ni-B buffer (20 mM pH 7.5 Tris-HCl, 0.3 M NaCl, 500 mM imidazole + 1% Triton X100). The sample eluted with 30% Ni-B buffer was collected to obtain the preliminarily purified sample.

[0139] The sample was purified using Superdex 200 Increase.

[0140] Equilibrate the Superdex 200 Increase column with SEC buffer (5mM pH 7.4 PB + 0.9% NaCl + 0.01% Tween 80). Cytiva (28990944), take 1 mL of the 100% eluted sample purified by the above Ni affinity chromatography for purification.

[0141] (2.4) Characterization of fatty acid-modified recombinant MakA protein

[0142] The purified MLMrkA sample was subjected to N-terminal sequencing using the Edman degradation method to determine whether the N-terminus of the recombinant protein was blocked. The molecular weight of MLMrkA was further determined using mass spectrometry (XevoG2-XS QTof (Waters)), and the fatty acid modification was inferred from the molecular weight increment. The morphology of the purified fatty acid-modified recombinant MrkA lipoprotein was observed using transmission electron microscopy (TEM).

[0143] Example 3: Evaluation of the immunogenicity and protective effect of fatty acid-modified recombinant MrkA protein (LipoMrkAST)

[0144] (3.1) Evaluation of the immunogenicity and protective effect of LipoMrkAST (intramuscular immunization)

[0145] Female BALB / c mice aged 6–8 weeks were randomly divided into the following groups (n = 5): Saline, MrkAST (1 μg), MrkAST (1 μg) + Al(OH)3, LipoMrkAST (1 μg), LipoMrkAST (1 μg) + Al(OH)3, MrkAST (5 μg), MrkAST (5 μg) + Al(OH)3, LipoMrkAST (5 μg), and LipoMrkAST (5 μg) + Al(OH)3. They were immunized by intramuscular injection on days 0, 14, and 28. Two weeks after immunization, blood was collected from the orbital venous sinus. The MrkAST protein prepared in Example 1 was used to coat the plates, and the titer of MrkAST-specific antibodies was detected by ELISA.

[0146] ELISA results showed that a single intramuscular injection of LipoMrkAST protein could induce high titers of MrkAST-specific IgG antibodies. There was no significant difference in antibody titers induced by the two immunization doses of 1 μg and 5 μg (Figure 8A), and the highest titer was reached after three immunizations (Figure 8B, Figure 8C). However, the seroconversion rate and antibody titer of mice immunized with Al(OH)3-adsorbed MrkAST were significantly lower than those of the LipoMrkAST group.

[0147] Two weeks after three immunizations, mice in the 1 μg dose group (3.98 × 10⁻⁶) were challenged intraperitoneally with the clinically resistant Kp355 isolate of Klebsiella pneumoniae. 7 The survival rate of mice in each group was observed using CFU (Figure 9). The results showed that the protective effect of LipoMrkAST was significantly better than that of the MrkAST group.

[0148] The results above demonstrate that, compared with the non-lipidated recombinant MrkA protein, the fatty acid-modified recombinant MrkA protein (LipoMrkAST) can induce a high-titer MrkAST-specific IgG antibody response more quickly and shows better protective effects in a mouse bacteremia model.

[0149] (3.2) Immunogenicity evaluation of LipoMrkAST (respiratory mucosal immunity)

[0150] Female BALB / c mice aged 6–8 weeks were randomly divided into three groups: Saline, MrkAST (5 μg), and LipoMrkAST (5 μg). Immunization was performed via respiratory mucosal nebulization on days 0, 14, and 28. Two weeks post-immunization, blood was collected from the orbital venous sinus, coated with MrkAST protein, and the serum MrkA-specific antibody titer was detected by ELISA. Results showed that after three respiratory mucosal immunizations, the fatty acid-modified recombinant MrkA protein (LipoMrkAST) induced MrkAST-specific serum IgG and IgA antibodies (Figure 10).

[0151] Example 4: Preparation of fatty acid-modified Klebsiella pneumoniae double antigen immunoassay composition

[0152] (4.1) SpyCatcher-OPS KpO2 (SC4573-OPS KpO2 Expression and preparation of )

[0153] Following the method described in patent CN111909951B, the Klebsiella pneumoniae O polysaccharide-protein conjugate SpyCatcher-OPS was prepared using a bioconjugation method. KpO2 (SC-OPS KpO2 The recombinant expression vector pETtac28-pglL-SC4573, containing the encoding sequence of the Neisseria meningitidis oligosaccharide transferase PglL (amino acid sequence as shown in SEQ ID NO. 15) and the encoding sequence of the recombinant SpyCatcher protein with an O-glycosylation sequence (amino acid sequence as shown in SEQ ID NO. 16), was introduced into the O antigen ligase-deficient Kp355 strain (CGMCC No. 19879ΔwaaL) to construct the recombinant engineered strain Kp355ΔwaaL / pET28tac-SC4573. The polysaccharide-protein conjugates of the engineered strain were cultured and purified to SC4573-OPS using Ni affinity chromatography, anion exchange chromatography, and size exclusion chromatography. KpO2 .

[0154] Alternatively, referring to the method described in patent CN105695497B, the Klebsiella pneumoniae O polysaccharide-protein conjugate SpyCatcher-OPS can be prepared using a bioconjugation method. KpO2 (SC-OPS KpO2 The recombinant expression vector pETtac28-pglL-SC4573, containing the coding sequence of the Neisseria meningitidis oligosaccharide transferase PglL and the coding sequence of the recombinant SpyCatcher protein with O-glycosylation, and the recombinant vector pACYC-OPS containing the Kp355 OPS locus, were used to express the protein. KpO2Recombinant engineered bacteria W3110ΔwaaL / pET28tac-SC4573 / pACYC-OPS were constructed by introducing the O antigen ligase-deficient E. coli (W3110ΔwaaL). KpO2 Engineered bacteria were cultured to express polysaccharide-protein conjugates, and the polysaccharide-protein conjugate SC4573-OPS was purified by Ni affinity chromatography, anion exchange chromatography, and size exclusion chromatography. KpO2 The results are shown in Figure 11.

[0155] SpyCatcher-OPS, a polysaccharide-protein conjugate of Klebsiella pneumoniae type O1, was prepared according to the above method. KpO1 (SC-OPS KpO1 The purification results are shown in Figure 12.

[0156] (4.2) LipoMrkAST and SC4573-OPS KpO2 connection

[0157] LipoMrkAST with SC4573-OPS KpO2 Mix according to a specific molar ratio (1:3 recommended) and react at 37°C for 2 hours, or at 4°C for 12 hours, to allow LipoMrkAST and SC4573-OPS to react. KpO2 The reaction was thorough, and the connection effect was detected by SDS-PAGE and WB. The results are shown in Figure 13.

[0158] The results showed that LipoMrkAST and SC4573-OPS KpO2 The molecular weight increased significantly after mixing, proving that the two molecules underwent a linkage reaction, and this was observed in SC4573-OPS. KpO2 Under excessive conditions, LipoMrkAST can be fully connected.

[0159] The ligation products were purified by size exclusion chromatography, and the Superdex 200 Increase column was equilibrated with SEC (5 mM pH 7.4 PB + 0.9% NaCl) buffer. Cytiva (28990944), 1 mL of the above ligation product was purified. SDS-PAGE and WB results showed that LipoMrkAST and SC4573-OPS were in the range of 8 mL to 12 mL. KpO2 The linker product (LipoMrkA-OPS) KpO2 ), 13 mL to 16 mL is unreacted SC4573-OPS KpO2(Figure 1). The ligation products were collected in the range of 8 mL to 12 mL, and their average diameter was measured by dynamic light scattering. The results showed that the dispersion coefficient of the ligation products was 0.25 and the average diameter was 19.4 nm (Figure 15).

[0160] (4.3) MrkAST and SC4573-OPS KpO1 connection

[0161] MrkAST with SC4573-OPS KpO1 Mix according to a specific molar ratio (1:3 recommended), and react at 37°C for 2 hours, or at 4°C for 12 hours, to allow MrkAST and SC4573-OPS to react. KpO1 The reaction was thorough, and the connection effect was detected by SDS-PAGE and WB. The results are shown in Figure 17.

[0162] (4.4) LipoMrkAST and SC4573-OPS KpO1 connection

[0163] LipoMrkAST with SC4573-OPS KpO1 Mix according to a specific molar ratio (1:3 recommended) and react at 37°C for 2 hours, or at 4°C for 12 hours, to allow LipoMrkAST and SC4573-OPS to react. KpO1 The reaction was thorough, and the connection effect was detected by SDS-PAGE and WB. The results are shown in Figure 18.

[0164] The results showed that LipoMrkAST and SC4573-OPS KpO1 The molecular weight increased significantly after mixing, proving that the two molecules underwent a linkage reaction, and this was observed in SC4573-OPS. KpO1 Under excessive conditions, LipoMrkAST can be almost completely connected.

[0165] The ligation products were purified by size exclusion chromatography, and the Superdex 200 Increase column was equilibrated with SEC (5 mM pH 7.4 PB + 0.9% NaCl) buffer. Cytiva (28990944), 1 mL of the above ligation product was purified. SDS-PAGE and WB results showed that LipoMrkAST and SC4573-OPS were in the range of 9 mL to 12 mL. KpO1 The linker product (LipoMrkA-OPS) KpO1 ), 13 mL to 16 mL is unreacted SC4573-OPS KpO1 (Figure 19) Collect the ligation product in the range of 9 mL to 12 mL for subsequent immunological evaluation.

[0166] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A fatty acid-modified recombinant MrkA protein.

2. The fatty acid-modified recombinant MrkA protein according to claim 1, characterized in that, It contains an N-terminal cysteine ​​(Cys) that has been fatty acid modified.

3. The fatty acid-modified recombinant MrkA protein according to claim 2, characterized in that, The fatty acid-modified N-terminal cysteine ​​(Cys) specifically refers to Cys whose thiol group is modified with diacylglycerol, or Cys whose thiol group is modified with diacylglycerol and whose α-amino group is modified with acyl group.

4. The fatty acid-modified recombinant MrkA protein according to claim 3, characterized in that, The acyl carbon chain length is C16 to C18.

5. The fatty acid-modified recombinant MrkA protein according to any one of claims 1-4, characterized in that, The recombinant MrkA protein comprises a polypeptide with the amino acid sequence shown in SEQ ID NO.

1.

6. An immune composition comprising the fatty acid-modified recombinant MrkA protein according to any one of claims 1-5.

7. The immune composition containing fatty acid-modified recombinant MrkA protein according to claim 6, characterized in that, It further contains a polysaccharide coupled to the fatty acid-modified recombinant MrkA protein.

8. The immune composition containing fatty acid-modified recombinant MrkA protein according to claim 7, characterized in that, The polysaccharide is covalently linked to fatty acid-modified recombinant MrkA protein via a linker.

9. The immune composition containing fatty acid-modified recombinant MrkA protein according to claim 8, characterized in that, The connector is the SpyCatcher / SpyTag connector.

10. The immune composition containing fatty acid-modified recombinant MrkA protein according to claims 7-9, characterized in that, The polysaccharide forms a polysaccharide-protein conjugate containing SpyCatcher protein with the recombinant fusion protein containing SpyCatcher protein, and the polysaccharide-protein conjugate containing SpyCatcher protein is covalently linked to the fatty acid-modified MrkA protein containing SpyTag polypeptide through the formation of isopeptide bonds.

11. The immune composition containing fatty acid-modified recombinant MrkA protein according to claim 10, characterized in that, The SpyCatcher protein is a polypeptide containing any of the amino acid sequences shown in SEQ ID NO. 11-13, and the SpyTag polypeptide is any of the amino acid sequences shown in SEQ ID NO. 2-4.

12. The immune composition according to claim 10 containing fatty acid-modified recombinant MrkA protein, characterized in that, The amino acid sequence of the fatty acid-modified recombinant MrkA protein containing the SpyTag polypeptide is shown in SEQ ID NO.

21.

13. The immune composition containing fatty acid-modified recombinant MrkA protein according to claim 10, characterized in that, The amino acid sequence of the polysaccharide-protein conjugate containing SpyCatcher protein is shown in SEQ ID NO.

22.

14. The immune composition containing fatty acid-modified recombinant MrkA protein according to claims 7-13, characterized in that, The polysaccharide is Klebsiella pneumoniae polysaccharide, which is Klebsiella pneumoniae O polysaccharide (OPS) and / or Klebsiella pneumoniae capsular polysaccharide (CPS).

15. A method for preparing recombinant MrkA protein modified with fatty acid as described in any one of claims 1-14, characterized in that, The method includes the following steps: 1) DNA fragments containing signal peptide coding sequences, MrkA protein coding sequences, and SpyTag polypeptide coding sequences, or with or without such sequences, were introduced into E. coli, and recombinant E. coli were cultured to express recombinant MrkA protein with N-terminal fatty acid modification. 2) Cultivate the recombinant Escherichia coli described in step 1) to express the fatty acid-modified recombinant MrkA protein; 3) The recombinant Escherichia coli was lysed, a surfactant was added, and the fatty acid-modified recombinant MrkA fusion protein was extracted and purified from the lysate.

16. The preparation method according to claim 15, characterized in that, The signal peptide is characterized by the amino acid sequence of the last four C-terminal positions being [L / V / I][A / S / T / V / I][G / A / S][C].

17. The preparation method according to claim 15, characterized in that, The amino acid sequence of the signal peptide is shown in any one of SEQ ID NO.5-7.

18. A method for preparing an immune composition containing fatty acid-modified recombinant MrA protein as described in any one of claims 7-14, characterized in that, The method includes the steps of mixing a fatty acid-modified recombinant MrA protein containing SpyTag polypeptide and a bacterial polysaccharide protein conjugate containing SpyCatcher protein in a certain proportion, and reacting it fully under suitable conditions to obtain an immune composition.

19. The use of a fatty acid-modified recombinant MrkA protein as described in any one of claims 1-5 in the preparation of a product capable of inducing the production of antigen-specific antibodies in animals, wherein the fatty acid-modified recombinant MrkA protein is administered via intramuscular injection, subcutaneous injection, nasal instillation, or nebulized inhalation.

20. The use of a composition containing fatty acid-modified recombinant MrkA protein as described in any one of claims 6-14 in the preparation of a product capable of inducing the production of antigen-specific antibodies in animals, wherein the immunizing composition is administered via intramuscular injection, subcutaneous injection, nasal instillation, or nebulized inhalation.

21. The application according to claims 19-20, characterized in that, The immune product mentioned is a vaccine.

22. An immunization vaccine, characterized in that, The vaccine comprises the fatty acid-modified recombinant MrkA protein as described in any one of claims 1-5, or the composition containing the fatty acid-modified recombinant MrkA protein as described in any one of claims 6-14.

23. A biomaterial, characterized in that, The biomaterial is any one of the following: C1) A nucleic acid molecule encoding the fatty acid-modified recombinant MrkA protein as described in any one of claims 1-14; C2) An expression cassette containing the nucleic acid molecule described in C1); C3) A recombinant vector containing the nucleic acid molecule described in C1), or a recombinant vector containing the expression cassette described in C2); C4) Recombinant microorganisms containing the nucleic acid molecules described in C1), or recombinant microorganisms containing the expression cassette described in C2), or recombinant microorganisms containing the recombinant vector described in C3); C5) Recombinant cells containing the nucleic acid molecule described in C1), or recombinant cells containing the expression cassette described in C2), or recombinant cells containing the recombinant vector described in C3).

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