Immune composition containing fatty acid modified recombinant MrkA protein and application thereof

Abstract

The invention discloses an immune composition containing a fatty acid modified recombinant MrkA protein and an application of the immune composition, particularly relates to an immune composition containing a fatty acid modified klebsiella pneumoniae recombinant MrkA protein and an application of the immune composition, and belongs to the technical field of biological medicines. Wherein the fatty acid modified recombinant MrkA protein can be self-polymerized into nanoparticles, and compared with the non-fatty acid modified recombinant MrkA protein, the antigen can induce faster and higher humoral immunity through injection immunity, and also can induce mucosal immunity and humoral immunity through respiratory nebulization immunity. According to the present invention, the polysaccharide antigen is further displayed on the fatty acid modified recombinant MrkA protein through the SpyTag / SpyCatcher connector so as to improve the immunogenicity of the polysaccharide, such that the immune composition containing the fatty acid modified klebsiella pneumoniae recombinant MrkA protein is provided;

Description

Technical Field

[0001] The invention belongs to the field of biomedical technology, and specifically relates to an immune composition containing fatty acid-modified recombinant MrkA protein and application thereof. Background Art

[0002] Klebsiella pneumoniae (Kp) is a Gram-negative bacterium with a capsule, which is widely distributed in the natural environment and the digestive tract of mammals. Kp is a conditional pathogen, which usually causes contact infection or nosocomial infection through wounds and invasive intubation of medical devices, thereby causing pneumonia, bacteremia, urinary tract infection, etc. Among them, ICU patients, the elderly and other people with weakened immune function are the main targets of infection. Literature reports that in cases of ventilator-associated pneumonia (VAP), deaths caused by Kp infection account for 83%. Klebsiella pneumoniae naturally carries the β-lactamase gene SHV-1, and continuously acquires antibiotic resistance genes through plasmids and other transferable genetic materials, such as aminoglycoside antibiotic resistance genes, quinolone antibiotic 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 carrier of resistance genes, transferring resistance genes from the environment to clinical pathogens. It is currently considered to be the main source and transmission tool of antibiotic resistance in the world.

[0003] Among the many strategies for preventing and treating bacterial infections, vaccination is considered to be the most economical and effective method. Compared with viruses, bacteria have larger genes, more virulence factors, and more complex pathogenic mechanisms, immune escape mechanisms, and drug resistance mechanisms. Therefore, the selection and design of bacterial vaccine antigens, delivery vectors, adjuvants, and their compatibility with each other require more consideration. The safety, accessibility, conservation, and abundance of antigens are the most important considerations for the selection of vaccine antigens. Type 3 fimbriae are distributed throughout the bacterial surface and are prevalent in Klebsiella pneumoniae. They mediate the adhesion of Klebsiella pneumoniae to the respiratory tract or other tissues. MrkA protein is the main component of type 3 fimbriae fibers and is related to the formation of biofilms. MrkA protein is highly conserved in Klebsiella and even Enterobacteriaceae, making it one of the ideal candidate antigens. Studies have shown that directly extracting type 3 fimbriae complexes from Klebsiella pneumoniae or immunizing mice with recombinantly expressed MrkA protein can induce mice to produce protective antibodies, and MrkA-specific monoclonal antibodies isolated from antiserum are also protective. However, the directly extracted type 3 fimbriae complex is highly heterogeneous, and the recombinantly expressed MrkA protein exists in the form of inclusion bodies. Subsequent problems such as protein renaturation, structure confirmation, stability and relatively weak immunogenicity exist, which prevents MrkA protein from becoming a candidate protective antigen.

[0004] There is a class of proteins in bacteria whose N-terminal cysteine ​​(Cys) is modified by fatty acid and fixed to the cell surface by fatty acid chains, called bacterial lipoproteins. The in vivo synthesis of bacterial lipoproteins is completed under the guidance of signal peptides. Bioinformatics and statistical analysis of the signal peptide sequences of bacterial natural fatty acid-modified and non-fatty acid-modified proteins revealed that the signal peptides of fatty acid-modified proteins are non-polar amino acids near the signal peptidase cleavage site and have a certain degree of conservation. In summary, it was found that the conservative sequence of the last four amino acids in this region is [L / V / I][A / S / T / V / I][G / A / S][C], among which the C-terminal cysteine ​​is the lipidation modification site. Under the guidance of the signal peptide, the nascent peptide chain is localized to the outside of the cell membrane through the Sec or TAT secretion pathway. The Lgt enzyme uses phosphatidylglycerol (PG) as a substrate to connect diacylglycerol to the sulfhydryl group of Cys. The LspA enzyme removes the signal peptide except the C-terminal Cys. The Lnt enzyme uses phosphatidylethanolamine (PE) as a substrate to transfer a fatty acid chain to the alpha-amino group of Cys. Bacterial fatty acid modification is heterogeneous, mainly in terms of the number and length of fatty acid chains. The length of the fatty acyl carbon chain of bacterial lipoproteins is usually between C14 and C18, but the vast majority of fatty acyl carbon chains are C16.

[0005] Displaying antigens on the surface of protein nanocarriers is an effective means to improve the immunogenicity of antigens. The preparation scheme (modular preparation scheme) of expressing and preparing nanoparticles and loaded antigens separately and connecting the carrier and loaded antigens through a universal, efficient and controllable connection method can effectively solve the problems of low expression efficiency, particle assembly failure, structural instability and loss of immunogenicity caused by the fusion expression of carrier and antigen. The SpyTag / SpyCatcher connection system is one of the most intensively studied protein connection systems. This system is widely used in vaccine development and research due to its advantages such as rapid reaction, specificity, wide range of reaction conditions and irreversible reaction. It can be fused with pathogen antigens and suitable carrier proteins to achieve precise covalent connection between antigens and carrier proteins, especially to achieve repeated display of antigens on the surface of self-polymerized nanocarrier proteins, which improves the presentation efficiency and immunogenicity of antigens. This connection system originates from the CnaB2 domain of the fibronectin-binding protein of Streptococcus pyogenes. Lys31 and Asp117 of the CnaB2 domain can form an intramolecular isopeptide bond. After splitting the CnaB2 domain into the N-terminal domain containing Lys31 (SpyCatcher, 116aa) and the C-terminal polypeptide containing Asp117 (SpyTag, 13aa), the two fragments can still form specific isopeptide bonds. To further optimize the SpyTag / SpyCatcher connection system, Mark Howarth's team analyzed the relationship between its structure and function and demonstrated the minimum SpyCatcher required to form an isopeptide bond. To improve the speed and efficiency of the connection reaction, the team artificially designed the SpyTag (13aa) / SpyCatcher (116aa) connection system and developed the SpyTag002 / SpyCatcher002 and SpyTag003 / SpyCatcher003 connection systems.

[0006] In the current Klebsiella pneumoniae subunit vaccine technology scheme, protein subunit vaccine and polysaccharide conjugate vaccine are the main candidate vaccines, and the antigen selection is limited to polysaccharide antigens (OPS or CPS) or protein antigens, which have weak immunogenicity and can only induce immune memory against polysaccharide antigens or protein antigens. In view of the characteristics of susceptible populations, in order to improve the immunogenicity of antigens and simultaneously induce strong immune responses and immune memory against polysaccharides and proteins of Klebsiella pneumoniae, the present invention selects the conservative protein MrkA distributed on the surface of Klebsiella pneumoniae as a protein antigen, and self-polymerizes it into nanoparticles through fatty acid modification. Compared with the MrkA protein not modified by fatty acid, the recombinant MrkA protein modified by fatty acid can induce faster and higher humoral immunity through injection immunization, and can also induce mucosal immunity and humoral immunity through respiratory atomization immunization. The polysaccharide antigen is displayed on the surface of the recombinant MrkA protein nanoparticles modified by fatty acid through a pluggable modular preparation technology, further providing a Klebsiella pneumoniae dual antigen immune composition. Compared with the existing Klebsiella pneumoniae subunit vaccine scheme, the immune composition provided by the present invention can not only induce immune memory against Klebsiella pneumoniae polysaccharide, but also induce immune memory against Klebsiella pneumoniae surface protein. In particular, it can induce mucosal immunity through respiratory tract aerosol immunization. Summary of the invention

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

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

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

[0010] According to the second aspect of the technical solution of the present invention, an immune composition containing a recombinant MrkA protein modified by fatty acid is provided. The immune composition contains a recombinant MrkA protein modified by fatty acid 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 bond linker, preferably a SpyCatcher / SpyTag linker.

[0013] The polysaccharide and the recombinant fusion protein containing the SpyCatcher protein form a polysaccharide-protein conjugate containing the SpyCatcher protein, and the polysaccharide-protein conjugate containing the SpyCatcher protein is covalently connected to the fatty acid-modified MrkA protein containing the SpyTag polypeptide by forming an isopeptide bond.

[0014] The SpyCatcher protein is a polypeptide containing an amino acid sequence shown in any one of SEQ ID NOs. 11-13, and the SpyTag polypeptide is an amino acid sequence shown in any one of SEQ ID NOs. 2-4.

[0015] When the recombinant fusion protein containing the SpyCatcher protein comprises 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 the SpyCatcher protein comprises 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 the SpyCatcher protein comprises 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 the SpyCatcher protein containing the amino acid sequence shown in SEQ ID NO.11 and the 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 the SpyTag polypeptide is shown in SEQ ID NO.21. The amino acid sequence of the polysaccharide-protein conjugate containing the SpyCatcher protein is shown in SEQ ID NO.22; in order 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 the SpyTag polypeptide and the His tag is shown in SEQ ID NO.18, and the amino acid sequence of the polysaccharide-protein conjugate containing the SpyCatcher protein and the His tag is shown in SEQ ID NO.16 The polysaccharide-protein conjugate containing SpyCatcher protein can be prepared by chemical coupling method or biological coupling method. The polysaccharide includes Klebsiella pneumoniae polysaccharide, and the Klebsiella pneumoniae polysaccharide is Klebsiella pneumoniae O polysaccharide (OPS) and / or Klebsiella pneumoniae capsular polysaccharide (CPS).

[0019] Taking Klebsiella pneumoniae O polysaccharide as an example, a Klebsiella pneumoniae O polysaccharide-protein conjugate containing SpyCatcher protein is prepared by a bioconjugation method. The preparation method is as follows: a DNA fragment containing a coding sequence of Neisseria meningitidis O-oligosaccharide transferase PglL and a DNA fragment containing a signal peptide, SpyCatcher protein and an O-glycosylation motif coding sequence are introduced into Klebsiella pneumoniae with a defective O antigen transferase gene, and the recombinant bacteria are cultured to obtain a conjugate of Klebsiella pneumoniae O polysaccharide and a recombinant SpyCatcher protein containing an O-glycosylation motif, wherein the amino acid sequence of the Neisseria meningitidis O-oligosaccharide transferase PglL is shown in SEQ ID NO.15, and the amino acid sequence of the recombinant SpyCatcher protein containing an O-glycosylation motif is shown in SEQ ID NO.16; Alternatively, a DNA fragment containing the coding sequence of Neisseria meningitidis O-oligosaccharide transferase PglL, a DNA fragment containing a signal peptide and SpyCatcher protein and O-glycosylation motif coding sequence, and a DNA sequence containing the Klebsiella pneumoniae O-polysaccharide synthesis locus are introduced into Escherichia coli with a defective O-antigen transferase gene, and the recombinant bacteria are cultured to obtain a conjugate of Klebsiella pneumoniae O-polysaccharide and a recombinant SpyCatcher protein containing an O-glycosylation motif.

[0020] According to the third aspect of the technical solution of the present invention, a method for preparing the above-mentioned fatty acid-modified recombinant MrkA protein is provided, the method comprising the following steps: 1) Introducing a DNA fragment containing a signal peptide coding sequence, a MrkA protein coding sequence, and a SpyTag polypeptide coding sequence or not into Escherichia coli, and culturing the recombinant Escherichia coli to express a recombinant MrkA protein with a fatty acid modification at the N-terminus; 2) culturing the recombinant Escherichia coli described in step 1) to express the fatty acid-modified recombinant MrkA protein; 3) Lysing the recombinant E. coli, adding a surfactant, and extracting and purifying the fatty acid-modified recombinant MrkA fusion protein from the lysate.

[0021] The signal peptide is characterized in that the amino acid sequence of the last four positions at the C-terminus is [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, and the SpyTag polypeptide coding sequence or not can be introduced into Escherichia coli in the form of a recombinant expression vector, or can be integrated into the Escherichia coli genome.

[0022] According to the fourth aspect of the technical solution of the present invention, a method for preparing a composition containing a fatty acid-modified recombinant MrkA protein as mentioned above is provided, the method comprising the steps of mixing a fatty acid-modified recombinant MrkA protein containing a SpyTag polypeptide and a bacterial polysaccharide-protein conjugate containing a SpyCatcher protein in a certain proportion, and fully reacting them under appropriate conditions to obtain an immune composition.

[0023] According to the fifth aspect of the technical solution of the present invention, there is provided a use of a recombinant MrkA protein modified by fatty acid as described above in the preparation of a product that can induce an animal to produce antigen-specific antibodies, wherein the recombinant MrkA protein modified by fatty acid is immunized by intramuscular injection, subcutaneous injection, nasal drops or aerosol inhalation.

[0024] According to the sixth aspect of the technical solution of the present invention, there is provided a composition containing fatty acid-modified recombinant MrkA protein as described above for use in preparing a product that can induce animals to produce antigen-specific antibodies. The immune composition is administered by intramuscular injection, subcutaneous injection, nasal drops or aerosol inhalation.

[0025] According to the seventh aspect of the technical solution of the present invention, an immune vaccine is provided, wherein the vaccine comprises the above-mentioned fatty acid-modified recombinant MrkA protein, or the above-mentioned composition containing the fatty acid-modified recombinant MrkA protein.

[0026] According to an eighth aspect of the technical solution of the present invention, a pharmaceutical composition is provided, wherein the pharmaceutical composition comprises the above-mentioned fatty acid-modified recombinant MrkA protein, or the above-mentioned composition containing the fatty acid-modified recombinant MrkA protein.

[0027] According to a ninth aspect of the technical solution of the present invention, a biomaterial is provided, wherein the biomaterial is any one of the following: C1) a nucleic acid molecule encoding a fatty acid-modified recombinant MrkA protein according to any one of claims 1 to 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) a recombinant microorganism containing the nucleic acid molecule described in C1), or a recombinant microorganism containing the expression cassette described in C2), or a recombinant microorganism containing the recombinant vector described in C3); C5) a recombinant cell containing the nucleic acid molecule described in C1), or a recombinant cell containing the expression cassette described in C2), or a recombinant cell containing the recombinant vector described in C3).

[0028] Compared with the prior art, the present invention has at least the following beneficial effects: The present invention discloses a fatty acid-modified recombinant MrkA protein of Klebsiella pneumoniae, which can be polymerized into nanoparticles. A single injection of immunized mice can produce high-titer MrkA-specific IgG antibodies, and three injections of immunization can protect mice from lethal attacks of clinically resistant strains of Klebsiella pneumoniae; after three immunizations of mice immunized by respiratory aerosol, high-titer MrkA-specific serum IgG and serum IgA antibodies can be detected. Compared with recombinant MrkA proteins that are not fatty acid-modified, the antigen can induce faster and higher humoral immunity through injection immunization, and can also induce mucosal immunity through respiratory aerosol immunization. The present invention further displays Klebsiella pneumoniae polysaccharide antigens on the fatty acid-modified recombinant MrkA protein through SpyTag / SpyCatcher connectors to improve the immunogenicity of the polysaccharide, thereby providing a Klebsiella pneumoniae multi-antigen immune composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is an SDS-PAGE identification diagram of the expression of the recombinant MrkAST protein modified by fatty acid containing SpyTag polypeptide; Figure 1 The left picture in the figure shows the result of Coomassie brilliant blue staining; Figure 1 The right picture shows the Anti-His tag monoclonal antibody detection results.

[0030] Figure 2 This is an SDS-PAGE identification diagram of the Ni affinity chromatography purified sample of the recombinant MrkA protein modified with fatty acid containing SpyTag polypeptide; Figure 3 It is a molecular exclusion chromatogram of the fatty acid-modified recombinant MrkA protein containing the SpyTag polypeptide; Figure 4 This is the SDS-PAGE identification diagram of the sample of the molecular exclusion chromatography purification of the recombinant MrkA protein modified with fatty acid containing SpyTag polypeptide; Figure 5This is the relative molecular weight detection diagram (Q-Tof) of the recombinant MrkA protein modified by fatty acid containing SpyTag polypeptide; Figure 6 This is a transmission electron microscopy (TEM) identification image of the recombinant MrkA protein modified with fatty acid containing SpyTag polypeptide; Figure 7 This is the SDS-PAGE identification image of the non-fatty acid-modified recombinant MrkA protein containing the SpyTag polypeptide; Fig. 8A , Figure 8B and Figure 8C They are respectively the detection of IgG antibody titer by intramuscular immunization of recombinant MrkA protein modified by fatty acid; Fig. 8A For one free two weeks; Figure 8B It is free for two weeks; Figure 8C It is three free weeks; Fig. 9 To evaluate the protective effect of intramuscular immunization of fatty acid modified recombinant MrkA protein in a lethal bacteremia model; Fig.10 To detect the titer of IgG and IgA antibodies in serum from mucosal immune test of recombinant MrkA protein modified by fatty acid; Fig.11 SDS-PAGE and WB identification of Klebsiella pneumoniae O2 type O polysaccharide protein conjugate; Fig.12 This is the SDS-PAGE and WB identification diagram of Klebsiella pneumoniae O1 type O polysaccharide protein conjugate; Fig.13 The figure shows the connection and electrophoresis identification of the fatty acid-modified recombinant MrkA protein and the Klebsiella pneumoniae O2 type O polysaccharide protein conjugate; Fig.14 LipoMrkA-OPS is a fatty acid modified Klebsiella pneumoniae double antigen immune composition KpO2 Size exclusion chromatography and electrophoresis identification diagram; Fig.15 LipoMrkA-OPS is a fatty acid modified Klebsiella pneumoniae double antigen immune composition KpO2 Dynamic light scattering (DLS) identification chart; Fig.16 LipoMrkA-OPS is a fatty acid modified Klebsiella pneumoniae double antigen immune composition KpO2 Transmission electron microscopy (TEM) identification image; Fig.17 The figure shows the connection and electrophoresis identification of the non-fatty acid modified recombinant MrkA protein and the Klebsiella pneumoniae O2 type O polysaccharide protein conjugate; Fig.18. The connection and electrophoresis identification of fatty acid modified recombinant MrkA protein and Klebsiella pneumoniae O1 type O polysaccharide protein conjugate; Fig.19 . LipoMrkA-OPS, a fatty acid-modified dual-antigen immune composition of Klebsiella pneumoniae KpO1 Molecular exclusion chromatography and electrophoresis identification diagram. DETAILED DESCRIPTION

[0031] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0032] The present invention will be further described below with reference to specific embodiments.

[0033] pET30a(+) was purchased from Novagen.

[0034] Escherichia coli BL21DE3 was purchased from Thermo Fisher Scientific Inc., catalog number EC0114.

[0035] Escherichia coli C43DEDE3 was purchased from Sigma, catalog number CMC0019.

[0036] Escherichia coli W3110 was purchased from ATCC, catalog number ATCC27325.

[0037] Klebsiella pneumoniae strain 355, the deposit number is CGMCC No.19879, the name of the depository: General Microbiology Center of China Culture Collection Administration, the address of the depository: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the classification and naming of the biological material sample: Klebsiella pneumoniae (Latin name "Klebsiella pneumoniae"), the deposit date: May 26, 2020.

[0038] Anti-His tag mouse monoclonal antibody was purchased from Sigma, catalog number A7058.

[0039] Chelating Sepharose FF chromatography medium was from Cytiva, catalog number 17057502.

[0040] Sephadex G25 fine chromatography medium was purchased from Cytiva, catalog number 17003202.

[0041] SOURCE30Q chromatography medium was purchased from Cytiva, product catalog number 17127503.

[0042] SOURCE 30S chromatography medium was purchased from Cytiva, catalog number 17127302.

[0043] Superdex TM 75 Increase 10 / 300 GL prepacked columns were purchased from Cytiva, catalog number 29148721.

[0044] Superdex TM 200 Increase 10 / 300 GL prepacked columns were purchased from Cytiva, catalog number 28990944.

[0045] BALB / c mice were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.

[0046] High-resolution mass spectrometer: XevoG2-XS QTof (Waters).

[0047] SEQ ID NO.1: Partial sequence of MrkA protein: TDVSCTVSVNGQGSDANVYLSPVTLTEVKAAAADTYLKPKSFTIDVSNCQAADGTKQDDVSKLGVNWTGGNLLAGATSKQQGYLANTEASGAQNIQLVLSTDNATALTNKIIPGDSTQPKAKGDASAVADGARFTYYVGYATSAPTTVTTGVVNSYATYEITYQ; SEQ ID NO.2: SpyTag polypeptide: AHIVMVDAYKPTK; SEQ ID NO.3: SpyTag002 polypeptide: VPTIVMVDAYKRYK; SEQ ID NO.4: SpyTag003 polypeptide: RGVPHIVMVDAYKRYK; SEQ ID NO.5: ML:MKATKLVLGAVILGSTLLAGC; SEQ ID NO.6: rAg:MKKLLIAAMMAAAALAAC; SEQ ID NO.7: P4: MKTTLKMTALAALSAFVLAGC; SEQ ID NO.8: MLMrkAST: CATATGaaagctactaaactggtactgggcgcggtaatcctgggttctactctgctggcaggttgcGGATCCGGTAGCGGCAGCggaggtagcACCGATGTGAGCTGCACCGTGAGCGTGAACGGCCAAGGCAGCGATGCGAACGTGTATCTGAGCCCGGTGACCCTGACCGAAGTGAAAGCCGCGGCGGCGGATACCTACCTGAAACCGAAAAGCTTTACCATTGACGTTAGCAACTGCCAAGCGGCGGATGGCACCAAACAAGATGATGTGAGCAAACTGGGCGTGAACTGGACGGGCGGTAATTTACTGGCGGGCGCGACGAGCAAACAGCAAGGCTATCTGGCGAACACCGAAGCGAGCGGCGCGCAGAACATTCAGCTGGTGCTGAGCACCGATAACGCGACCGCGCTGACCAACAAAATTATTCCGGGCGATAGCACGCAGCCGAAAGCGAAAGGCGATGCGAGTGCCGTGGCGGATGGCGCGCGCTTTACCTATTATGTGGGCTATGCGACGAGCGCGCCGACCACCGTGACCACCGGCGTGGTGAACAGCTATGCGACCTATGAAATTACCTATCAAGGCGGTGGCAGCGCGGCGCATGCGGCGGATACGACCGTGGGCGGCGGTCAAGTGAACTTTTTTGGCAAAGTGGGCggtGGaAGCggaGGCgggtccGCGCATATTGTGATGGTGGATGCGTATAAACCGACCAAAgggtccCATCACCATCACCATCACCACCATTAAGCGGCCGC; SEQ ID NO.9:rAgMrkAST: CATATGaaaaaattattgattgccgcaatgatggcggctgccttggcagcttgcGGATCCGGTAGCGGCAGCggaggtagcACCGATGTGAGCTGCACCGTGAGCGTGAACGGCCAAGGCAGCGATGCGAACGTGTATCTGAGCCCGGTGACCCTGACCGAAGTGAAAGCCGCGGCGGCGGATACCTACCTGAAACCGAAAAGCTTTACCATTGACGTTAGCAACTGCCAAGCGGCGGATGGCACCAAACAAGATGATGTGAGCAAACTGGGCGTGAACTGGACGGGCGGTAATTTACTGGCGGGCGCGACGAGCAAACAGCAAGGCTATCTGGCGAACACCGAAGCGAGCGGCGCGCAGAACATTCAGCTGGTGCTGAGCACCGATAACGCGACCGCGCTGACCAACAAAATTATTCCGGGCGATAGCACGCAGCCGAAAGCGAAAGGCGATGCGAGTGCCGTGGCGGATGGCGCGCGCTTTACCTATTATGTGGGCTATGCGACGAGCGCGCCGACCACCGTGACCACCGGCGTGGTGAACAGCTATGCGACCTATGAAATTACCTATCAAGGCGGTGGCAGCGCGGCGCATGCGGCGGATACGACCGTGGGCGGCGGTCAAGTGAACTTTTTTGGCAAAGTGGGCggtGGaAGCggaGGCgggtccGCGCATATTGTGATGGTGGATGCGTATAAACCGACCAAAgggtccCATCACCATCACCATCACCACCATTAAGCGGCCGC; SEQ ID NO.10:P4-MrkAST: CATATGAAAACCACCCTGAAAATGACCGCTCTGGCTGCTCTGTCTGCTTTCGTTCTGGCTGGTtgcGGATCCGGTAGCGGCAGCggaggtagcACCGATGTGAGCTGCACCGTGAGCGTGAACGGCCAAGGCAGCGATGCGAACGTGTATCTGAGCCCGGTGACCCTGACCGAAGTGAAAGCCGCGGCGGCGGATACCTACCTGAAACCGAAAAGCTTTACCATTGACGTTAGCAACTGCCAAGCGGCGGATGGCACCAAACAAGATGATGTGAGCAAACTGGGCGTGAACTGGACGGGCGGTAATTTACTGGCGGGCGCGACGAGCAAACAGCAAGGCTATCTGGCGAACACCGAAGCGAGCGGCGCGCAGAACATTCAGCTGGTGCTGAGCACCGATAACGCGACCGCGCTGACCAACAAAATTATTCCGGGCGATAGCACGCAGCCGAAAGCGAAAGGCGATGCGAGTGCCGTGGCGGATGGCGCGCGCTTTACCTATTATGTGGGCTATGCGACGAGCGCGCCGACCACCGTGACCACCGGCGTGGTGAACAGCTATGCGACCTATGAAATTACCTATCAAGGCGGTGGCAGCGCGGCGCATGCGGCGGATACGACCGTGGGCGGCGGTCAAGTGAACTTTTTTGGCAAAGTGGGCggtGGaAGCggaGGCgggtccGCGCATATTGTGATGGTGGATGCGTATAAACCGACCAAAgggtccCATCACCATCACCATCACCACCATTAAGCGGCCGC; SEQ ID NO.11:miniSC: DSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNG; SEQ ID NO.12: (SpyCatcher002 amino acid sequence): GAMVTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGEATKGDAHT; SEQ ID NO.13: (SpyCatcher003 amino acid sequence): GAMVTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDAHT; SEQ ID NO.14: pelB-MrkAST: catATGAAATATCTGCTGCCGACCGCGGCGGCGGGCCTGCTGCTGCTGGCGGCGCAGCCGGCGATGGCGACCGATGTGAGCTGCACCGTGAGCGTGAACGGCCAAGGCAGCGATGCGAACGTGTATCTGAGCCCGGTGACCCTGACCGAAGTGAAAGCCGCGGCGGCGGATACCTACCTGAAACCGAAAAGCTTTACCATTGACGTTAGCAACTGCCAAGCGGCGGATGGCACCAAACAAGATGATGTGAGCAAACTGGGCGTGAACTGGACGGGCGGTAATTTACTGGCGGGCGCGACGAGCAAACAGCAAGGCTATCTGGCGAACACCGAAGCGAGCGGCGCGCAGAACATTCAGCTGGTGCTGAGCACCGATAACGCGACCGCGCTGACCAACAAAATTATTCCGGGCGATAGCACGCAGCCGAAAGCGAAAGGCGATGCGAGTGCCGTGGCGGATGGCGCGCGCTTTACCTATTATGTGGGCTATGCGACGAGCGCGCCGACCACCGTGACCACCGGCGTGGTGAACAGCTATGCGACCTATGAAATTACCTATCAAGGCGGTGGCAGCGCGGCGCATGCGGCGGATACGACCGTGGGCGGCGGTCAAGTGAACTTTTTTGGCAAAGTGGGCggtGGaAGCggaGGCgggtccGCGCATATTGTGATGGTGGATGCGTATAAACCGACCAAAgggtccCATCACCATCACCATCACCACCATTAAGCGGCCGC; SEQ ID NO.15: Amino acid sequence of PglL: ; SEQ ID NO.16: Amino acid sequence of recombinant SpyCatcher protein with O-glycosylation sequence (4573SC amino acid sequence): HHHHHHHHHHSAVTEYYLNHGEWPGNNTSAGVATSSEIKGAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHI; SEQ ID NO.17: Nucleotide sequence of recombinant SpyCatcher protein with O-glycosylation sequence: ATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCGATGGCCCAcCAtCAtCAtCAcCAcCAcCAtCACCAtAGCGCCGTGACCGAGTACTATCTGAACCATGGCGAGTGGCCGGGTAATAACACCAGCGCCGGCGTGGCCACAAGCAGTGAGATCAAGggcgccatggttgataccttatcaggtttatcaagtgagcaaggtcagtccggtgatatgacaattgaagaagatagtgctacccatattaaattctcaaaacgtgatgaggacggcaaagagttagctggtgcaactatggagttgcgtgattcatctggtaaaactattagtacatggatttcagatggacaagtgaaagatttctacctgtatccaggaaaatatacatttgtcgaaaccgcagcaccagacggttatgaggtagcaactgctattacctttacagttaatgagcaaggtcaggttactgtaaatggcaaagcaactaaaggtgacgctcatattTGA; SEQ ID NO.18 Amino acid sequence of fatty acylated recombinant MrkAST protein: CGSGSGSGGSTDVSCTVSVNGQGSDANVYLSPVTLTEVKAAAADTYLKPKSFTIDVSNCQAADGTKQDDVSKLGVNWTGGNLLAGATSKQQGYLANTEASGAQNIQLVLSTDNATALTNKIIPGDSTQPKAKGDASAVADGARFTYYVGYATSAPTTVTTGVVNSYATYEITYQGGGSAAHAADTTVGGGQVNFFGKVGGGSGGGSAHIVMVDAYKPTKGSHHHHHHHH; SEQ ID NO.19 Amino acid sequence of recombinant MrkAST: TDVSCTVSVNGQGSDANVYLSPVTLTEVKAAAADTYLKPKSFTIDVSNCQAADGTKQDDVSKLGVNWTGGNLLAGATSKQQGYLANTEASGAQNIQLVLSTDNATALTNKIIPGDSTQPKAKGDASAVADGARFTYYVGYATSAPTTVTTGVVNSYATYEITYQGGGSAAHAADTTVGGGQVNFFGKVGGGSGGGSAHIVMVDAYKPTKGSHHHHHHHH; SEQ ID NO.20:MLMrkA: CATATGaaagctactaaactggtactgggcgcggtaatcctgggttctactctgctggcaggttgcGGATCCGGTAGCGGCAGCggaggtagcACCGATGTGAGCTGCACCGTGAGCGTGAACGGCCAAGGCAGCGATGCGAACGTGTATCTGAGCCCGGTGACCCTGACCGAAGTGAAAGCCGCGGCGGCGGATACCTACCTGAAACCGAAAAGCTTTACCATTGACGTTAGCAACTGCCAAGCGGCGGATGGCACCAAACAAGATGATGTGAGCAAACTGGGCGTGAACTGGACGGGCGGTAATTTACTGGCGGGCGCGACGAGCAAACAGCAAGGCTATCTGGCGAACACCGAAGCGAGCGGCGCGCAGAACATTCAGCTGGTGCTGAGCACCGATAACGCGACCGCGCTGACCAACAAAATTATTCCGGGCGATAGCACGCAGCCGAAAGCGAAAGGCGATGCGAGTGCCGTGGCGGATGGCGCGCGCTTTACCTATTATGTGGGCTATGCGACGAGCGCGCCGACCACCGTGACCACCGGCGTGGTGAACAGCTATGCGACCTATGAAATTACCTATCAAGGCGGTGGCAGCGCGGCGCATGCGGCGGATACGACCGTGGGCGGCGGTCAAGTGAACTTTTTTGGCAAAGTGgggtccCATCACCATCACCATCACCACCATTAAGCGGCCGC; SEQ ID NO.21: MLMrkAST (without His tag): CGSSGSGGSTDVSCTVSVNGQGSDANVYLSPVTLTEVKAAAADTYLKPKSFTIDVSNCQAADGTKQDDVSKLGVNWTGGNLLAGATSKQQGYLANTEASGAQNIQLVLS TDNATALTNKIIPGDSTQPKAKGDASAVADGARFTYYVGYATSAPTTVTTGVVNSYATYEITYQGGGSAAHAADTTVGGGQVNFFGKVGGGSGGGSAHIVMVDAYKPTK; SEQ ID NO.22: Amino acid sequence of recombinant SpyCatcher protein with O-glycosylation sequence (4573SC amino acid sequence, without His tag): SAVTEYYLNHGEWPGNTSAGVATSSEIKGAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHI.

[0048] Example 1: Preparation of fatty acid-modified recombinant MrkA protein containing SpyTag polypeptide (1.1) Construction of a fatty acid-modified recombinant MrkA protein expression vector containing SpyTag peptide According to the Klebsiella pneumoniae type 3 fimbriae MrkA protein sequence (type 3 fimbriaemajor subunit, GenBank: EU682505.2) published in GenBank, the recombinant MrkA protein was designed using SWISS-MODEL or other artificial intelligence tools, and 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, respectively, and SpyTag polypeptide (SEQ ID NO.2) and His tag were added. They were named MLMrkAST, rAgMrkAST, and P4MrkAST, respectively, and the DNA sequences were optimized and synthesized by Sangon Biotech (Shanghai) Co., Ltd. according to the preferred codons of Escherichia coli.

[0049] The optimized nucleotide sequence of MLMrkAST is shown in SEQ ID NO.8, the optimized nucleotide sequence of rAgMrkAST is shown in SEQ ID NO.9, and the optimized nucleotide sequence of P4SC is shown in SEQ ID NO.10; In SEQ ID NO.8, positions 1 to 6 are NdeⅠ restriction sites, positions 4 to 66 are signal peptide coding sequences shown in SEQ ID NO.5, positions 67 to 93 are linker coding sequences, positions 94 to 657 are recombinant MrkA protein coding sequences, positions 658 to 681 are linker coding sequences, positions 682 to 720 are SpyTag polypeptide tag coding sequences, positions 721 to 750 are linker and His tag coding sequences, and positions 754 to 761 are NotⅠ restriction sites; In SEQ ID NO.9, positions 1 to 6 are NdeⅠ restriction sites, positions 4 to 54 are signal peptide coding sequences shown in SEQ ID NO.6, positions 55 to 81 are linker coding sequences, positions 82 to 645 are recombinant MrkA protein coding sequences, positions 646 to 669 are linker coding sequences, positions 670 to 708 are SpyTag polypeptide tag coding sequences, positions 709 to 738 are linker and His tag coding sequences, and positions 742 to 749 are NotⅠ restriction sites; In SEQ ID NO.10, positions 1 to 6 are NdeⅠ restriction sites, positions 4 to 66 are signal peptide coding sequences shown in SEQ ID NO.7, positions 67 to 93 are linker coding sequences, positions 94 to 657 are recombinant MrkA protein coding sequences, positions 658 to 681 are linker coding sequences, positions 682 to 720 are SpyTag polypeptide tag coding sequences, positions 721 to 750 are linker and His tag coding sequences, and positions 754 to 761 are NotⅠ restriction sites; The optimized sequences were cloned into expression vectors respectively. Taking pET30a vector as an example, the obtained DNA fragments were inserted between the NdeⅠ and NotⅠ restriction sites of pET30a to construct pET30-MLMrkAST, pET30-rAgMrkAST, and pET30-P4MrkAST expression vectors.

[0050] (1.2) Construction of a strain expressing recombinant MrkA protein containing fatty acid-modified SpyTag peptide Taking SEQ ID NO.8 as an example, the constructed expression vector pET30-MLMrkAST was introduced into Escherichia coli C43DE3 (purchased from Sigma, Catalog No. CMC0019) or BL21DE3 (Thermo Fisher Scientific Inc., Catalog No. EC0114) host cells, and LB solid culture medium containing kanamycin with a final concentration of 50 μg / mL was coated. The positive clones were the expression strains C43DE3 / pET30-MLMrkAST or BL21DE3 / pET30-MLMrkAST.

[0051] (1.3) Expression and purification of fatty acid-modified recombinant MrkA protein containing SpyTag peptide The recombinant bacteria C43DE3 / pET30-MLMrkAST or BL21DE3 / pET30-MLMrkAST single clone was selected and inoculated into LB medium containing kanamycin with a final concentration of 50 μg / mL, cultured at 37°C until OD600 was about 0.6, IPTG with a final concentration of 0.5 mM was added, and the temperature was lowered to 25°C for induction for 6 to 20 hours.

[0052] The next day, the bacterial suspensions induced at 25°C were taken, and the cells were centrifuged and suspended in distilled water at a ratio of 1:30 (W / V). The cells were ultrasonically disrupted, and the precipitate and supernatant were separated by centrifugation. The precipitate was resuspended in an equal volume of water, and the sample was prepared with 5X reducing buffer (250mM pH6.8 Tris-HCl, 10% SDS, 0.5% bromophenol blue, 50% glycerol, 500mM DTT), and the sample was prepared by boiling water bath for 10min, and 15% SDS-PAGE electrophoresis was performed. After the electrophoresis, the cells were transferred to a PVDF membrane, and the membrane was transferred at a constant voltage of 20V for 1h. The Anti-His tag monoclonal antibody (Sigma, A7058) was used for detection. The results are shown as follows: Figure 1 shown. Figure 1 The left picture in the figure shows the result of Coomassie brilliant blue staining; Figure 1 The right picture shows the detection result of Anti-His tag monoclonal antibody (Sigma, A7058).

[0053] 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.

[0054] The sample was purified using a Chelating affinity chromatography column (Φ1.6 cm × 15 cm).

[0055] After harvesting the bacteria, suspend them in Ni-A1 buffer (20mM pH7.5 Tris-HCl + 0.3M NaCl + 5mMimidazole + 1% Triton X100) at a ratio of 1:20 (W / V), disrupt them with ultrasound, take the supernatant by centrifugation, and purify them by Chelating affinity chromatography.

[0056] First, rinse the column bed with a 0.5 M NaOH aqueous solution for at least 3 column volumes, then equilibrate with deionized water to neutral pH, then equilibrate with a 0.2 M NiSO4 aqueous solution for at least 3 column volumes, then equilibrate with Ni-B buffer (20 mM pH 7.5 Tris-HCl, 0.3 M NaCl, 500 mM imidazole + Triton X100) for 1 column volume, and finally equilibrate with Ni-A1 buffer (20 mM pH 7.5 Tris-HCl + 0.3 M NaCl + 5 mM imidazole + 1% Triton X100) for at least 1 column volume. The sample containing MLMrkAST was purified by Chelating affinity chromatography column, and the unbound protein was washed away with Ni-A1 buffer (20mM pH7.5 Tris-HCl + 0.3M NaCl + 5mM imidazole + 1% Triton X100) and balanced for at least 5 column volumes; then eluted with 10%, 30%, and 100% Ni-B (20mM pH7.5 Tris-HCl, 0.3MNaCl, 500mM imidazole + 1% Triton X100) buffer; the samples eluted with 30% Ni-B or 100% Ni-B buffer were collected to obtain the preliminary purified sample ( Figure 2 ).

[0057] The samples were purified using Superdex 200 Increase.

[0058] A Superdex200Increase column (φ1×30 cm, Cytiva, 28990944) was equilibrated with SEC (5mM pH7.4 PB + 0.9% NaCl + 0.01% Tween 80) buffer, and 1 mL of the 100% elution sample from the above Ni affinity chromatography was taken for purification. The results showed that the target protein was eluted in the range of 8 mL to 16 mL ( Figure 3 ), SDS-PAGE electrophoresis ( Figure 4 ) showed that the protein purity in the retention volume range of 10 mL to 16 mL was higher and suitable for subsequent experimental requirements.

[0059] (1.4) Characterization of fatty acid-modified recombinant MrkA protein containing SpyTag peptide In order to characterize the MLMrkAST protein, Shanghai Zhongke New Life Biotechnology Co., Ltd. was commissioned to perform N-terminal sequencing of the purified sample using the Edman degradation method. The results showed that the sequencing failed and did not match the theoretical sequence. It was speculated that the N-terminus of the recombinant protein was blocked. The molecular weight of MLMrkAST was further detected using mass spectrometry (XevoG2-XS Q-Tof (Waters)). Figure 5 ), the theoretical molecular weight of the unmodified recombinant MrkA protein (without N-terminal Cys) is 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, which were consistent with the molecular weight of recombinant MrkAST containing Cys modified with sulfhydryl groups by diacylglycerol and α-amino groups by acyl groups. The lengths of the three corresponding fatty acid chains were slightly different, among which 23827.00 Da was (C16, C16, C16), 23841.25 Da was (C17, C16, C16), and 23855.25 Da was (C18, C16, C16 or C17, C17, C16). There are also a small number of peaks at 23867.25 Da and 23878.75 Da, corresponding to fatty acid chain forms such as (C18, C17, C16) and (C18, C17, C17). That is, MLMrkAST contains an N-terminal Cys with a thiol group modified by diacylglycerol and an α-amino group modified by an acyl group, and the fatty acid chain lengths of the three acyl groups are C16-C18.

[0060] Based on the above calculations and the technical solution of the present invention, the N-terminus of the recombinant MrkA protein prepared by the present invention is modified by fat, and the recombinant MrkA protein prepared by this method is named LipoMrkAST.

[0061] The appearance of the purified LipoMrkAST lipoprotein was observed using transmission electron microscopy (TEM), and the results showed that the obtained LipoMrkAST was irregular particles.

[0062] (1.5) Preparation of non-fatty acid-modified recombinant MrkA protein (MrkAST) containing SpyTag peptide The signal peptide of the recombinant MrkA protein was replaced with the bacterial pelB signal peptide. The amino acid sequence of the recombinant MrkAST mature peptide is shown in SEQ ID NO.19, and the nucleotide sequence is shown in SEQ ID NO.14.

[0063] In SEQ ID NO.14, positions 1 to 6 are NdeⅠ restriction sites, positions 4 to 69 are pelB signal peptide coding sequences, positions 70 to 633 are MrkA protein coding sequences, positions 634 to 696 are SpyTag polypeptide coding sequences, positions 697 to 726 are linker and His tag coding sequences, and positions 730 to 737 are NotⅠ restriction sites; The recombinant MrkA protein (MrkAST) without fatty acid modification was prepared according to the expression and purification method described in this example. The purification results are shown in FIG. Figure 7 shown.

[0064] Example 2: Preparation of fatty acid-modified recombinant MrkA protein without SpyTag polypeptide (2.1) Construction of expression vector for fatty acid-modified recombinant MrkA protein (MLMrkA) According to the Klebsiella pneumoniae type 3 fimbriae MrkA protein sequence published in GenBank (type 3 fimbriaemajor subunit, GenBank: EU682505.2), the 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 preferred codons of Escherichia coli.

[0065] 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 signal peptide coding sequences shown in SEQ ID NO.5, positions 67 to 93 are linker coding sequences, positions 94 to 657 are recombinant MrkA protein coding sequences, positions 658 to 687 are linker and His tag coding sequences, and positions 691 to 698 are NotⅠ restriction sites; The optimized sequence was cloned between the NdeⅠ and NotⅠ restriction sites of the pET30a vector to construct the pET30-MLMrkA expression vector.

[0066] (2.2) Construction of expression strain of fatty acid-modified recombinant MrkA protein (MLMrkA) The constructed expression vector pET30-MLMrkA was introduced into the host cells of Escherichia coli C43DE3 (purchased from Sigma, catalog number CMC0019), and LB solid culture medium containing kanamycin with a final concentration of 50 μg / mL was coated. The positive clone was the expression strain C43DE3 / pET30-MLMrkA.

[0067] (2.3) Expression and purification of fatty acid-modified recombinant Klebsiella pneumoniae MrkA protein (MLMrkA) The recombinant bacteria C43DE3 / pET30-MLMrkA single clone was picked and inoculated into LB medium containing kanamycin with a final concentration of 50 μg / mL, cultured at 37°C until OD600 was about 0.6, IPTG with a final concentration of 0.5 mM was added, and the temperature was lowered to 25°C for induction for 6h-20h.

[0068] The sample was purified using a Chelating affinity chromatography column (Φ1.6 cm × 15 cm).

[0069] After harvesting the bacteria, suspend them in Ni-A1 buffer (20mM pH7.5 Tris-HCl + 0.3M NaCl + 5mMimidazole + 1% Triton X100) at a ratio of 1:20 (W / V), disrupt them with ultrasound, take the supernatant by centrifugation, and purify them by Chelating affinity chromatography.

[0070] First, rinse the column bed with a 0.5 M NaOH aqueous solution for at least 3 column volumes, then equilibrate with deionized water to neutral pH, then equilibrate with a 0.2 M NiSO4 aqueous solution for at least 3 column volumes, then equilibrate with Ni-B buffer (20 mM pH 7.5 Tris-HCl, 0.3 M NaCl, 500 mM imidazole + Triton X100) for one column volume, and finally equilibrate with Ni-A1 buffer (20 mM pH 7.5 Tris-HCl + 0.3 M NaCl + 5 mM imidazole + 1% Triton X100) for at least 1 column volume. The sample containing MLMrkA was purified using a Chelating affinity chromatography column, and the unbound proteins were washed away with Ni-A1 buffer (20mM pH7.5 Tris-HCl + 0.3M NaCl + 5mM imidazole + 1% Triton X100) and equilibrated for at least 5 column volumes; then eluted with 10%, 30%, and 100% Ni-B (20mM pH7.5 Tris-HCl, 0.3MNaCl, 500mM imidazole + 1%Triton X100) buffer; the sample eluted with 30% Ni-B buffer was collected to obtain a preliminarily purified sample.

[0071] The samples were purified using Superdex 200 Increase.

[0072] A Superdex200 Increase column (φ1×30 cm, Cytiva, 28990944) was equilibrated with SEC (5 mM pH7.4 PB + 0.9% NaCl + 0.01% Tween 80) buffer, and 1 mL of the 100% elution sample from the above Ni affinity chromatography was taken for purification.

[0073] (2.4) Characterization of fatty acid-modified recombinant MakA protein The purified MLMrkA samples were sequenced by N-terminal sequencing using the Edman degradation method to detect whether the N-terminus of the recombinant protein was blocked. The molecular weight of MLMrkA was further detected by mass spectrometry (XevoG2-XS QTof (Waters)), and the fatty acid modification form was estimated by the molecular weight increment. The appearance of the purified fatty acid modified recombinant MrkA lipoprotein was observed by transmission electron microscopy (TEM).

[0074] Example 3: Evaluation of the immunogenicity and protective effect of fatty acid-modified recombinant MrkA protein (LipoMrkAST) (3.1) Evaluation of the immunogenicity and protective effect of LipoMrkAST (intramuscular immunization) Female BALB / c mice aged 6 to 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), LipoMrkAST (5 μg) + Al(OH)3, and immunized by intramuscular injection on days 0, 14, and 28. Two weeks after immunization, blood was collected from the orbital venous sinus and coated with the MrkAST protein prepared in Example 1, and the MrkAST-specific antibody titer was detected by ELISA.

[0075] ELISA results showed that a single intramuscular injection of LipoMrkAST protein could induce high titer MrkAST-specific IgG antibodies, and there was no significant difference in the antibody titer induced by the two immunization doses of 1μg and 5μg ( Fig. 8A ), reaching the highest titer after three immunizations ( Figure 8B , Figure 8C ); The antibody conversion rate and antibody titer of mice immunized with Al(OH)3-adsorbed MrkAST were significantly lower than those of the LipoMrkAST group.

[0076] Two weeks after the third immunization, mice in the 1 μg group (3.98×10 7 CFU) to observe the survival rate of mice in each group ( Fig. 9 ). The results showed that the protective effect of LipoMrkAST was significantly better than that of MrkAST group.

[0077] The above results prove that compared with the unlipidated recombinant MrkA protein, the fatty acid-modified recombinant MrkA protein (LipoMrkAST) can induce high-titer MrkAST-specific IgG antibody response more quickly and show better protective effect in the mouse bacteremia model.

[0078] (3.2) Evaluation of the immunogenicity of LipoMrkAST (respiratory mucosal immunity) Female BALB / c mice aged 6 to 8 weeks were randomly divided into the following groups: Saline, MrkAST (5 μg), and LipoMrkAST (5 μg). They were immunized by respiratory mucosal aerosolization on days 0, 14, and 28. Blood was collected from the orbital venous sinus two weeks after immunization, and the plates were coated with MrkAST protein. ELISA was used to detect the titer of MrkA-specific antibodies in the serum. The results showed that after three respiratory mucosal immunizations, the fatty acid-modified recombinant MrkA protein (LipoMrkAST) induced MrkAST-specific serum IgG and IgA antibodies ( Fig.10 ).

[0079] Example 4: Preparation of fatty acid-modified Klebsiella pneumoniae dual antigen immune composition (4.1) SpyCatcher-OPS KpO2 (SC4573-OPS KpO2 ) expression and preparation Referring to the method described in patent CN111909951B, the Klebsiella pneumoniae O polysaccharide protein conjugate SpyCatcher-OPS was prepared by bioconjugation method KpO2 (SC-OPS KpO2 The recombinant expression vector pETtac28-pglL-SC4573 containing the coding sequence of oligosaccharide transferase PglL of Neisseria meningitidis (amino acid sequence as shown in SEQ ID NO.15) and the coding sequence of recombinant SpyCatcher protein with 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 bacteria Kp355ΔwaaL / pET28tac-SC4573. The polysaccharide-protein conjugate of the engineered bacteria was cultured and purified by Ni affinity chromatography, anion exchange chromatography, and molecular exclusion chromatography to obtain SC4573-OPS KpO2 .

[0080] Or, referring to the method described in patent CN105695497B, the Klebsiella pneumoniae O polysaccharide protein conjugate SpyCatcher-OPS is prepared by bioconjugation method KpO2 (SC-OPS KpO2 The recombinant expression vector pETtac28-pglL-SC4573 containing the coding sequence of the oligosaccharide transferase PglL of Neisseria meningitidis and the coding sequence of the recombinant SpyCatcher protein with an O-glycosylation sequence, and the recombinant vector pACYC-OPS containing the Kp355 OPS locus were KpO2Introduced into O antigen ligase-deficient Escherichia coli (W3110ΔwaaL) to construct the recombinant engineering bacteria W3110ΔwaaL / pET28tac-SC4573 / pACYC-OPS KpO2 Cultivate engineered bacteria to express polysaccharide-protein conjugates, and purify polysaccharide-protein conjugate SC4573-OPS by Ni affinity chromatography, anion exchange chromatography, and size exclusion chromatography KpO2 , the results are as follows Fig.11 shown.

[0081] SpyCatcher-OPS, a Klebsiella pneumoniae O1 type O polysaccharide protein conjugate, was prepared according to the above method. KpO1 (SC-OPS KpO1 ), the purification results are as follows Fig.12 .

[0082] (4.2) LipoMrkAST and SC4573-OPS KpO2 Connections Using LipoMrkAST with SC4573-OPS KpO2 Mix according to a certain molar ratio (recommended 1:3), place at 37℃ for 2 hours, or place at 4℃ for 12 hours to make LipoMrkAST and SC4573-OPS KpO2 The reaction was complete, and the connection effect was detected by SDS-PAGE and WB. The results are as follows Fig.13 .

[0083] The results showed that LipoMrkAST and SC4573-OPS KpO2 The molecular weight increased significantly after mixing, proving that the two molecules had a connection reaction, and in SC4573-OPS KpO2 Under excess conditions, LipoMrkAST can be fully ligated.

[0084] The ligation product was purified by size exclusion chromatography. A Superdex 200 Increase column (φ1×30 cm, Cytiva, 28990944) was equilibrated with SEC (5mM pH7.4 PB + 0.9% NaCl) buffer. 1 mL of the ligation product was purified. SDS-PAGE and WB results showed that the ligation product was present in the range of 8 mL to 12 mL. KpO2 The ligation product (LipoMrkA-OPS KpO2 ), 13mL to 16mL 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 detected 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 ( Fig.15 ).

[0085] (4.3) MrkAST and SC4573-OPS KpO1 Connections Using MrkAST with SC4573-OPS KpO1 Mix them in a certain molar ratio (recommended 1:3), place them at 37℃ for 2 hours, or place them at 4℃ for 12 hours to make MrkAST and SC4573-OPS KpO1 The reaction was complete, and the connection effect was detected by SDS-PAGE and WB. The results are as follows Fig.17 .

[0086] (4.4) LipoMrkAST and SC4573-OPS KpO1 Connections Using LipoMrkAST with SC4573-OPS KpO1 Mix according to a certain molar ratio (recommended 1:3), place at 37℃ for 2 hours, or place at 4℃ for 12 hours to make LipoMrkAST and SC4573-OPS KpO1 The reaction was complete, and the connection effect was detected by SDS-PAGE and WB. The results are as follows Fig.18 .

[0087] The results showed that LipoMrkAST and SC4573-OPS KpO1 The molecular weight increased significantly after mixing, proving that the two molecules had a connection reaction, and in SC4573-OPS KpO1 Under excess conditions, LipoMrkAST can be almost completely ligated.

[0088] The ligation product was purified by size exclusion chromatography. A Superdex 200 Increase column (φ1×30 cm, Cytiva, 28990944) was equilibrated with SEC (5mM pH7.4 PB + 0.9% NaCl) buffer. 1 mL of the ligation product was purified. SDS-PAGE and WB results showed that the ligation product was present in the range of 9 mL to 12 mL. KpO1 The ligation product (LipoMrkA-OPS KpO1 ), 13mL to 16mL is unreacted SC4573-OPS KpO1 ( Fig.19), collect 9 mL to 12 mL of ligation product for subsequent immunological evaluation.

[0089] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A recombinant MrkA protein modified by fatty acid.

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

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 a Cys thiol group modified by diacylglycerol, or a Cys thiol group modified by diacylglycerol and an α-amino group modified by an acyl group.

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

5. The fatty acid-modified recombinant MrkA protein according to any one of claims 1 to 4, characterized in that: The recombinant MrkA protein comprises a polypeptide having an 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 to 5.

7. The immune composition containing fatty acid-modified recombinant MrkA protein according to claim 6, characterized in that: The invention further comprises 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 the 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 a SpyCatcher / SpyTag connector.

10. The immune composition containing fatty acid-modified recombinant MrkA protein according to claims 7-9, characterized in that: The polysaccharide and the recombinant fusion protein containing the SpyCatcher protein form a polysaccharide-protein conjugate containing the SpyCatcher protein, and the polysaccharide-protein conjugate containing the SpyCatcher protein is covalently connected to the fatty acid-modified MrkA protein containing the SpyTag polypeptide by forming an isopeptide bond.

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 an amino acid sequence shown in any one of SEQ ID NOs. 11-13, and the SpyTag polypeptide is an amino acid sequence shown in any one of SEQ ID NOs. 2-4.

12. The immune composition containing fatty acid-modified recombinant MrkA protein according to claim 10, 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, and the Klebsiella pneumoniae polysaccharide is Klebsiella pneumoniae O polysaccharide (OPS) and / or Klebsiella pneumoniae capsular polysaccharide (CPS).

15. A method for preparing a fatty acid-modified recombinant MrkA protein according to any one of claims 1 to 14, characterized in that: The method comprises the following steps: 1) Introducing a DNA fragment containing a signal peptide coding sequence, a MrkA protein coding sequence, and a SpyTag polypeptide coding sequence or not into Escherichia coli, and culturing the recombinant Escherichia coli to express a recombinant MrkA protein with a fatty acid modification at the N-terminus; 2) culturing the recombinant Escherichia coli described in step 1) to express the fatty acid-modified recombinant MrkA protein; 3) Lysing the recombinant E. coli, adding a surfactant, and extracting and purifying the fatty acid-modified recombinant MrkA fusion protein from the lysate.

16. The preparation method according to claim 15, characterized in that: The signal peptide is characterized in that the last four amino acid sequences at the C-terminus are [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 a fatty acid-modified recombinant MrkA protein according to any one of claims 7 to 14, characterized in that: The method comprises the steps of mixing a fatty acid-modified recombinant MrkA protein containing a SpyTag polypeptide and a bacterial polysaccharide-protein conjugate containing a SpyCatcher protein in a certain ratio, and fully reacting them under appropriate conditions to obtain an immune composition.

19. Use of the fatty acid modified recombinant MrkA protein according to any one of claims 1 to 5 in the preparation of a product capable of inducing an animal to produce antigen-specific antibodies, wherein the fatty acid modified recombinant MrkA protein is immunized by intramuscular injection, subcutaneous injection, nasal drops or aerosol inhalation.

20. Use of a composition containing fatty acid-modified recombinant MrkA protein as claimed in any one of claims 6 to 14 in the preparation of a product capable of inducing animals to produce antigen-specific antibodies, wherein the immune composition is administered by intramuscular injection, subcutaneous injection, nasal drops or aerosol inhalation.

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

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

23. Biomaterial, characterized in that The biological material is any of the following: C1) a nucleic acid molecule encoding the fatty acid-modified recombinant MrkA protein according to any one of claims 1 to 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) a recombinant microorganism containing the nucleic acid molecule described in C1), or a recombinant microorganism containing the expression cassette described in C2), or a recombinant microorganism containing the recombinant vector described in C3); C5) a recombinant cell containing the nucleic acid molecule described in C1), or a recombinant cell containing the expression cassette described in C2), or a recombinant cell containing the recombinant vector described in C3).

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