Klebsiella pneumoniae truncated outer membrane protein and application thereof

Abstract

The invention belongs to the technical field of molecular biology, and discloses a truncated outer membrane protein of klebsiella pneumoniae and application thereof. The invention provides an outer membrane protein Lol B of klebsiella pneumoniae, which can be used for immunizing to induce a protective antibody aiming at various serotypes of klebsiella pneumoniae and killing the klebsiella pneumoniae. Specifically, the outer membrane protein Lol B immune serum can efficiently kill Klebsiella pneumoniae, and when a mouse immunized by the outer membrane protein Lol B immune serum is attacked by K1, K2 and K6 serotype Klebsiella pneumoniae, the mouse can be completely protected to survive, and the trend of weight loss of the mouse is remarkably reduced. Therefore, the outer membrane protein Lol B provided by the invention has a clinical application value for preventing and treating klebsiella pneumoniae infection.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to a truncated outer membrane protein of Klebsiella pneumoniae and its uses. Background Technology

[0002] In 2017, the World Health Organization released its first list of 12 key pathogens that seriously endanger human health, among which Klebsiella pneumoniae (Klebsiella pneumoniae) was listed. Klebsiella pneumoniae Enterobacteriaceae, represented by Klebsiella pneumoniae (KP), are classified as extremely important. KP is a Gram-negative bacterium and one of the most common opportunistic pathogens in clinical practice. KP can colonize the intestines, nasopharynx, and axilla, with the digestive tract being the most common site of colonization. Colonization rates vary considerably by region; in Western countries, the colonization rate in the colon is 5%-35%, while in some Asian countries (such as Malaysia), it can reach over 85%. KP can cause infections in various parts of the body, and is commonly seen in elderly patients, those with malnutrition, chronic diseases, and those experiencing systemic failure. It can cause systemic or local infections such as pneumonia, urinary tract infections, meningitis, and sepsis. Based on virulence and pathogenic characteristics, KP is currently divided into two categories: classic Klebsiella pneumoniae (cKP), which mainly causes hospital-acquired infections such as pneumonia, urinary tract infections, and sepsis, and has a high drug resistance rate, commonly seen in people with underlying diseases or weakened immune systems. Another type is highly virulent Klebsiella pneumoniae (hvKP), which mainly causes infection in healthy people in the community without underlying diseases. The most common cause is liver abscess, which is caused by KP infection in 66% of cases.

[0003] In recent years, the resistance of Klebsiella pneumoniae (KP) to various common antibiotics has become increasingly severe. Bacterial resistance monitoring results show that KP isolation rates have jumped to second place among Gram-negative bacilli, second only to Escherichia coli, reaching 19.8% in 2021. Its resistance rate to ampicillin has reached as high as 91.8%, and its resistance rate to piperacillin is close to 50%. In particular, with the widespread clinical use of carbapenems, the detection rate of carbapenem-resistant Klebsiella pneumoniae (CRKP) has been increasing year by year. Furthermore, KP infection has an extremely high mortality rate. Statistics show that 22%-32% of KP-related community pneumonia patients require ICU treatment, with a mortality rate as high as 45%-72%. In addition, KP accounts for 5%-20% of sepsis cases caused by Gram-negative bacterial infections, with a mortality rate as high as 27.4%-37%. More importantly, studies have shown that the mortality rate of KP infection, which is sensitive to carbapenems, is 20%-30%, while the mortality rate of CRKP infection is significantly higher, reaching 40%-70%. Due to its extremely strong drug resistance and pathogenicity, CRKP is known as the "king of superbugs." Considering the already severe situation of KP drug resistance, especially the widespread prevalence of CRKP, antibiotic treatment is extremely difficult, making the development of new and effective prevention and control methods urgent. Vaccine development is one of the most promising strategies.

[0004] Since the emergence of Klebsiella pneumoniae (KP) vaccine research in the 1970s, various types of vaccine research have continued to develop. Early research focused on inactivated, attenuated, and bacterial lysis vaccines. These vaccines had complex compositions, were difficult to control in terms of quality, and may have residual toxicity, thus failing to enter clinical trials due to safety concerns. Later, research on ribosomal vaccines began, but due to their intracellular components and limited immunoprotective effects, they were not studied in depth. Next came polysaccharide vaccines. The antigens used in polysaccharide vaccines mainly include capsular polysaccharides and LPS (lipopolysaccharide). However, because there are more than 80 serotypes of Klebsiella pneumoniae capsular polysaccharide (K-antigen) and 12 serotypes of LPS (O-antigen), and the K-antigen and O-antigen vary greatly among different serotypes, although studies have shown that these vaccines exhibit good immunogenicity and safety in human trials and have good protective effects in passive immunization, their cross-protective efficacy against other serotypes is limited, objectively restricting their application. Recent research has primarily focused on recombinant protein vaccines. Various secreted proteins and outer membrane proteins have shown some protective effects against Klebsiella pneumoniae infection, such as outer membrane proteins (OmpA, OmpK36, FepA, OmpK17, OmpW), collicin I receptor protein, adhesin MrkD protein, fimbriae, cell surface iron regulatory proteins, and toxoids. These represent the most promising vaccine types currently. However, progress in developing highly specific antigens for Klebsiella pneumoniae has been slow, resulting in no breakthroughs in vaccine development for the prevention and treatment of Klebsiella pneumoniae infection, and no effective drugs are currently available. Therefore, the development of a broad-spectrum, highly effective protein vaccine against Klebsiella pneumoniae is urgently needed. Summary of the Invention

[0005] The present invention aims to at least solve one of the technical problems existing in the prior art, and to develop a Klebsiella pneumoniae protein vaccine. To this end, the present invention proposes an outer membrane protein of Klebsiella pneumoniae, Lol B, whose immunization can produce Klebsiella pneumoniae-specific antibodies to resist Klebsiella pneumoniae infection. Therefore, the Klebsiella pneumoniae outer membrane protein Lol B provided by the present invention is particularly suitable as a vaccine for the prevention and treatment of Klebsiella pneumoniae infection and / or diseases related to Klebsiella pneumoniae infection.

[0006] The first aspect of the present invention is to provide an outer membrane protein, Lol B.

[0007] The second aspect of the present invention aims to provide molecular materials related to the outer membrane protein Lol B of the first aspect of the present invention.

[0008] The third aspect of this invention is to provide a method for preparing the outer membrane protein Lol B according to the first aspect of this invention.

[0009] The fourth aspect of this invention aims to provide the application of the outer membrane protein Lol B of the first aspect of this invention or the biomaterial of the second aspect of this invention.

[0010] The fifth aspect of this invention aims to provide a product.

[0011] The sixth aspect of this invention aims to provide a method for detecting Klebsiella pneumoniae.

[0012] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides an outer membrane protein Lol B, wherein the amino acid sequence of the outer membrane protein Lol B is as follows: a1) SEQ ID NO:2; or b1) An amino acid sequence that is at least 90% homologous to SEQ ID NO: 2 and has the same or similar function; or c1) An amino acid sequence with the same or similar function after one or more amino acids have been substituted, deleted or added to the amino acid sequence shown in SEQ ID NO:2.

[0013] A second aspect of the invention provides a biomaterial related to the outer membrane protein Lol B of the first aspect of the invention, said biomaterial comprising at least one of d1)-d12): d1) A nucleic acid molecule encoding the outer membrane protein Lol B of the first aspect of the present invention; d2) An expression cassette containing the nucleic acid molecules described in d1); d3) A carrier containing the nucleic acid molecule described in d1); d4) A carrier containing the expression box described in d2); d5) Cell lines containing the nucleic acid molecules described in d1); d6) Cell lines containing the expression cassette described in d2); d7) Cell lines containing the vector described in d3); d8) Cell lines containing the vector described in d4); d9) Microorganisms containing the nucleic acid molecules described in d1); d10) Microorganisms containing the expression cassette described in d2); d11) Microorganisms containing the vector described in d3); d12) Microorganisms containing the carrier described in d4).

[0014] In some embodiments of the present invention, the nucleic acid molecule described in d1) may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.

[0015] In some embodiments of the present invention, the nucleotide sequence of the nucleic acid molecule described in d1) is as follows: 1) As shown in SEQ ID NO:1; 2) A nucleotide sequence of SEQ ID NO.:1 with one or more nucleotide substitutions and / or deletions and / or additions that have the same function as SEQ ID NO.:4; 3) A nucleotide sequence that has 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91% or 90% homology with SEQ ID NO:1 and has the same function as SEQ ID NO:1.

[0016] In some embodiments of the present invention, the expression cassette refers to DNA capable of expressing the outer membrane protein Lol B in host cells. This DNA may include not only a promoter to initiate transcription of the gene encoding the protein, but also a terminator to terminate transcription of the gene encoding the protein. Furthermore, the expression cassette may also include a replication start site, a transcription start sequence, an enhancer sequence, a selection element, or a reporter gene.

[0017] In some embodiments of the present invention, the vector includes a promoter that is operatively linked to the nucleic acid molecule.

[0018] In some embodiments of the present invention, the vector is independently selected from non-pathogenic viral vectors and viral vectors.

[0019] In some embodiments of the present invention, the viral vector includes at least one of lentiviral vector, adenovirus vector, baculovirus vector, retrovirus vector, poxvirus vector, Sendai virus vector, and herpes simplex virus vector.

[0020] In some embodiments of the present invention, the non-viral vector includes at least one of plasmid vectors, cationic polymer vectors, chitosan, polyethyleneimine, nanoparticle vectors, and liposomes.

[0021] In some embodiments of the present invention, the vector may be a plasmid vector, a phage particle, a viral vector, a cell vector, a bacteriophage, a visceron, an F visceron, or an artificial chromosome. The vector may be a cloning vector or an expression vector.

[0022] In some embodiments of the present invention, the cell line may be bacteria (such as Escherichia coli or Bacillus subtilis), algae, fungi (such as yeast or Aspergillus), insect cells (such as S2 Drosophila cells or Sf9 cells), or animal cells (such as CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, or HEK293 cells). The biological cell line does not include reproductive materials.

[0023] A third aspect of the present invention provides a method for preparing the outer membrane protein Lol B of the first aspect of the present invention, comprising the following steps: culturing the microorganisms and / or cell lines of the second aspect of the present invention to express the outer membrane protein Lol B.

[0024] In some embodiments of the present invention, the preparation method includes introducing plasmid pET28a-Lol B into Escherichia coli BL21 to obtain a microorganism expressing the protein shown in SEQ ID NO:2, wherein the plasmid pET28a-Lol B is obtained by ligating the nucleotide sequence shown in SEQ ID NO:1 to plasmid pET28a.

[0025] In some embodiments of the present invention, the expression is induced expression.

[0026] In some embodiments of the present invention, the induced expression is induced by IPTG.

[0027] A fourth aspect of the invention provides the use of the outer membrane protein Lol B of the first aspect of the invention or the biological material of the second aspect of the invention in at least one of e1)-e8): e1) Prepare products for the prevention or treatment of diseases related to Klebsiella pneumoniae infection; e2) Detection of Klebsiella pneumoniae; e3) Prepare products for detecting Klebsiella pneumoniae; e4) Inhibits the activity of Klebsiella pneumoniae; e5) Prepare products that inhibit the activity of Klebsiella pneumoniae; e6) Prepare products for diagnosing Klebsiella pneumoniae infection; e7) Neutralizes the virulence of Klebsiella pneumoniae; e8) Prepare products for neutralizing the virulence of Klebsiella pneumoniae; The above applications are for purposes other than disease diagnosis and treatment.

[0028] In some embodiments of the present invention, the product comprises at least one of a drug, an immune antigen, a reagent, a detection plate, a reagent kit, and a detection chip.

[0029] In some embodiments of the invention, the medicine includes a vaccine, such as a subunit vaccine for the prevention or treatment of Klebsiella pneumoniae infection.

[0030] In some embodiments of the present invention, the Klebsiella pneumoniae includes K1, K2 and / or K6 serotypes of Klebsiella pneumoniae.

[0031] A fifth aspect of the present invention provides a product comprising the outer membrane protein Lol B of the first aspect of the present invention and the biomaterial of the second aspect of the present invention.

[0032] In some embodiments of the present invention, the product has at least one function among f1)-f5): f1) Prevention or treatment of diseases related to Klebsiella pneumoniae infection; f2) Detection of Klebsiella pneumoniae; f3) Inhibits the activity of Klebsiella pneumoniae; f4) Diagnosis of Klebsiella pneumoniae infection; f5) neutralizes the virulence of Klebsiella pneumoniae.

[0033] In some embodiments of the present invention, the Klebsiella pneumoniae includes K1, K2 and / or K6 serotypes of Klebsiella pneumoniae.

[0034] In some embodiments of the present invention, the product comprises at least one of a drug, an immune antigen, a reagent, a detection plate, a reagent kit, and a detection chip.

[0035] In some embodiments of the present invention, the drug includes a vaccine.

[0036] In some embodiments of the present invention, the vaccine is a subunit vaccine for the prevention or treatment of Klebsiella pneumoniae infection.

[0037] In some embodiments of the invention, the vaccine further includes a pharmaceutically acceptable adjuvant.

[0038] In some embodiments of the present invention, the adjuvant is an aluminum adjuvant, an oil-emulsion adjuvant such as oil-in-water, water-in-oil, or bidirectional emulsion, a microbial adjuvant such as peptidoglycan (PG), lipopolysaccharide (LPS) from the outer membrane of Gram-negative bacteria, mycobacteria and their components (such as muramyl dipeptide), GpG oligonucleotides (GpG ODN), cholera toxin (CT), a particulate antigen delivery system such as liposomes, polymeric microspheres, inert nanospheres, immunostimulating complexes (ISCOM), cytokines, polysaccharides such as inulin (MPI), and at least one of natural sources such as propolis and saponins.

[0039] In some embodiments of the present invention, the adjuvant is selected from any one of aluminum hydroxide adjuvant, aluminum phosphate adjuvant, immunostimulatory complex (ISCOMS) adjuvant, aluminum monostearate adjuvant, MF59, complete Freund's adjuvant, incomplete Freund's adjuvant, and mycobacterial BCG adjuvant.

[0040] The vaccines described above are administered in a dosage-compatible manner, and at amounts such as therapeutically effective doses and immunogenically effective doses. The dosage depends on the recipient, the recipient's immune system's ability to synthesize antibodies, and the expected level of protection. The exact amount of active ingredient to be administered depends on the physician's judgment and varies from person to person. The appropriate schedule for initial administration and booster vaccinations can also vary, but typically a second injection or other administration is given at a certain interval (days, weeks, or months) after the first administration.

[0041] In some embodiments of the present invention, the vaccine can be used alone or in combination, or in combination with other active agents (e.g., polysaccharide vaccines, other protein vaccines).

[0042] In some embodiments of the present invention, the medicament may further comprise a pharmaceutically acceptable adjuvant or immunomodulator, selected from poly-ICLC, 1018ISS, Amplivax, MF59, AS03, AS04, AS15, BCG, CP-870, CP-893, CpG7909, CyaA, cyclic dinucleotides (such as STING), dSLIM, GM-CSF, IL-2, IC30, IC31, MontanideISA51, etc.

[0043] In some embodiments of the present invention, the product further includes pharmaceutically acceptable excipients.

[0044] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one selected from fillers, disintegrants, diluents, dispersants, excipients, stabilizers, lubricants, binders, humectants, flavoring agents, solubilizers, suspending agents, solvents, sustained-release agents, emulsifiers, absorption enhancers, surfactants, preservatives, pigments, fragrances, and solvents.

[0045] In some embodiments of the present invention, the filler is selected from starch, sucrose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, or glucose, etc.; the binder is selected from cellulose derivatives, alginate, starch, water, dextrin, gelatin, or polyvinylpyrrolidone, etc.; the disintegrant is selected from microcrystalline cellulose, sodium carboxymethyl starch, croscarmellose, low-substituted hydroxypropyl cellulose, or croscarmellose sodium; the lubricant is selected from stearic acid, polyethylene glycol, calcium carbonate, sodium bicarbonate, microcrystalline silica, talc, or magnesium stearate; and the suspending agent is selected from microcrystalline silica, beeswax, cellulose, solid polyethylene glycol, etc. Alcohol: The wetting agent is selected from glycerin, Tween-80, hydrogenated castor oil, or lecithin; the solvent is selected from ethanol, liquid polyethylene glycol, isopropanol, Tween-80, glycerin, propylene glycol, or vegetable oil, wherein the vegetable oil is selected from soybean oil, castor oil, peanut oil, blended oil, etc.; the surfactant is selected from sodium dodecylbenzenesulfonate, stearic acid, polyoxyethylene-polyoxypropylene copolymer, fatty acid sorbitan, or polysorbate (Tween), etc.; the flavoring agent is selected from aspartame, sucralose, flavoring, stevia, acesulfame potassium, citric acid, or sodium saccharin; the preservative is selected from at least one of methylparaben or propylparaben.

[0046] In some embodiments of the present invention, the dosage form of the drug includes a gastrointestinal dosage form or a non-gastrointestinal dosage form.

[0047] In some embodiments of the present invention, the gastrointestinal dosage form includes at least one of powder, tablet, granule, capsule, sustained-release, solution, dry suspension, effervescent tablet, emulsion, suspension, syrup, drops, and chewable tablet.

[0048] In some embodiments of the present invention, the gastrointestinal dosage forms include, but are not limited to, enteric-coated tablets, coated tablets, film-coated tablets, sugar-coated tablets, dispersible tablets, sucking tablets, chewable tablets, effervescent tablets, scratch tablets, sustained-release and controlled-release dosage forms, sustained-release tablets, sustained-release coated tablets, controlled-release tablets, orally disintegrating tablets, lozenges, and oral patches.

[0049] In some embodiments of the present invention, the non-gastrointestinal drug delivery dosage form includes at least one of injection dosage form, respiratory dosage form, skin dosage form, mucosal dosage form, and cavity dosage form.

[0050] In some embodiments of the present invention, the injectable dosage forms include, but are not limited to, injection solutions, solutions for injection, injection solutions for intravenous infusion, suspensions for injection, sterile powders for injection, intravenous injections, water injections, emulsions for injection, powder injections, injections, sterile powder injections, lyophilized powder injections, etc.

[0051] In some embodiments of the present invention, the drug may be administered via local administration at a pharmaceutically acceptable dose.

[0052] The outer membrane protein Lol B can effectively stimulate the body to produce a high humoral immune response and can provide significant protection against lethal doses of Klebsiella pneumoniae infection, making it a candidate antigen for Klebsiella pneumoniae vaccines.

[0053] In some embodiments of the present invention, the product is an immune antigen.

[0054] The aforementioned immunoantigens induce a specific antibody response against Klebsiella pneumoniae by immunizing animals, such as inducing bactericidal antibodies against different serotypes of Klebsiella pneumoniae (capable of killing K1, K2 and K6 serotypes of Klebsiella pneumoniae), in order to prevent and treat Klebsiella pneumoniae infection.

[0055] A sixth aspect of the present invention provides a method for detecting Klebsiella pneumoniae, comprising contacting a sample containing or suspected of containing Klebsiella pneumoniae with the outer membrane protein Lol B of the first aspect of the present invention or the product of the fifth aspect of the present invention, and detecting the formation of a complex of the outer membrane protein Lol B with Klebsiella pneumoniae, said method being used for non-disease diagnostic purposes.

[0056] A seventh aspect of the invention provides a method for preventing or treating Klebsiella pneumoniae infection or Klebsiella pneumoniae infection-related diseases in a subject, comprising administering to the subject in need a preventive or therapeutically effective amount of the outer membrane protein Lol B of the first aspect of the invention and the product (pharmaceutical) of the fifth aspect of the invention.

[0057] In some embodiments of the present invention, the subject is a mammal, such as a human.

[0058] In some embodiments of the present invention, the outer membrane protein Lol B of the first aspect of the present invention and the product (medication) of the fifth aspect of the present invention may be administered to a subject via any suitable route of administration. Such routes of administration include, but are not limited to, oral, intramuscular, intraperitoneal, oral, sublingual, local, parenteral, rectal, intrathecal, or nasal routes.

[0059] In some embodiments of the present invention, the disease includes pneumonia.

[0060] The beneficial effects of this invention are: This invention provides an outer membrane protein, Lol B, of Klebsiella pneumoniae, which, when immunized, can induce protective antibodies against multiple serotypes of Klebsiella pneumoniae, killing the bacteria. Specifically, Lol B-immunized serum can efficiently kill Klebsiella pneumoniae; mice immunized with this serum can completely survive challenges from K1, K2, and K6 serotypes of Klebsiella pneumoniae, and significantly reduce the tendency for weight loss in mice. Therefore, the outer membrane protein Lol B provided by this invention has clinical application value in the prevention and treatment of Klebsiella pneumoniae infection.

[0061] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

[0062] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 This is an SDS-PAGE electrophoresis image of the purified Lol B protein expressed by *E. coli*; lane M: protein molecular weight standard (marker); lane 1: *Klebsiella pneumoniae* Lol B protein expressed and purified by *E. coli*, the amino acid sequence of the expressed protein is SEQ ID NO:1.

[0063] Figure 2 To determine the binding activity of mouse immune serum against K2 serotype Klebsiella pneumoniae in an enzyme-linked immunosorbent assay (ELISA), five mice were immunized intramuscularly with aluminum adjuvant mixed with Lol B protein. Two immunizations were administered, 14 days apart. Serum was collected 14 days after the last immunization for ELISA experiments. The x-axis represents the dilution factor of the mouse serum, and the y-axis represents the OD value. 450 nm value.

[0064] Figure 3 This study describes a complement-mediated bactericidal assay using Lol B protein-immunized serum. The serum was the immunized serum after two injections of Lol B protein, diluted 100-fold. Figure A shows the bactericidal effect of serum against Klebsiella pneumoniae K1 serotype, and Figure B shows the bactericidal effect of serum against Klebsiella pneumoniae K2 serotype. The vertical axis represents the number of colonies. The control group consisted of negative serum as a bactericidal control. Statistical analysis between groups was performed using the t-test. .

[0065] Figure 4 To immunize mice with two doses of Lol B, 14 days after the second dose, the mice were infected with 1×10 6 Analysis of body weight and survival rate of mice infected with K1 serotype Klebsiella pneumoniae at a dose of CFU / mouse; Figure A shows the body weight curve of mice, Figure B shows the survival rate curve of mice, and the control group is the mice immunized with PBS; In Figure A, the area under each curve was calculated first using the AUC method, and then the statistical differences between the groups were compared.

[0066] Figure 5 To immunize mice with two doses of Lol B, 14 days after the second dose, the mice were infected with 1×10 6Analysis of body weight and survival rate of mice infected with K2 serotype Klebsiella pneumoniae at a dose of CFU / mouse; Figure A shows the body weight curve of mice, Figure B shows the survival rate curve of mice, and the control group is the mice immunized with PBS; In Figure A, the area under each curve was first calculated using the AUC method, and then the statistical differences between the groups were compared.

[0067] Figure 6 To immunize mice with two doses of Lol B, 14 days after the second dose, the mice were infected with 1×10 6 Analysis of body weight and survival rate of mice infected with K6 serotype Klebsiella pneumoniae at a dose of CFU / mouse; Figure A shows the body weight curve of mice, Figure B shows the survival rate curve of mice, and the control group is the mice immunized with PBS; In Figure A, the area under each curve was first calculated using the AUC method, and then the statistical differences between the groups were compared. Detailed Implementation

[0068] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0069] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0070] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in this invention are generally performed in accordance with the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F.M. Ausubel et al., A Concise Guide to Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995. Where specific conditions are not specified in the examples, they are performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0071] Unless otherwise specified, "room temperature" in this invention means 25℃±5℃.

[0072] The term "vaccine" refers to a biological product made from various pathogenic microorganisms for preventive inoculation. Vaccines are preparations used to prevent infectious diseases by artificially attenuating, inactivating, or genetically modifying pathogenic microorganisms (such as bacteria, rickettsiae, viruses, etc.) and their metabolic products. Vaccines retain the characteristic of stimulating the animal's immune system. When an animal comes into contact with this harmless pathogen, the immune system produces protective substances such as immune hormones, active physiological substances, and specific antibodies. When the animal is exposed to the same pathogen again, its immune system, following its previous memory, produces more protective substances to prevent harm from the pathogen.

[0073] The term "protein vaccine" refers to a vaccine prepared by constructing a gene for a specific antigen on an expression vector, transforming the constructed expression vector into bacterial, yeast, mammalian, or insect cells, expressing a large amount of antigen protein under certain induction conditions, and then purifying the protein.

[0074] The term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains (each pair consisting of a "light" (L) chain and a "heavy" (H) chain). Antibody light chains can be classified as κ and λ light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both light and heavy chains, variable and constant regions are linked by "J" regions of approximately 12 or more amino acids, and the heavy chain also contains "D" regions of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant regions of antibodies mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable regions (VH and VL) of each heavy / light chain pair form the antibody binding sites. The allocation of amino acids to various regions or domains follows the definitions in Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J.Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883.

[0075] The term "Da" is a commonly used unit of molecular weight. Da stands for Dalton, and it is the algebraic sum of the atomic masses of all atoms in a molecule. Proteins are large molecules, so they are often represented by kDa (kilodaltons). The Dalton is a unit of atomic mass, and it is frequently used as D or KD in biochemistry, molecular biology, and proteomics.

[0076] In this invention, amino acids are typically represented using single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0077] In order to obtain a Klebsiella pneumoniae vaccine with immunoprotective effect, the inventors of this application expressed and purified Lol B protein, immunized mice, and verified the bactericidal activity of the serum of immunized mice and the ability of immunized mice to resist the attack of multiple serotypes of Klebsiella pneumoniae. The results showed that the serum induced by Lol B immunization can effectively kill bacteria, and the mice immunized with it can resist infection of K1, K2 and K6 serotypes of Klebsiella pneumoniae.

[0078] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0079] Example 1: Synthesis, subcloning, and protein expression of the Lol B gene 1. The synthesis of the DNA sequence (SEQ ID NO:1) encoding the Lol B protein of Klebsiella pneumoniae, the ligation of this sequence with pET28a, and the expression strain pET28a-Lol-BL21 were all synthesized and constructed by Shanghai Sangon Biotech Co., Ltd.

[0080] The DNA sequence of Lol B protein is: (SEQ ID NO:1).

[0081] 2. Lol B-induced expression Add 100 μL of overnight cultured pET28a-Lol-BL21 bacterial culture to 10 mL of Kana-resistant LB medium, and incubate at 37°C for 3 h at 180 rpm until OD500 is reached. 600 When the concentration is 0.6-0.8, add IPTG to make a final concentration of 200 μM, and then incubate the culture in a shaker at 25℃ for 5 h to induce expression. Take out the induced bacterial culture, centrifuge at 12000 rpm for 5 min, discard the supernatant, add 1 mL of bacterial lysis buffer (50 mM citric acid, pH 5.0), mix well, sonicate for 3 min, and then centrifuge at 14000 rpm for 15 min at 4℃ to separate the supernatant and precipitate.

[0082] 3. Scale-up culture to obtain protein Take 400 μL of pET28a-Lol-BL21 bacterial culture stored at 4℃ and add it to 30 mL of LB medium containing Kana resistance for primary activation. After incubation at 37℃ for 5-6 h at 200 rpm, take 20 mL of the primary activated bacterial culture and add it to 2000 mL of LB medium containing Kana resistance for secondary activation. Incubate at 37℃ for 3-4 h until OD reaches 0.5%. 600 When the concentration was 1.0, 200 μL LIPTG (final concentration 200 μM) was added and the mixture was incubated in a shaker at 25℃ for 5 h. After centrifugation at 12000 rpm for 15 min, the bacterial cells were collected. The bacterial cells were then resuspended in 50 mL of bacterial lysis buffer (50 mM citric acid, pH 5.0). The bacterial solution was then sonicated for 3 min (200 V), and the supernatant was collected for subsequent purification.

[0083] 4. Preliminary purification of BoNT-Ag by Ni–NTA affinity chromatography Take 5 mL of Ni–NTA affinity chromatography packing material and equilibrate it with Ni-NTA lysis buffer (pH 8.0, 17.3 mM Na2HPO4, 2.7 mM NaH2PO4, 300 mM NaCl and 10 mM imidazole). Then, add the supernatant from step 3 to the affinity chromatography packing material and elute impurities and non-specifically bound proteins with Ni-NTA wash buffer (pH 8.0, 17.3 mM Na2HPO4, 2.7 mM NaH2PO4, 300 mM NaCl and 20 mM imidazole). Finally, elute the target protein with Ni-NTA elution buffer (pH 8.0, 17.3 mM Na2HPO4, 2.7 mM NaH2PO4, 300 mM NaCl and 250 mMimidazole).

[0084] 5. SDS-PAGE electrophoresis Pour 10% separating gel into the gel casting plate, add distilled water to flatten the gel, and let it solidify at room temperature for 30 min. Pour off the top layer of distilled water, then pour in the stacking gel, immediately insert the comb, and let it solidify at room temperature for 30 min. Load 10 μL of the prepared samples for SDS-PAGE electrophoresis. Initially, electrophoresis at 80 V for 30 min, then adjust to 180 V and electrophoresis for 1-2 h. After electrophoresis, remove the gel, stain it with Coomassie Brilliant Blue solution, and then destain it with destaining solution. Observe the results under an imaging system.

[0085] The results are as follows Figure 1As shown, the eluted target protein (Lol B protein) has a high purity.

[0086] The amino acid sequence of Lol B protein is as follows: TELSLTAQPGSVQLIDNKGQTYTAADAEEMIGRLTGMPIPLNSLRQWIIGLPGDATDYSLDDRYRLRELNYTQNGKTWHVTYGGYTSDTQPALPSNVELNNGAQRIKLKMDNWIVK (SEQ ID NO: 2).

[0087] Example 2: Immune mice with Lol B and determination of ELISA titer in mouse serum The Lol B antigen (i.e., the Lol B protein in Example 1) was diluted with PBS and Al(OH)3 at a concentration of 1 mg / mL was added to prepare the vaccine. BALB / c mice (n=5 per group) were immunized by intramuscular injection in both thighs using a No. 5 half-needle on days 0 and 14. The injection volume per mouse was 100 μL, and the antigen content was 10 μg / 50 μL. The blank control group was immunized with the same volume of PBS. On day 14 after the last immunization, ocular blood was collected from BALB / c mice, and the level of specific IgG response against K2 serotype Klebsiella pneumoniae was detected by ELISA.

[0088] 1. Preparation of liquid 1) Preparation of coating solution: Weigh 1.6 g of Na2CO3 and 2.9 g of NaHCO3, dissolve them in 1 L of ddH2O, and adjust the pH to 9.6 using a pH meter; 2) Preparation of blocking solution: 1g bovine serum albumin, dissolved in 100 mL antibody dilution solution (1:100). 3) Preparation of antibody dilution solution: Dissolve phosphate in 1L ddH2O, then add 500 μL Tween 20, and then adjust the pH to 7.4 using a pH meter; 4) Preparation of washing solution: Same as antibody dilution solution 5) The colorimetric solution (TMB) is a product of Sangon Biotech (Shanghai) Co., Ltd. 6) Preparation of the stop solution (2M H2SO4): Pour 22.2 mL of concentrated sulfuric acid into 177.8 mL of ddH2O.

[0089] 2. ELISA detection of antibody titers produced by mice immunized with Lol B recombinant protein. 1) Coat 10 with coating solution 5 CFU was diluted from clinically collected K2 serotype Klebsiella pneumoniae to a total volume of 10 mL; 2) Coating: Add 100 μL of Klebsiella pneumoniae dilution to the microplate, incubate overnight at 4°C, wash 3 times with washing buffer, air dry, wrap with plastic wrap, and store in a refrigerator at 4°C for later use. 3) Blocking: Add 200 μL of blocking buffer to each well of the ELISA plate, incubate at 37℃ for 2 h, and wash 3 times; 4) Dilute mouse serum serially at ratios of 1:1000, 1:2000, 1:4000, and 1:8000; 5) Take the sealed ELISA plate, add diluted serum in sequence, 100 μL / well, incubate at 37℃ for 1 h, wash 3 times, and air dry; 6) Dilute the HRP-labeled goat anti-mouse IgG antibody preservation solution 1:10000 to prepare the antibody working solution; 7) Add diluted antibody working solution, 100 μL / well, incubate at 37℃ for 40 min, wash three times, and air dry; 8) Add 100 μL of substrate development solution (TMB) per well and react at room temperature in the dark for 5 min; 9) Add stop solution (2M H2SO4) and immediately place on a microplate reader to measure the OD value at a wavelength of 450 nm; 10) Result interpretation: A positive result is defined as A sample / A negative value ≥ 2.1 (the negative control is a 1:1000 dilution of mouse pre-immunization serum).

[0090] The results showed that the titers of specific IgG antibodies against Klebsiella pneumoniae produced by the five mice immunized with the Lol B protein antigen were all 1:51200. Figure 2 This indicates that the Lol B protein constructed in this invention has good immunogenicity, and its immunization can induce specific antibodies against Klebsiella pneumoniae.

[0091] Example 3: Bactericidal experiment of Lol B protein-immunized serum (1) Prepare the Klebsiella pneumoniae collected from the clinic for the experiment, culture it to the logarithmic growth phase, wash it twice with sterile phosphate solution and adjust it to the experimental concentration.

[0092] (2) Induction of HL-60 cells (human promyelocytic leukemia cells) into neutrophils: HL-60 cells in good condition were centrifuged. The collected cells were centrifuged again, the supernatant was discarded, and the cells were resuspended in 1 mL of sterile PBS and counted. A sterile T75 cell culture flask was filled with 25 mL of 15% complete culture medium, and DMSO was added, adjusting the final DMSO concentration to 1.25%. 3 × 10⁻⁶ cells were collected. 7 After mixing the HL 60 cells with the above culture medium, they were placed in a constant temperature culture at 37 ℃ and 5% CO2 for 6 days to complete differentiation.

[0093] (3) On the 6th day after HL-60 cell differentiation, the cells were collected in a 50 mL centrifuge tube, centrifuged at 1,000 rpm at room temperature for 5 min to collect the cells, and washed once with RPMI 1640 basal medium.

[0094] (4) After resuspending the neutrophils, count the cells, then take 10 μL of cell suspension, dry it on a glass slide, and stain the cell nuclei with Giemsa stain to observe whether the single cell nucleus has differentiated into multiple cell nuclei. The staining process strictly follows the recommended staining procedure of Giemsa stain (Beyotime, product number: C0133-100ml). The overall cell differentiation density reaches more than 90% and is included in the subsequent steps of this experiment. The cells are photographed and preserved under a microscope.

[0095] (5) Antibody and bacteria mixing: Take a sterile U-shaped plate and add the antibody into each well sequentially, 40 μL / well. Then, mix the diluted bacteria with the antibody in the well, 10 μL / well. Add the sample by submerging the pipette tip below the liquid surface, ensuring that the number of bacteria in each well is equal. Replace the pipette tip after each well. Place the plate on a shaker and mix thoroughly for 20 min.

[0096] (6) Cell and complement mixing: Take diluted HL-60 cells and guinea pig serum complement (BIOBYING, catalog number: BY-9531) and mix them thoroughly at a volume ratio of 4:1. Inactivated complement (i.e., control group, inactivated at 56℃ for 30 min) is mixed with cells in the same way. Set aside.

[0097] (7) Place the 96-well plate in a sterile environment and mix it at a speed of 200 rpm on a shaker for 15 min.

[0098] (8) Incubate the 96-well cell culture plates at 37°C and 5% CO2 for 45 min. Do not stack the 96-well plates. To maintain the CO2 concentration and humidity, do not open the door of the CO2 incubator.

[0099] (9) After incubation, place the 96-well cell culture plate on ice for 20 min to terminate the phagocytic reaction.

[0100] (10) Gently blow and mix the reaction mixture, take an appropriate amount of the mixture, dilute it in a gradient, spread it on a plate, count the results, and calculate the sterilization rate of each group. Note that the dilution and spreading time should not be too long, as it will affect the overall experimental results.

[0101] The results showed that, compared with the inactivated complement control group, serum immunized with Lol B protein significantly killed K1 serotype ( Figure 3 Serotypes A and K2 ( Figure 3 B) Klebsiella pneumoniae.

[0102] Example 4: Animal Immunization and Challenge Protection Experiment The Lol B antigen (i.e., the Lol B protein from Example 1) was diluted with PBS, and Al(OH)3 at a concentration of 1 mg / mL was added to prepare the vaccine. BALB / c mice were immunized bilaterally via intramuscular injection in the thighs using a No. 5 half-needle at days 0 and 14. Each mouse received 100 μL of the antigen, with a concentration of 10 μg / 50 μL. The blank control group was immunized with the same volume of PBS. On day 14 after the last immunization, a vaccine was administered using 5 × 10⁻⁶ PBS. 6 Mice were challenged with CFU doses of different serotypes of Klebsiella pneumoniae (K1, K2, and K6 serotypes) collected clinically via intratracheal instillation. The mice were observed for weight changes and survival status for 16 days. The weight and number of deaths of the mice were recorded daily. The survival rate and weight changes of the mice were calculated at the end of the observation period.

[0103] Animal experiment results such as Figure 4 , Figure 5 and Figure 6 As shown. Compared with the control group, mice immunized with Lol B protein showed a significantly reduced trend of weight loss when exposed to K1 serotype Klebsiella pneumoniae infection ( ). Figure 4 (A), and protects 100% of mice survival ( Figure 4 In contrast, the survival rate of mice in the control group against K1 serotype Klebsiella pneumoniae infection was 80%. When challenged with K2 serotype Klebsiella pneumoniae, the weight loss trend in the Lol B protein immunized group was also significantly less severe compared to the control group. Figure 5 In the immunized group (A), the survival rate of mice in both the immunized group and the control group was 80%. However, mice in the immunized group died on day 11 after challenge, while mice in the control group died on day 2 after challenge. Figure 5 (See data for Lol B), indicating that Lol B immunization prolonged the survival time of mice. Regarding K6 serotype Klebsiella pneumoniae infection, due to the relatively weak virulence of this strain, both the Lol B immunized group and the control group mice achieved 100% survival. Figure 6 (A), but compared to the control group mice, the trend of weight loss in the Lol B immunized group mice was significantly alleviated ( Figure 6 (B)

[0104] The above results indicate that the Lol B protein of Example 1 has clinical application value in the prevention and treatment of Klebsiella pneumoniae infection.

[0105] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. An outer membrane protein Lol B, the amino acid sequence of which is: al) SEQ ID NO: 2; or bl) an amino acid sequence that is at least 90% homologous to SEQ ID NO: 2 and functionally identical or similar; or cl) an amino acid sequence that is modified from the amino acid sequence shown in SEQ ID NO: 2 by substitution, deletion or addition of one or more amino acids and functionally identical or similar.

2. A biological material related to the outer membrane protein Lol B of claim 1, comprising at least one of dl) - d12): dl) a nucleic acid molecule encoding the outer membrane protein Lol B of claim 1; d2) an expression cassette comprising the nucleic acid molecule of dl); d3) a vector comprising the nucleic acid molecule of dl); d4) a vector comprising the expression cassette of d2); d5) a cell line comprising the nucleic acid molecule of dl); d6) a cell line comprising the expression cassette of d2); d7) a cell line comprising the vector of d3); d8) a cell line comprising the vector of d4); d9) a microorganism comprising the nucleic acid molecule of dl); d10) a microorganism comprising the expression cassette of d2); dl l) a microorganism comprising the vector of d3); d12) a microorganism comprising the vector of d4). The microorganism and / or cell line described in claim 2 is cultured to express the outer membrane protein Lol B.

4. Use of the outer membrane protein Lol B of claim 1 or the biological material of claim 2 in at least one of el) - e8): el) preparing a product for preventing or treating a disease related to Klebsiella pneumoniae infection; e2) detecting Klebsiella pneumoniae; e3) preparing a product for detecting Klebsiella pneumoniae; e4) inhibiting the activity of Klebsiella pneumoniae; e5) preparing a product for inhibiting the activity of Klebsiella pneumoniae; e6) preparing a product for diagnosing Klebsiella pneumoniae infection; e7) neutralizing the virulence of Klebsiella pneumoniae; e8) preparing a product for neutralizing the virulence of Klebsiella pneumoniae; The above uses are non-disease diagnosis and treatment purposes. The product comprises at least one of a drug, an immunogen, a reagent, a detection plate, a kit, a detection chip.

6. A product comprising the outer membrane protein Lol B of claim 1 or the biological material of claim 2. The product comprises at least one of a drug, an immunogen, a reagent, a detection plate, a kit, a detection chip. The product has at least one of the following functions: fl) preventing or treating a disease related to Klebsiella pneumoniae infection; f2) detecting Klebsiella pneumoniae; f3) inhibiting the activity of Klebsiella pneumoniae; f4) diagnosing Klebsiella pneumoniae infection; f5) neutralizing the virulence of Klebsiella pneumoniae. The drug comprises a vaccine. ​ ​ ​ ​ ​ ​ ​ ​ 3. The method of producing the outer membrane protein Lol B according to claim 1, comprising the steps of: ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 5. Use according to claim 4, characterized in that, ​ ​ 7. The product of claim 6, wherein, ​ 8. The product of claim 6, wherein, ​ ​ ​ ​ ​ ​ 9. The product according to claim 7 or 8, characterized in that, ​ 10. A method for detecting Klebsiella pneumoniae comprising contacting a sample containing or suspected of containing Klebsiella pneumoniae with the outer membrane protein Lol B of claim 1 or the product of any one of claims 6 to 9 and detecting the formation of a complex of the outer membrane protein Lol B and Klebsiella pneumoniae, the method being for non-diagnostic purposes.

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