CN113648409A - Application of L-rhamnose antibody in preparation of medicine for preventing and/or treating drug-resistant bacterial infection - Google Patents
Application of L-rhamnose antibody in preparation of medicine for preventing and/or treating drug-resistant bacterial infection Download PDFInfo
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- CN113648409A CN113648409A CN202110696762.XA CN202110696762A CN113648409A CN 113648409 A CN113648409 A CN 113648409A CN 202110696762 A CN202110696762 A CN 202110696762A CN 113648409 A CN113648409 A CN 113648409A
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- rhamnose
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- washing
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
- A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
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- A—HUMAN NECESSITIES
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- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Pharmacology & Pharmacy (AREA)
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- Immunology (AREA)
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- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention relates to application of an L-rhamnose antibody in preparing a medicament for preventing and/or treating drug-resistant bacterial infection. The L-rhamnose antibody specifically recognizes L-rhamnose, and is a human L-rhamnose natural antibody or a murine L-rhamnose monoclonal antibody; the drug-resistant bacteria refer in particular to drug-resistant bacteria containing L-rhamnose synthesis approach in genome. The invention discovers that L-rhamnose exists in polysaccharide structures on the surfaces of a plurality of drug-resistant bacteria due to the existence of an L-rhamnose synthesis way in the genome of a considerable part of the drug-resistant bacteria, and an L-rhamnose antibody can recognize and combine the L-rhamnose structures on the surfaces of the drug-resistant bacteria, so that the L-rhamnose antibody has extremely strong capability of inhibiting and killing a plurality of drug-resistant bacteria including pseudomonas aeruginosa, escherichia coli and streptococcus pneumoniae, and has the advantages of broad spectrum and good killing effect.
Description
Technical Field
The invention relates to application of an L-rhamnose antibody in preparation of a medicament for preventing and/or treating drug-resistant bacterial infection, belonging to the technical field of medicaments.
Background
Resistant bacteria refer to bacteria that develop a resistance to the corresponding antibiotic after a long-term antibiotic selection. The long-term, widespread, and overuse of antibiotics has led to the mutation of some bacteria into drug-resistant bacteria, which are either passed on to the next generation or acquired by other bacteria.
Bacterial resistance has become a serious problem of common concern in the global medical community. Currently, the clinical means for treating drug-resistant bacterial infections are very limited, mainly by adjusting the type or dosage of antibiotics used through sputum culture and/or drug sensitivity tests. However, for some super-resistant bacteria, there are often times when no drug is available. In addition, a great deal of research has been conducted on drug-resistant bacteria, and it is mainly expected to overcome drug-resistant bacterial infection by: 1) the novel antibiotic is directly developed aiming at drug-resistant bacteria, however, the drug resistance of the bacteria is far faster than the research and development speed of the novel antibiotic, and the research and development difficulty of the antibiotic is more and more increased. 2) The method overcomes a drug resistance mechanism and restores the sensitivity of bacteria to antibacterial drugs, for example, a synthetase inhibitor is researched aiming at beta-lactamase produced by drug resistant bacteria, the enzyme inhibitor and antibiotics are combined for use, the bacterial resistance is overcome and the sterilization effect of the antibiotics is exerted, but only the beta-lactamase inhibitor and penicillin (or cephalosporin) which are clinically available are applied in a compound way so far, and other drug resistance mechanisms cannot be overcome. 3) Antibiotic substitute products, such as antibacterial polypeptides, bacteriophages and the like, mostly stay in the experimental research stage and are far away from clinical application.
L-rhamnose (L-rhamnose, L-Rha), i.e. 6-deoxy-L-mannose, has two synthetic pathways in nature: the rml pathway and the gdp pathway. Among them, the rml pathway is present in the genomes of 42% of bacteria and 21% of archaea, and the gdp pathway is present in the genomes of some bacteria (mainly pseudomonas aeruginosa and bacillus), plants, fungi and algae. Among these, there are a considerable number of drug-resistant bacteria whose genomes contain L-rhamnose synthesis genes, such as all or part of the serotypes of Acinetobacter baumannii, Pseudomonas aeruginosa, Mycobacterium tuberculosis, Mycobacterium leprae, Escherichia coli, Klebsiella pneumoniae, Streptococcus pneumoniae, Shigella flexneri, Neisseria gonorrhoeae, enterococcus faecium, enterococcus faecalis, Salmonella enterica, Clostridium difficile, Salmonella typhi, and Streptococcus pyogenes. L-rhamnose is used as an antigenic determinant and is often involved in the composition of saccharide antigens on the surface of bacteria, thereby helping the identification and diagnosis of pathogenic bacteria.
Mammals, including humans, do not contain the synthetic pathway for L-rhamnose. In contrast, a large number of L-rhamnose antibodies are naturally present in healthy humans, which is found by Gildersleve JC et al. There has since been much work to exploit the fact that L-rhamnose antibodies are naturally and abundantly present in humans to enhance the immunogenicity of antigens (by linking L-rhamnose). For example, after a tumor antigen linked with L-rhamnose is injected into an L-rhamnose antibody mouse model, the L-rhamnose binds to L-rhamnose antibodies abundantly present in the mouse, thereby enhancing the recognition and uptake of the tumor antigen by antigen presenting cells.
Although natural antibodies have been reported in the literature to be involved in innate immunity, they play an important role in the clearance of pathogenic bacteria. One of the implications of the natural and abundant presence of L-rhamnose antibodies in humans may be the protection against infection by bacteria containing the L-rhamnose antigen. Weaker levels of native L-rhamnose antibodies may not provide the person with an effective ability to combat this part of the bacterial attack. Therefore, the L-rhamnose antibody level is improved for the population with low L-rhamnose natural antibody level, and the broad-spectrum drug-resistant bacterial infection prevention and/or treatment effect can be achieved.
Based on the principle, the invention provides a medicine with a novel action mechanism and/or a novel chemical structure for preventing and/or treating drug-resistant bacterial infection, and has extremely important and profound significance.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides the application of the L-rhamnose antibody in preparing a medicament for preventing and/or treating drug-resistant bacterial infection.
Description of terms:
room temperature: having a meaning well known in the art, typically 25. + -. 2 ℃.
NHS: n-hydroxysuccinimide.
ELISA: enzyme linked immunosorbent assay.
Column volume: the volume of 1 column is the volume of agarose gel in the L-rhamnose affinity chromatography column, wherein the column is an empty column of the affinity chromatography column, the column body is made of polypropylene material, and a sieve plate is arranged at the lower outlet of the column and used for preventing the agarose gel from seeping out.
PBS: phosphate buffered saline, wherein 1 × PBS refers to 0.01M PBS solution; 3 XPBS refers to 0.03M PBS solution.
The technical scheme of the invention is as follows:
the application of the L-rhamnose antibody in preparing a medicament for preventing and/or treating drug-resistant bacterial infection is characterized in that the L-rhamnose antibody specifically recognizes L-rhamnose, and the L-rhamnose antibody is a human L-rhamnose natural antibody or a murine L-rhamnose monoclonal antibody; the drug-resistant bacteria refer in particular to drug-resistant bacteria containing L-rhamnose synthesis approach in genome.
According to the invention, the preferable preparation method of the human L-rhamnose natural antibody comprises the following steps:
(1) synthesis of L-rhamnose-NH2
Performing acetyl protection and thiophenol glycosylation on L-rhamnose to obtain an intermediate product 1, connecting the intermediate product 1 with 3-azido propanol to obtain an intermediate product 2, and performing deacetylation protection and reduction reaction on the intermediate product 2 to obtain L-rhamnose-NH2；
(2) Affinity chromatographic column for preparing L-rhamnose
Loading NHS activated agarose gel into a column, washing and balancing, and adding the L-rhamnose-NH prepared in the step (1)2Uniformly mixing, slowly shaking for reaction for 3-5 hours at room temperature, then adding confining liquid, slowly shaking for reaction for 0.5-1.5 hours at room temperature, washing with a washing solution A and a washing solution B to obtain an L-rhamnose affinity chromatographic column, and storing in 20% ethanol at 4 ℃;
(3) purification of L-rhamnose Natural antibodies
And (3) washing and balancing the L-rhamnose affinity chromatographic column prepared in the step (2), adding serum, uniformly mixing, incubating at room temperature for 3-4 hours, washing the L-rhamnose affinity chromatographic column again, eluting, collecting eluent, adjusting the pH of the eluent to be neutral, and finally performing ultrafiltration and concentration on the eluent to obtain the humanized L-rhamnose natural antibody.
Preferably, according to the invention, in the step (1), the molar ratio of the L-rhamnose to the thiophenol is 1: 1.1; the molar ratio of the intermediate product 1 to the 3-azidopropanol is 1: 2.
Preferably, in the step (2), the flushing liquid is pre-cooled 1mM HCl, and the volume of the flushing liquid is 10-15 column volumes; the equilibrium solution is 0.2M NaHCO30.5M NaCl solution, pH 8-9, and the volume of the equilibrium solution is 3-5 column volumes.
Preferably, in step (2), the L-rhamnose-NH is2The addition amount of (a) is 1-2 column volumes, and the concentration is 5-15 mg/mL.
Preferably, in step (2), the blocking solution is 0.1M Tris-HCl solution, pH is 8.5, and the addition amount is 3-5 column volumes.
Preferably, in the step (2), the washing solution A is 0.1M Tris-HCl and 0.5M NaCl solution, and the pH value is 8-9; the washing solution B is 0.1M HCOOH and 0.5M NaCl solution, and the pH value is 3-5; washing liquid A and B wash 3 ~ 6 times in turn, and the volume of washing liquid A is 2 ~ 4 column volumes in washing process every time, and the volume of washing liquid B is 2 ~ 4 column volumes, washes 1 ~ 2 times with the deionized water of 2 ~ 4 column volumes at last.
According to the invention, in the step (3), the rinsing liquid for rinsing is deionized water, and the volume of the rinsing liquid is 10-15 column volumes; the balanced equilibrium solution is 1 multiplied by PBS buffer solution, the pH value is 7.4, and the volume of the balanced solution is 10-15 column volumes.
Preferably, in the step (3), the serum is healthy adult serum, and the addition amount of the serum is 10-15 column volumes.
Preferably, in the step (3), the eluent to be eluted is 0.2M glycine solution, and the pH value is 2-3; and carrying out ultrafiltration concentration by using an 8-12 kDa ultrafiltration tube.
According to the preferred method, the preparation method of the murine L-rhamnose monoclonal antibody comprises the following steps:
(1) synthesis of L-rhamnose-NHS
Adding L-rhamnose-NH2Reacting with succinyl to obtain an intermediate product 3, and activating the intermediate product 3 by 2-succinimidyl-1, 1,3, 3-tetramethyluronium tetrafluoroborate to obtain L-rhamnose-NHS;
(2) preparation of L-rhamnose-OVA
Dissolving the L-rhamnose-NHS prepared in the step (1) in 3 XPBS buffer solution to obtain an L-rhamnose-NHS solution with the concentration of 5-15 mg/mL; dissolving egg albumin (OVA) in 3 XPBS buffer solution to obtain OVA solution with the concentration of 5-15 mg/mL; mixing the two solutions in equal volumes, slowly stirring at room temperature for 0.5-1.5 hours, transferring the reaction solution into a 15mL 3kDa ultrafiltration tube, centrifuging for 25-35 minutes at 3000-4000 revolutions, adding 1 XPBS buffer solution, continuously centrifuging for 25-35 minutes at 3000-4000 revolutions, repeating for 3-4 times, and obtaining an L-rhamnose-OVA solution;
(3) preparation of mouse splenocyte suspension
Immunizing female Balb/c mice with 6-8 weeks old by using the immunogen, and carrying out immunization treatment; on the 37 th day after the immune treatment, taking mouse serum for ELISA detection, selecting a mouse with the antiserum titer being more than 1:10000, taking out the spleen by aseptic operation, and extruding and grinding the spleen in a plate to obtain a mouse spleen cell suspension;
(4) preparation of murine monoclonal hybridoma cells
Uniformly mixing the mouse spleen cell suspension prepared in the step (3) and mouse homologous myeloma cells, adding polyethylene glycol to form hybridoma cells, screening out positive clones from the hybridoma cells through ELISA, and performing expanded culture to obtain monoclonal hybridoma cells;
(5) preparation of murine L-rhamnose monoclonal antibody
Sensitizing a female Balb/c mouse with the age of 6-8 weeks by adopting a Freund incomplete adjuvant, injecting the monoclonal hybridoma cell prepared in the step (4) into the abdominal cavity of the female Balb/c mouse after 3-10 days, collecting ascites after 8-12 days, and purifying the obtained ascites to obtain the murine L-rhamnose monoclonal antibody.
Preferably, in step (1), the L-rhamnose-NH is2The molar ratio to succinyl is 1: 1.1; the molar ratio of the intermediate product 3 to the 2-succinimidyl-1, 1,3, 3-tetramethyluronium tetrafluoroborate is 1: 1.1.
Preferably, in step (1), the L-rhamnose-NH is2The molar ratio to succinyl is 1: 1.1; the molar ratio of the intermediate product 3 to the 2-succinimidyl-1, 1,3, 3-tetramethyluronium tetrafluoroborate is 1: 1.1.
preferably, in step (3), the method of the immunological treatment is as follows: immunizing female Balb/c mice with 6-8 weeks old with immunogen which is a solution of L-rhamnose-OVA and Freund's adjuvant mixed in equal volume, and adopting subcutaneous multipoint injection in an immunization mode; the dosage of the L-rhamnose-OVA is 20-40 mu g per mouse on the 1 st, 15 th and 30 th immune cycles; the Freund's adjuvant used Freund's complete adjuvant for the first immunization (day 1), Freund's incomplete adjuvant for the second immunization (day 15) and the third immunization (day 30).
Preferably, in step (4), the ratio of the number of mouse spleen cells to the number of mouse isoline myeloma cells is 1:1.
Preferably, in step (5), the ascites fluid is purified by protein A or protein G method.
Preferably according to the invention, the drug-resistant bacteria are bacteria containing the rml pathway and/or gdp pathway, including but not limited to Acinetobacter baumannii, Pseudomonas aeruginosa, Mycobacterium tuberculosis, Mycobacterium leprae, Escherichia coli, Klebsiella pneumoniae, Streptococcus pneumoniae, Shigella flexneri, Neisseria gonorrhoeae, enterococcus faecium, enterococcus faecalis, Salmonella enterica, Clostridium difficile, Salmonella typhi, Streptococcus pyogenes.
According to the invention, the preferable application is intravenous injection after a solution of a human source L-rhamnose natural antibody or a mouse source L-rhamnose monoclonal antibody is diluted to 5-15 mg/mL.
According to the preferable application of the invention, the human source L-rhamnose natural antibody or the mouse source L-rhamnose monoclonal antibody is diluted into a solution of 5-15 mg/mL, and then complement, vaseline and glycerol are added to prepare an ointment which is directly applied for use.
The intravenous injection in the application of the invention can effectively aim at the blood stream infection caused by drug-resistant bacteria, and the direct smearing can effectively aim at the skin infection caused by the drug-resistant bacteria. The invention can also carry out subcutaneous or intramuscular injection by matching L-rhamnose-OVA with corresponding adjuvant.
The invention synthesizes L-rhamnose-NH2And L-rhamnose-NHS as follows:
both the acetyl protection and the deacetylation protection in the present invention are carried out according to the prior art.
Has the advantages that:
1. the invention provides a new application of an L-rhamnose antibody, namely an application of a human L-rhamnose natural antibody or a mouse L-rhamnose monoclonal antibody in preparing a medicament for preventing and/or treating drug-resistant bacterial infection. The invention discovers that L-rhamnose exists in polysaccharide structures on the surfaces of a plurality of drug-resistant bacteria due to the existence of an L-rhamnose synthesis way in the genome of a considerable part of the drug-resistant bacteria, and an L-rhamnose antibody can recognize and combine the L-rhamnose structures on the surfaces of the drug-resistant bacteria, so that the L-rhamnose antibody has extremely strong capability of inhibiting and killing a plurality of drug-resistant bacteria including pseudomonas aeruginosa, escherichia coli and streptococcus pneumoniae, and has the advantages of broad spectrum and good killing effect.
2. The L-rhamnose antibody exists in human body, so that no side effect or little side effect exists in injection or smearing, and compared with antibiotics, the L-rhamnose antibody does not generate drug resistance.
3. The L-rhamnose-OVA in the process of preparing the murine L-rhamnose monoclonal antibody is an L-rhamnose conjugate, and the L-rhamnose-OVA can be matched with a corresponding adjuvant to be used for injecting the population with low L-rhamnose natural antibody level so as to improve the L-rhamnose antibody level of the population with low L-rhamnose natural antibody level and further prevent the infection of various drug-resistant bacteria containing L-rhamnose in a broad spectrum manner.
Drawings
FIG. 1 is an SDS-PAGE electrophoresis of purified natural L-rhamnose antibody of human origin.
In the figure: RGM: a natural antibody of human L-rhamnose comprising IgM and IgG; RG: IgG of natural human L-rhamnose antibodies.
FIG. 2 is an ELISA titer test chart of purified human L-rhamnose natural antibody.
In the figure: FIG. A shows IgG and IgM titers in a human L-rhamnose natural antibody; FIG. B shows IgG and IgM titers in IgG of the human L-rhamnose natural antibody.
FIG. 3 is an SDS-PAGE electrophoresis of L-rhamnose-NHS and OVA after conjugation.
In the figure: 1, OVA; 2, L-rhamnose-OVA; m: protein molecular weight standard, unit kDa.
FIG. 4 is a Western blot assay of L-rhamnose-NHS and OVA after conjugation.
In the figure: the primary antibody uses a human L-rhamnose natural antibody, and the secondary antibody uses a goat anti-human IgG; m: protein molecular weight standard, unit kDa.
FIG. 5 is an ELISA titer assay of L-rhamnose antibodies in serum of L-rhamnose-OVA-immunized mice.
In the figure, the titre is taken as log10Log when the titer is 1:100001010000＝4。-
FIG. 6 is a schematic diagram of the structure of O antigen repeat units of a partial serotype of E.coli.
FIG. 7 is an SDS-PAGE/silver stain assay of lipopolysaccharide of a partial serotype of E.coli.
FIG. 8 is a Western blot of lipopolysaccharide of a partial serotype of E.coli.
In the figure, the primary antibody is human L-rhamnose natural antibody, and the secondary antibody is goat anti-human IgG.
FIG. 9 is a schematic structural diagram of a P.aeruginosa partial serotype O antigen repeat unit.
FIG. 10 is an SDS-PAGE/silver stain assay of a portion of the serotype lipopolysaccharide of Pseudomonas aeruginosa.
FIG. 11 is a Western blot of a partial serotype of lipopolysaccharide from Pseudomonas aeruginosa.
In the figure, the primary antibody is human L-rhamnose natural antibody, and the secondary antibody is goat anti-human IgG.
FIG. 12 is a diagram of in vitro killing experiment of human L-rhamnose natural antibody.
In the figure: panel (A) is a partial serotype of E.coli; panel (B) is a partial serotype of P.aeruginosa; the ordinate is the percent kill; the abscissa represents the dilution factor of the natural human L-rhamnose antibody.
Detailed Description
The technical solutions of the present invention are further described below with reference to examples, but the scope of the present invention is not limited thereto. The raw materials mentioned in the examples are all common commercial products unless otherwise specified.
Human serum cat. No. h4522, Sigma-Aldrich in the examples; OVA cat. No. A5503, Sigma-Aldrich; NHS-activated Sepharose 4 Fast Flow cat. No.17090601, GE healthcare; rabbit complement, available from Cedarlane Labs; CCK8 cells, available from Dojindo; lipopolysaccharide extraction kit, iNtRON Biotechnology company.
Example 1
The preparation method of the human L-rhamnose natural antibody comprises the following steps:
(1) synthesis of L-rhamnose-NH2
Performing acetyl protection and 1.1mol thiophenol glycosylation reaction on 1mol of L-rhamnose to obtain an intermediate product 1, then connecting 1mol of the intermediate product 1 with 2mol of 3-azido propanol to obtain an intermediate product 2, and performing deacetylation protection and reduction reaction on the intermediate product 2 to obtain L-rhamnose-NH2；
(2) Affinity chromatographic column for preparing L-rhamnose
The NHS activated Sepharose column was packed, the column was washed with 10 column volumes of pre-cooled, 1mM HCl, and 5 column volumes of 0.2M NaHCO3The column was equilibrated with 0.5M NaCl solution (pH 8.3); 2mL of L-rhamnose-NH with a concentration of 10mg/mL was added2Then evenly mixing, slowly shaking for reaction for 4 hours at room temperature, continuously adding 3 column volumes of 0.1M Tris-HCl solution (pH 8.5), evenly mixing, and slowly shaking for reaction for 1 hour at room temperature; the column was washed alternately with 3 column volumes of 0.1M Tris-HCl, 0.5M NaCl solution (pH 8) and 3 column volumes of 0.1M HCOOH, 0.5M NaCl solution (pH 4) for 3 cycles; finally, washing with deionized water with 3 column volumes to obtain an L-rhamnose affinity chromatographic column, and storing in 20% ethanol at 4 ℃;
(3) preparation of human L-rhamnose natural antibody
Washing the L-rhamnose affinity chromatographic column prepared in the step (2) with 10 column volumes of deionized water, then balancing with 10 column volumes of 1 XPBS buffer (pH 7.4), adding 10 column volumes of human serum, uniformly mixing, incubating at room temperature for 3 hours, washing the L-rhamnose affinity chromatographic column with 10 column volumes of 1 XPPBS buffer (pH 7.4), eluting with 0.2M glycine solution (pH 2.5), collecting the eluent, adjusting the pH of the eluent to be neutral with 1M Tris-HCl solution (pH 9.0), and finally performing ultrafiltration and concentration on the eluent through a 10kDa ultrafiltration tube to obtain the L-rhamnose natural antibody.
SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoresis detection and ELISA (enzyme-linked immunosorbent assay) antibody titer detection are carried out on the L-rhamnose natural antibody prepared in the embodiment, and the detection results are shown in figures 1-2.
As can be seen from FIGS. 1-2, the human L-rhamnose natural antibody was successfully purified from human serum.
EXAMPLE 2 preparation of murine L-rhamnose monoclonal antibody
The preparation method of the murine L-rhamnose monoclonal antibody comprises the following steps:
(1) synthesis of L-rhamnose-NHS
1mol of L-rhamnose-NH2Reacting with 1.1mol of succinyl to obtain an intermediate product 3, and activating 1.1mol of the intermediate product 3 by 1.1mol of 2-succinimidyl-1, 1,3, 3-tetramethyluronium tetrafluoroborate to obtain L-rhamnose-NHS;
(2) preparation of L-rhamnose-OVA
10mg of L-rhamnose-NHS were dissolved in 1mL of 3 × PBS buffer (pH 7.4); another 10mg of OVA was dissolved in 1mL of 3 × PBS buffer (pH 7.4); mixing the two solutions in equal volume, and slowly stirring at room temperature for 1 hour; transferring the reaction system into a 3kDa ultrafiltration tube, centrifuging for 30 minutes at 4 ℃ under 3500 rpm, adding a proper amount of 1 XPBS buffer solution into a sleeve, centrifuging for 30 minutes at 4 ℃ under 3500 rpm, repeating for 3 times, wherein the solution obtained in the sleeve is L-rhamnose-OVA;
(3) preparation of mouse splenocyte suspension
Taking 5 female Balb/c mice with the age of 6 weeks, injecting 30 mu g L-rhamnose-OVA into each mouse at subcutaneous multiple points, wherein L-rhamnose-OVA is fully mixed and emulsified with 75 mu L Freund's adjuvant uniformly in advance, and the immune cycle is 1 day, 15 days and 30 days; the Freund's adjuvant complete Freund's adjuvant is used for the first immunization (day 1), the Freund's incomplete adjuvant is used for the second immunization (day 15) and the third immunization (day 30), ELISA detection is carried out on mouse serum on day 37, a mouse with the antiserum titer larger than 1:10000 is selected, the mouse is killed by an eyeball removal bloodletting method, the spleen is taken out through aseptic operation, and the mouse splenocyte suspension is obtained through extrusion grinding in a flat dish;
(4) preparation of monoclonal hybridoma cells
And (3) mixing the mouse spleen cell suspension prepared in the step (3) and mouse homologous myeloma cells according to the ratio of 1:1, then adding polyethylene glycol to form hybridoma cells, screening out positive clones from the hybridoma cells by ELISA, and performing expanded culture to obtain monoclonal hybridoma cells;
(5) preparation of murine L-rhamnose monoclonal antibody
Sensitizing 5 female Balb/c mice with the age of 6 weeks by adopting a Freund incomplete adjuvant, injecting the monoclonal hybridoma prepared in the step (4) into the abdominal cavity of the female Balb/c mice after 7 days, collecting ascites after 10 days, and purifying the obtained ascites by a protein A method to obtain the murine L-rhamnose monoclonal antibody.
SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoresis detection and Western blot detection are carried out on the L-rhamnose-OVA prepared in the embodiment, and the detection results are shown in FIGS. 3-4.
The L-rhamnose-OVA prepared in the example is immunized with mice, the antiserum obtained is subjected to ELISA to detect the titer of anti-rhamnose antibodies, the detection result is shown in figure 5, and the titer of antibodies in the L-rhamnose-OVA immune group is greater than 1: 10000.
As can be seen from FIGS. 3 to 5, the L-rhamnose-OVA was successfully prepared in this example, and the L-rhamnose-OVA immunized mice had produced high-titer L-anti-rhamnose antibodies. Therefore, the L-rhamnose-OVA prepared by the embodiment can be used for injecting the population with low L-rhamnose natural antibody level to improve the L-rhamnose antibody level of the population with low L-rhamnose natural antibody level, thereby preventing the infection of various bacteria containing L-rhamnose synthetic pathways in a broad spectrum manner.
Example 3 bactericidal Effect test of human L-rhamnose Natural antibody
Extracting bacterial lipopolysaccharide and detecting SDS-PAGE/silver staining, and the specific method comprises the following steps:
(1) extracting bacterial lipopolysaccharide: inoculating 5 μ L of glycerol strain into 5mL LB liquid culture medium, culturing overnight at 37 deg.C under 220 rpm; centrifuging and collecting overnight cultured thalli, and extracting lipopolysaccharide by using a lipopolysaccharide extraction kit; the glycerol bacteria is escherichia coli or pseudomonas aeruginosa;
(2) SDS-PAGE/silver stain detection: taking 5 mu L of extracted bacterial lipopolysaccharide, adding 1 mu L of 6 xSDS loading buffer solution, mixing uniformly, and boiling for 6 minutes; the resulting samples were subjected to 12% SDS-PAGE electrophoresis; after completion of electrophoresis, the mixture was placed in a fixative (35mL H)2O, 15mL acetic acid, 50mL isopropanol) for 2 hours, and then washing the gel twice with 200mL of 7.5% glacial acetic acid, each for 10 minutes; in an oxidizing solution (139mL H)2O, 11mL acetic acid, 1.059g periodic acid) for 3 minutes; then washing the gel with 200mL of deionized water for three times, each time for 30 minutes; in silver staining solution (140mL H2O, 1.4mL of 2M NaOH, 2mL of ammonia water and 5mL of 20% (w/v) silver nitrate) for 10 minutes, and then washing the gel with 200mL of deionized water for three times, 10 minutes each time; in a color developing solution (250mL H)2O, 12.5mg citric acid, 125 μ L formaldehyde) until the band is fully developed, then the development is stopped with L% glacial acetic acid and the silver stain results are recorded by scanning the gel.
The in vitro killing experiment of the human L-rhamnose natural antibody comprises the following specific steps:
human L-rhamnose natural antibody gradient dilutions (from 1:20 to 1:320 double dilutions in 1 × PBS buffer, pH 7.4) were added to 96-well plates at 50 μ L/well; 40. mu.L of 1X 10 aliquots were added to each well4Coli or pseudomonas aeruginosa (suspended in 1% (w/v) trypsin powder-containing 1 × PBS buffer, pH 7.4, filter sterilized with 0.22 μm filter) and 10 μ L rabbit complement, and mixed well with shaking; the control group was reacted at 37 ℃ for 1 hour with 1 XPBS buffer (pH 7.4) instead of the antibody; 10. mu.L of CCK8 was added to each well and reacted at 37 ℃ for 4 hours; OD determination with microplate reader450Absorbance of (a); the kill rate was calculated according to the following formula: rate of killing 1- (OD)450 antibody group/OD450 control group)/100％。
The lipopolysaccharide of Escherichia coli and Pseudomonas aeruginosa of various serotypes prepared and extracted in the embodiment is subjected to SDS-PAGE/silver staining detection and Western blotting detection, and the detection results are shown in FIGS. 6-11, and the humanized L-rhamnose natural antibody can be combined with Escherichia coli serotypes O1, O25b ST131 and O26 of L-rhamnose in a lipopolysaccharide structure and Pseudomonas aeruginosa serotypes O4 and O10.
In vitro killing experiments are carried out on the human L-rhamnose natural antibody prepared in the embodiment, the detection result is shown in fig. 12, and the human L-rhamnose natural antibody can effectively kill Escherichia coli serotypes O1 and O25b ST131 and Pseudomonas aeruginosa serotypes O4 and O10 with L-rhamnose in a lipopolysaccharide structure.
As can be seen from FIGS. 6 to 12, the human L-rhamnose natural antibody prepared in the embodiment can effectively bind and kill bacteria with L-rhamnose in a lipopolysaccharide structure.
Experimental example: typical treatment cases
The effect of the present invention will be further described below with reference to three typical cases of clinical application of the L-rhamnose antibody prepared in the examples.
Case 1: the patient has urinary tract infection and even bacteremia symptoms, urine or blood is identified as the Escherichia coli O25b ST131 through bacteria culture and 16s rRNA, and the Escherichia coli O25b ST131 is infected by drug-resistant bacteria.
The application method of the L-rhamnose antibody comprises the following steps: the human L-rhamnose natural antibody prepared in example 1 was dissolved in physiological saline at a concentration of 10mg/mL and then injected intravenously at a rate of 10mL per injection every 3 days.
The application effect of the L-rhamnose antibody is as follows: urinary tract infection and even bacteremia symptoms of the patient are reduced or disappeared, and Escherichia coli O25b ST131 is not detected in urine or blood by bacteria culture or 16s rRNA.
Case 2: the patient is hospitalized for a long time due to severe illness, and uses various antibiotics for a long time, so that the immunity is low. The patient blood was drawn and the level of L-rhamnose antibodies in the blood was found to be lower than normal and was considered to be at risk of infection with drug-resistant bacteria.
The application method of the L-rhamnose antibody comprises the following steps: the human L-rhamnose natural antibody prepared in example 1 was dissolved in physiological saline at a concentration of 5mg/mL and then injected intravenously at 10mL each for 3 days.
The application effect of the L-rhamnose antibody is as follows: patients are free of or at reduced risk of drug-resistant bacterial infection during hospitalization.
Case 3: the skin surface of the burn patient has blue-green or yellow-green lesions, and a lesion part sample is taken and identified as pseudomonas aeruginosa through bacteria culture and 16s rRNA, so that the skin of the burn patient is infected by the pseudomonas aeruginosa.
The application method of the L-rhamnose antibody comprises the following steps: dissolving the murine L-rhamnose monoclonal antibody prepared in the example 1 in normal saline, adding rabbit complement, vaseline and glycerol, and preparing into ointment to ensure that the final concentration of the murine L-rhamnose monoclonal antibody in the ointment is 5 mg/mL; the prepared ointment is uniformly applied to the skin infection position of pseudomonas aeruginosa of a patient 3 times a day.
The application effect of the L-rhamnose antibody is as follows: the blue-green or yellow-green lesions on the burned skin surface of the burn patient are weakened or disappeared, and pseudomonas aeruginosa is not detected by taking samples of the lesion parts through bacteria culture and 16s rRNA.
Claims (10)
- The application of an L-rhamnose antibody in preparing a medicament for preventing and/or treating drug-resistant bacterial infection is characterized in that the L-rhamnose antibody specifically recognizes L-rhamnose, and the L-rhamnose antibody is a human L-rhamnose natural antibody or a murine L-rhamnose monoclonal antibody; the drug-resistant bacteria are those with a genome containing an L-rhamnose synthesis way.
- 2. The use of claim 1, wherein the human L-rhamnose natural antibody is prepared by the following steps:(1) synthesis of L-rhamnose-NH2Performing acetyl protection and thiophenol glycosylation on L-rhamnose to obtain an intermediate product 1, connecting the intermediate product 1 with 3-azido propanol to obtain an intermediate product 2, and performing deacetylation protection and reduction reaction on the intermediate product 2 to obtain L-rhamnose-NH2；
(2) Affinity chromatographic column for preparing L-rhamnoseLoading NHS activated agarose gel into a column, washing and balancing, and adding the L-rhamnose-NH prepared in the step (1)2Uniformly mixing, slowly shaking for reaction for 3-5 hours at room temperature, then adding confining liquid, slowly shaking for reaction for 0.5-1.5 hours at room temperature, washing with a washing solution A and a washing solution B to obtain an L-rhamnose affinity chromatographic column, and storing in 20% ethanol at 4 ℃;(3) purification of L-rhamnose Natural antibodiesAnd (3) washing and balancing the L-rhamnose affinity chromatographic column prepared in the step (2), adding serum, uniformly mixing, incubating at room temperature for 3-4 hours, washing the L-rhamnose affinity chromatographic column again, eluting, collecting eluent, adjusting the pH of the eluent to be neutral, and finally performing ultrafiltration and concentration on the eluent to obtain the humanized L-rhamnose natural antibody. - 3. The use of claim 2, wherein in step (1), the molar ratio of L-rhamnose to thiophenol is 1: 1.1; the molar ratio of the intermediate product 1 to the 3-azidopropanol is 1: 2.
- 4. The use of claim 2, wherein in step (2), the washing solution is pre-cooled 1mM HCl, and the volume of the washing solution is 10-15 column volumes; the equilibrium solution is 0.2M NaHCO30.5M NaCl solution, pH 8-9, and the volume of the equilibrium solution is 3-5 column volumes; the L-rhamnose-NH2The addition amount of (a) is 1-2 column volumes, and the concentration is 5-15 mg/mL; the confining liquid is a 0.1M Tris-HCl solution, the pH value is 8.5, and the adding amount is 3-5 column volumes; the washing solution A is 0.1M Tris-HCl and 0.5M NaCl solution, and the pH value is 8-9; the washing solution B is 0.1M HCOOH and 0.5M NaCl solution, and the pH value is 3-5; washing the washing liquid A and the washing liquid B for 3-6 times alternately, wherein the volume of the washing liquid A is 2-4 column volumes in each washing process, the volume of the washing liquid B is 2-4 column volumes, and finally, washing for 1-2 times by using deionized water with 2-4 column volumes;in the step (3), the flushing liquid is deionized water, and the volume of the flushing liquid is 10-15 column volumes; the balanced equilibrium solution is 1 multiplied by PBS buffer solution, the pH value is 7.4, and the volume of the balanced solution is 10-15 column volumes; the serum is healthy adult serum, and the addition amount of the serum is 10-15 column volumes. The eluent for elution is 0.2M glycine solution, and the pH value is 2-3; and carrying out ultrafiltration concentration by using an 8-12 kDa ultrafiltration tube.
- 5. The use of claim 1, wherein the murine L-rhamnose monoclonal antibody is prepared by the following steps:(1) synthesis of L-rhamnose-NHSAdding L-rhamnose-NH2Reacting with succinyl to obtain an intermediate product 3, and activating the intermediate product 3 by 2-succinimidyl-1, 1,3, 3-tetramethyluronium tetrafluoroborate to obtain L-rhamnose-NHS;
(2) preparation of L-rhamnose-OVADissolving the L-rhamnose-NHS prepared in the step (1) in 3 XPBS buffer solution to obtain an L-rhamnose-NHS solution with the concentration of 5-15 mg/mL; dissolving egg albumin (OVA) in 3 XPBS buffer solution to obtain OVA solution with the concentration of 5-15 mg/mL; mixing the two solutions in equal volumes, slowly stirring at room temperature for 0.5-1.5 hours, transferring the reaction solution into a 15mL 3kDa ultrafiltration tube, centrifuging for 25-35 minutes at 3000-4000 revolutions, adding 1 XPBS buffer solution, continuously centrifuging for 25-35 minutes at 3000-4000 revolutions, repeating for 3-4 times, and obtaining an L-rhamnose-OVA solution;(3) preparation of mouse splenocyte suspensionImmunizing female Balb/c mice with 6-8 weeks old by using the immunogen, and carrying out immunization treatment; on the 37 th day after the immune treatment, taking mouse serum for ELISA detection, selecting a mouse with the antiserum titer being more than 1:10000, taking out the spleen by aseptic operation, and extruding and grinding the spleen in a plate to obtain a mouse spleen cell suspension;(4) preparation of murine monoclonal hybridoma cellsUniformly mixing the mouse spleen cell suspension prepared in the step (3) and mouse homologous myeloma cells, adding polyethylene glycol to form hybridoma cells, screening out positive clones from the hybridoma cells through ELISA, and performing expanded culture to obtain monoclonal hybridoma cells;(5) preparation of murine L-rhamnose monoclonal antibodySensitizing a female Balb/c mouse with the age of 6-8 weeks by adopting a Freund incomplete adjuvant, injecting the monoclonal hybridoma cell prepared in the step (4) into the abdominal cavity of the female Balb/c mouse after 3-10 days, collecting ascites after 8-12 days, and purifying the obtained ascites to obtain the murine L-rhamnose monoclonal antibody. - 6. The use of claim 5, wherein in step (1), the L-rhamnose-NH is present2The molar ratio to succinyl is 1: 1.1; the molar ratio of the intermediate product 3 to the 2-succinimidyl-1, 1,3, 3-tetramethyluronium tetrafluoroborate is 1: 1.1; the L-rhamnose-NH2The molar ratio to succinyl is 1: 1.1; the molar ratio of the intermediate product 3 to the 2-succinimidyl-1, 1,3, 3-tetramethyluronium tetrafluoroborate is 1: 1.1.
- 7. the use of claim 5, wherein in step (3), the method of the immunization treatment is as follows: immunizing female Balb/c mice with 6-8 weeks old with immunogen which is a solution of L-rhamnose-OVA and Freund's adjuvant mixed in equal volume, and adopting subcutaneous multipoint injection in an immunization mode; the dosage of the L-rhamnose-OVA is 20-40 mu g per mouse on the 1 st, 15 th and 30 th immune cycles; the Freund's adjuvant used Freund's complete adjuvant for the first immunization (day 1), Freund's incomplete adjuvant for the second immunization (day 15) and the third immunization (day 30);in the step (4), the number ratio of the mouse spleen cells to the mouse homologous myeloma cells is 1: 1;in the step (5), the ascites is purified by a protein A or protein G method.
- 8. The use according to claim 1, wherein the drug-resistant bacteria are bacteria comprising the rml pathway and/or the gdp pathway, in particular including acinetobacter baumannii, pseudomonas aeruginosa, mycobacterium tuberculosis, mycobacterium leprae, escherichia coli, klebsiella pneumoniae, streptococcus pneumoniae, shigella flexneri, neisseria gonorrhoeae, enterococcus faecium, enterococcus faecalis, salmonella enterica, clostridium difficile, salmonella typhi, streptococcus pyogenes.
- 9. The use of claim 1, wherein the use is intravenous injection after dilution of a solution of a human L-rhamnose natural antibody or a murine L-rhamnose monoclonal antibody to 5-15 mg/mL.
- 10. The use of claim 1, wherein the use is to dilute a solution of a human L-rhamnose natural antibody or a murine L-rhamnose monoclonal antibody to 5-15 mg/mL, add complement, vaseline and glycerol, prepare an ointment, and directly apply the ointment.
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| JPH06306092A (en) * | 1993-02-26 | 1994-11-01 | D D S Kenkyusho:Kk | Compound having specific bonding ability to adhesive molecule elam-1 |
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