CN113648409B - 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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- CN113648409B CN113648409B CN202110696762.XA CN202110696762A CN113648409B CN 113648409 B CN113648409 B CN 113648409B CN 202110696762 A CN202110696762 A CN 202110696762A CN 113648409 B CN113648409 B CN 113648409B
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Classifications
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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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- 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
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- 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
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/08—Solutions
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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
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- 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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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
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- Molecular Biology (AREA)
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- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Dermatology (AREA)
- Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- 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 the L-rhamnose antibody is a natural human L-rhamnose antibody or a cloned L-rhamnose Li Tangshan antibody; the drug-resistant bacteria particularly refer to drug-resistant bacteria containing an L-rhamnose synthetic pathway in a genome. The invention discovers that because of the synthetic way of L-rhamnose in the genome of a considerable part of drug-resistant bacteria, the polysaccharide structure on the surface of many drug-resistant bacteria has L-rhamnose, and the L-rhamnose antibody can identify and combine with the L-rhamnose structure on the surface of the drug-resistant bacteria, so that the drug-resistant bacteria have extremely strong killing-inhibiting capability on various drug-resistant bacteria including pseudomonas aeruginosa, escherichia coli and streptococcus pneumoniae, and have the advantages of broad spectrum and good killing effect.
Description
Technical Field
The invention relates to an application of an L-rhamnose antibody in preparing a medicament for preventing and/or treating drug-resistant bacterial infection, and belongs to the technical field of medicaments.
Background
By drug resistant bacteria is meant bacteria that develop after a prolonged antibiotic selection a tolerance to the corresponding antibiotic. The long-term, extensive and overuse of antibiotics has resulted in the mutation of some bacteria into drug-resistant bacteria, which can be transmitted to the next generation and acquired by other bacteria.
Bacterial resistance has become a serious concern for common concern in the global medical community. At present, the means for treating drug-resistant bacterial infection are very limited clinically, and the type or dosage of antibiotics is mainly adjusted through sputum culture and/or drug sensitivity test. However, for some super-resistant bacteria, there is often a non-drug usable environment. Furthermore, there is a great deal of research on drug-resistant bacteria, and it is mainly desirable to overcome drug-resistant bacterial infections by: 1) The novel antibiotics are directly developed for drug-resistant bacteria, however, the speed of bacterial drug resistance generation far exceeds the development speed of the novel antibiotics, and the development difficulty of the antibiotics is increased. 2) Overcoming the drug resistance mechanism, restoring the sensitivity of bacteria to antibacterial drugs, for example, researching a synthetase inhibitor aiming at beta-lactamase produced by drug-resistant bacteria, combining the enzyme inhibitor with antibiotics, and playing the bactericidal action of the antibiotics while overcoming the drug resistance of bacteria, but so far, only the beta-lactamase inhibitor and penicillin (or cephalosporin) compound application is clinically available, and other numerous drug resistance mechanisms cannot be overcome. 3) Antibiotic replacement products, such as antimicrobial polypeptides, phages, etc., are mostly in the experimental research stage, and are far away from clinical use.
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 genome of 42% bacteria, 21% archaea, and the gdp pathway is present in the genome of part of bacteria (mainly pseudomonas aeruginosa and bacillus), plants, fungi, algae. Among them, there are L-rhamnose-synthesizing genes in the genome of a considerable part of drug-resistant bacteria, such as all or part of 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, streptococcus pyogenes. L-rhamnose is taken as an antigenic determinant and is often involved in the composition of bacterial surface sugar antigens, so that the identification and diagnosis of pathogenic bacteria are facilitated.
Mammals, including humans, do not contain the synthetic pathway of L-rhamnose. In contrast, a large number of L-rhamnose antibodies are naturally occurring in healthy humans, which are found by Gildesleeve JC et al. Much work has been done hereafter to enhance the immunogenicity of antigens (by linking L-rhamnose) by exploiting the fact that L-rhamnose antibodies are naturally and abundantly present in humans. For example, after injection of a tumor antigen linked to L-rhamnose into a L-rhamnose antibody mouse model, L-rhamnose binds to L-rhamnose antibodies present in a large amount in the mouse, thereby enhancing the recognition and uptake of tumor antigens by antigen presenting cells.
Although it is reported in the literature that natural antibodies can participate in natural immunity, they play an important role in the elimination of pathogenic bacteria. One of the meanings of L-rhamnose antibodies naturally and in large quantities in humans may be to combat infections by bacteria containing the L-rhamnose antigen. Weaker L-rhamnose natural antibody levels may not provide an effective ability to fight this part of the bacterial attack to humans. Therefore, the L-rhamnose antibody level is improved for people with low L-rhamnose natural antibody level, and the L-rhamnose antibody level can play a role in preventing and/or treating broad-spectrum drug-resistant bacterial infection.
Based on the principle, the invention provides a medicine for preventing and/or treating drug-resistant bacterial infection with a novel action mechanism and/or a novel chemical structure, and the medicine has extremely important and profound significance.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides application of an L-rhamnose antibody in preparing a medicament for preventing and/or treating drug-resistant bacterial infection.
Description of the terminology:
room temperature: has a meaning known in the art and generally means 25.+ -. 2 ℃.
NHS: n-hydroxysuccinimide.
ELISA: and (5) measuring an enzyme-linked immunosorbent.
Column volume: the volume of 1 column refers to the volume of agarose gel in the L-rhamnose affinity chromatography column, wherein the column refers to an empty column of the affinity chromatography column, the column is made of polypropylene material, and a sieve plate is arranged at the lower outlet of the column and used for blocking the agarose gel from exuding.
PBS: phosphate buffered saline, wherein 1 XPBS refers to a 0.01M PBS solution; 3 XPBS refers to a 0.03M PBS solution.
The technical scheme of the invention is as follows:
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 natural human L-rhamnose antibody or a cloned L-murine Li Tangshan antibody; the drug-resistant bacteria particularly refer to drug-resistant bacteria containing an L-rhamnose synthetic pathway in a genome.
According to the invention, the preparation method of the humanized L-rhamnose natural antibody comprises the following steps of:
(1) Synthesis of L-rhamnose-NH 2
The L-mouse Li Tangjing acetyl protection and thiophenol glycosylation reaction are carried out to obtain an intermediate product 1, the intermediate product 1 is connected with 3-azidopropanol to obtain an intermediate product 2, and the intermediate product 2 is subjected to deacetylation protection and reduction reaction to obtain L-rhamnose-NH 2 ；
(2) Preparation of L-rhamnose affinity chromatography column
Loading the NHS activated agarose gel into a column, washing and balancing, and adding the L-rhamnose-NH prepared in the step (1) 2 Mixing uniformly, slowly shaking at room temperature for reaction for 3-5 hours, then adding a sealing solution, slowly shaking at room temperature for reaction for 0.5-1.5 hours, washing by 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) taking the L-rhamnose affinity chromatography column prepared in the step (2), adding serum after washing and balancing, incubating for 3-4 hours at room temperature after uniformly mixing, washing the L-rhamnose affinity chromatography column again, eluting, collecting eluent, adjusting the pH of the eluent to be neutral, and finally obtaining the humanized L-rhamnose natural antibody after ultrafiltration and concentration of the eluent.
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.
According to the invention, in the step (2), the flushing liquid is 1mM HCl after precooling, and the volume of the flushing liquid is 10-15 column volumes; the balance liquid is 0.2M NaHCO 3 0.5M NaCl solution, pH 8-9, and balancing liquid volume of 3-5 column volumes.
According to a preferred embodiment of the present invention, in step (2), the L-rhamnose-NH 2 The addition amount of the catalyst is 1-2 column volumes, and the concentration is 5-15 mg/mL.
According to a preferred embodiment of the present invention, in the step (2), the blocking solution is a 0.1M Tris-HCl solution, pH 8.5, and the amount of the blocking solution added is 3 to 5 column volumes.
According to the invention, in the step (2), the washing solution A is 0.1M Tris-HCl, 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; the washing liquid A and the washing liquid B are alternately washed for 3 to 6 times, the volume of the washing liquid A is 2 to 4 column volumes in each washing process, the volume of the washing liquid B is 2 to 4 column volumes, and finally the washing liquid A is washed by deionized water with 2 to 4 column volumes for 1 to 2 times.
According to the invention, in the step (3), the flushing liquid is deionized water, and the volume of the flushing liquid is 10-15 column volumes; the balancing solution is 1 XPBS buffer solution, the pH value is 7.4, and the volume of the balancing solution is 10-15 column volumes.
According to a preferred embodiment of the present invention, in the step (3), the serum is healthy adult serum, and the amount of the serum added is 10 to 15 column volumes.
Preferably, in step (3), the eluted eluent is 0.2M glycine solution with pH of 2-3; the ultrafiltration concentration is performed by using an ultrafiltration tube of 8-12 kDa.
According to the invention, the preparation method of the murine L-mouse Li Tangshan clone antibody comprises the following steps:
(1) Synthesis of L-rhamnose-NHS
L-rhamnose-NH 2 Reacting with succinyl to obtain an intermediate product 3, and activating the intermediate product 3 by 2-succinimidyl-1, 3-tetramethylurea tetrafluoroborate to obtain L-rhamnose-NHS;
(2) Preparation of L-rhamnose-OVA
Dissolving the L-rhamnose-NHS prepared in the step (1) in a 3X PBS buffer solution to obtain an L-rhamnose-NHS solution with the concentration of 5-15 mg/mL; dissolving chicken Ovalbumin (OVA) in 3 XPBS buffer solution to obtain OVA solution with the concentration of 5-15 mg/mL; mixing the two with equal volume, slowly stirring at room temperature for 0.5-1.5 hours, transferring the reaction solution into a 3kDa ultrafiltration tube of 15mL, centrifuging for 25-35 minutes at 3000-4000 rpm, adding 1 XPBS buffer, continuously centrifuging for 25-35 minutes at 3000-4000 rpm, and repeating for 3-4 times to obtain the solution which is L-rhamnose-OVA;
(3) Preparation of mouse spleen cell suspension
Immunizing female Balb/c mice with immunogen of 6-8 weeks old, and performing immune treatment; on day 37 after immune treatment, taking mouse serum for ELISA detection, selecting mice with antiserum titer larger than 1:10000, taking out spleen by aseptic operation, squeezing and grinding in a plate to obtain mouse spleen cell suspension;
(4) Preparation of murine monoclonal hybridoma cells
Uniformly mixing the mouse spleen cell suspension prepared in the step (3) with the mouse syngeneic myeloma cells, adding polyethylene glycol to form hybridoma cells, screening positive clones from the hybridoma cells through ELISA, and performing amplification culture to obtain monoclonal hybridoma cells;
(5) Preparation of murine L-murine Li Tangshan clone antibody
Sensitized 6-8 week old female Balb/c mice by adopting Freund's incomplete adjuvant, injecting the monoclonal hybridoma cells prepared in the step (4) into the abdominal cavity of the female Balb/c mice after 3-10 days, collecting ascites after 8-12 days, and purifying the obtained ascites to obtain the murine L-mouse Li Tangshan clone antibody.
According to a preferred embodiment of the present invention, in step (1), the L-rhamnose-NH 2 The molar ratio of the succinic acid to the succinyl is 1:1.1; the molar ratio of the intermediate 3 to the 2-succinimidyl-1, 3-tetramethylurea tetrafluoroborate is 1:1.1.
According to a preferred embodiment of the present invention, in step (1), the L-rhamnose-NH 2 The molar ratio of the succinic acid to the succinyl is 1:1.1; the molar ratio of the intermediate 3 to the 2-succinimidyl-1, 3-tetramethylurea tetrafluoroborate is 1:1.1.
according to a preferred embodiment of the present invention, in step (3), the method of immunization treatment is as follows: taking an immunogen for immunizing a female Balb/c mouse with the age of 6-8 weeks, wherein the immunogen is a solution prepared by mixing L-rhamnose-OVA with Freund's adjuvant in an equal volume, and the immunization mode adopts subcutaneous multipoint injection; the dosage of the L-rhamnose-OVA is 20-40 mug per mouse, wherein the immunization period is 1 day, 15 days and 30 days; the Freund's adjuvant used Freund's complete adjuvant for one-day (day 1), freund's incomplete adjuvant for two-day (day 15) and three-day (day 30).
According to a preferred embodiment of the invention, in step (4), the ratio of the number of mouse spleen cells to the number of mouse syngeneic myeloma cells is 1:1.
According to a preferred embodiment of the present invention, in step (5), the ascites is purified by the protein A or protein G method.
According to a preferred embodiment of the present invention, the drug resistant bacteria are bacteria containing 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 application is preferably intravenous injection after diluting the natural human L-rhamnose antibody or the cloned L-mouse Li Tangshan antibody to a solution of 5-15 mg/mL.
According to the invention, preferably, the application is that the natural human L-rhamnose antibody or the cloned L-mouse Li Tangshan antibody is diluted to a solution of 5-15 mg/mL, and then complement, vaseline and glycerol are added to prepare ointment which is directly smeared for use.
In the application of the invention, intravenous injection can effectively aim at blood flow infection caused by drug-resistant bacteria, and direct coating can effectively aim at skin infection caused by drug-resistant bacteria. The invention can also be used for subcutaneous or intramuscular injection by matching L-rhamnose-OVA with corresponding adjuvant.
The invention synthesizes L-rhamnose-NH 2 And L-rhamnose-NHS as follows:
in the present invention both acetyl protection and deacetylation protection are carried out according to the prior art.
The beneficial effects are that:
1. the invention provides a new application of an L-rhamnose antibody, namely an application of a humanized L-rhamnose natural antibody or a murine L-mouse Li Tangshan clone antibody in preparing a medicament for preventing and/or treating drug-resistant bacterial infection. The invention discovers that because of the synthetic way of L-rhamnose in the genome of a considerable part of drug-resistant bacteria, the polysaccharide structure on the surface of many drug-resistant bacteria has L-rhamnose, and the L-rhamnose antibody can identify and combine with the L-rhamnose structure on the surface of the drug-resistant bacteria, so that the drug-resistant bacteria have extremely strong killing-inhibiting capability on various drug-resistant bacteria including pseudomonas aeruginosa, escherichia coli and streptococcus pneumoniae, and have the advantages of broad spectrum and good killing effect.
2. The L-rhamnose antibody is in a human body, so that the L-rhamnose antibody has no side effect or little side effect when being injected or smeared, and the L-rhamnose antibody does not generate drug resistance compared with the use of antibiotics.
3. The L-rhamnose-OVA in the process of preparing the murine L-Li Tangshan clone antibody is an L-rhamnose conjugate, and the L-rhamnose-OVA is matched with a corresponding adjuvant and can be used for injecting people with low L-rhamnose natural antibody level so as to improve the L-rhamnose antibody level of the people with low L-rhamnose natural antibody level and further prevent the infection of various drug-resistant bacteria containing L-rhamnose in a broad spectrum.
Drawings
FIG. 1 is an SDS-PAGE electrophoresis of purified natural human L-rhamnose antibodies.
In the figure: RGM: a natural antibody of human L-rhamnose, comprising IgM and IgG; RG: igG of natural antibody of human L-rhamnose.
FIG. 2 is an ELISA titer chart of the purified human L-rhamnose natural antibody.
In the figure: panel (a) shows the titres of IgG and IgM in the human L-rhamnose natural antibody; panel (B) shows the IgG and IgM titers of the natural antibodies of human L-rhamnose.
FIG. 3 is an SDS-PAGE electrophoresis after L-rhamnose-NHS and OVA have been coupled.
In the figure: 1, OVA;2, l-rhamnose-OVA; m: protein molecular weight standard, unit kDa.
FIG. 4 is a Western blot analysis after L-rhamnose-NHS and OVA have been coupled.
In the figure: the primary antibody uses a humanized L-rhamnose natural antibody, and the secondary antibody uses goat anti-human IgG; m: protein molecular weight standard, unit kDa.
FIG. 5 is a graph of ELISA titer of L-rhamnose antibodies in serum of L-rhamnose-OVA immunized mice.
In the graph, log is taken from titer 10 Log when the potency is 1:10000 10 10000＝4。-
FIG. 6 is a schematic representation of the structure of the O antigen repeat unit of a partial serotype of E.coli.
FIG. 7 is a SDS-PAGE/silver staining of partial serotype lipopolysaccharides of E.coli.
FIG. 8 is a Western blot analysis of partial serotype lipopolysaccharides from E.coli.
In the figure, the primary antibody uses a natural human L-rhamnose antibody, and the secondary antibody uses goat anti-human IgG.
FIG. 9 is a schematic representation of the structure of the serotype O antigen repeat unit of the Pseudomonas aeruginosa moiety.
FIG. 10 is a SDS-PAGE/silver staining of partial serotype lipopolysaccharides from P.aeruginosa.
FIG. 11 is a Western blot analysis of partial serotype lipopolysaccharides from P.aeruginosa.
In the figure, the primary antibody uses a natural human L-rhamnose antibody, and the secondary antibody uses goat anti-human IgG.
FIG. 12 is a graph of an in vitro killing experiment of a human L-rhamnose natural antibody.
In the figure: panel (A) shows part of the E.coli serotype; panel (B) shows a Pseudomonas aeruginosa partial serotype; the ordinate is the killing percentage; the abscissa is the dilution factor of the humanized L-rhamnose natural antibody.
Detailed Description
The following describes the technical scheme of the present invention with reference to examples, but the scope of the present invention is not limited thereto. The raw materials used in the examples are all commercially available products unless otherwise specified.
Human serum cat No. H4522, sigma-Aldrich; OVA cat No. A5503, sigma-Aldrich; NHS-activated Sepharose 4 Fast Flow cat# Cat.No.17090601, GE healthcare; rabbit complement, sold by Cedarlane Labs; CCK8 cells, available from Dojindo corporation; lipopolysaccharide extraction kit, available from iNtRON Biotechnology.
Example 1
The preparation method of the humanized L-rhamnose natural antibody comprises the following steps:
(1) Synthesis of L-rhamnose-NH 2
1mol of L-mouse Li Tangjing acetyl protection and 1.1mol of thiophenol are subjected to glycosylation reaction to obtain an intermediate 1, then 1mol of the intermediate 1 is connected with 2mol of 3-azidopropanol to obtain an intermediate 2, and the intermediate 2 is subjected to deacetylation protection and reduction reaction to obtain L-rhamnose-NH 2 ；
(2) Preparation of L-rhamnose affinity chromatography column
Taking NHS activated agarose gel column, flushing with 10 column volumes of pre-chilled 1mM HClThe column was washed with 5 column volumes of 0.2M NaHCO 3 The column was equilibrated with 0.5M NaCl solution (ph=8.3); adding 2mL of L-rhamnose-NH with the concentration of 10mg/mL 2 Mixing uniformly, slowly shaking at room temperature for reaction for 4 hours, continuously adding 3 column volumes of 0.1M Tris-HCl solution (pH 8.5), mixing uniformly, and slowly shaking at room temperature for reaction for 1 hour; the column was alternately washed 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), and cycled 3 times; finally, washing with deionized water with 3 column volumes to obtain an L-rhamnose affinity chromatography column, and preserving in 20% ethanol at 4 ℃;
(3) Preparation of humanized L-rhamnose Natural antibody
Taking the L-rhamnose affinity chromatography column prepared in the step (2), washing with 10 column volumes of deionized water, balancing with 10 column volumes of 1 XPBS buffer solution (pH=7.4), adding 10 column volumes of human serum, mixing uniformly, incubating for 3 hours at room temperature, washing the L-rhamnose affinity chromatography column with 10 column volumes of 1 XPBS buffer solution (pH=7.4), eluting with 0.2M glycine solution (pH=2.5), collecting the eluent, regulating the pH of the eluent to be neutral with 1M Tris-HCl solution (pH=9.0), and finally performing ultrafiltration concentration on the eluent through a 10kDa ultrafiltration tube to obtain the L-rhamnose natural antibody.
SDS-PAGE electrophoresis detection and ELISA 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 fig. 1 to 2, the present example successfully purified human-derived L-rhamnose natural antibodies from human serum.
EXAMPLE 2 preparation of murine L-murine Li Tangshan clone antibody
The preparation method of the murine L-mouse Li Tangshan clone antibody comprises the following steps:
(1) Synthesis of L-rhamnose-NHS
1mol of L-rhamnose-NH 2 With 1.1mol of succinyl to obtain an intermediate 3,1mol of intermediate 3 is activated by 1.1mol of 2-succinimidyl-1, 3-tetramethylurea tetrafluoroborate to obtain L-rhamnose-NHS;
(2) Preparation of L-rhamnose-OVA
10mg of L-rhamnose-NHS was dissolved in 1mL of 3 XPBS buffer (pH=7.4); another 10mg of OVA was dissolved in 1mL of 3 XPBS buffer (pH=7.4); mixing the two materials uniformly in equal volume, and slowly stirring at room temperature for 1 hour; transferring the reaction system into a 3kDa ultrafiltration tube, centrifuging at a temperature of 4 ℃ for 30 minutes at 3500 revolutions, adding a proper amount of 1X PBS buffer solution into the sleeve, centrifuging at the temperature of 4 ℃ for 30 minutes at 3500 revolutions, and repeating for 3 times, wherein the solution obtained in the sleeve is L-rhamnose-OVA;
(3) Preparation of mouse spleen cell suspension
Taking 5 female Balb/c mice with 6 weeks of age, and injecting 30 mu g L-rhamnose-OVA into each of the female Balb/c mice subcutaneously at multiple points, wherein the L-rhamnose-OVA is fully and uniformly mixed with 75 mu L Freund's adjuvant in advance for emulsification, and the immunization period is 1 day, 15 days and 30 days; the Freund's adjuvant is prepared by using Freund's complete adjuvant for first-day (day 1), freund's incomplete adjuvant for second-day (day 15) and third-day (day 30), ELISA detection is carried out on mouse serum on day 37, mice with antiserum titer larger than 1:10000 are selected, the mice are killed by adopting an eyeball removing bloodletting method, spleens are taken out through aseptic operation, and the spleens are extruded and ground in a plate to obtain a mouse spleen cell suspension;
(4) Preparation of monoclonal hybridoma cells
The mouse spleen cell suspension prepared in the step (3) and the mouse syngeneic myeloma cells are prepared according to the following steps of 1:1, adding polyethylene glycol to form hybridoma cells, screening positive clones from the hybridoma cells by ELISA, and performing amplification culture to obtain monoclonal hybridoma cells;
(5) Preparation of murine L-murine Li Tangshan clone antibody
And (3) sensitized 5 female Balb/c mice with 6 weeks old by adopting a Freund incomplete adjuvant, and 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-mouse Li Tangshan clone antibody.
The detection results of SDS-PAGE electrophoresis detection and Western blotting detection of the L-rhamnose-OVA prepared in the embodiment are shown in figures 3-4.
The L-rhamnose-OVA prepared in the embodiment is subjected to mouse immunization, and the obtained antiserum is subjected to ELISA detection on the anti-rhamnose antibody titer, and the detection result is shown in figure 5, wherein the L-rhamnose-OVA immune group antibody titer is greater than 1:10000.
As can be seen from fig. 3 to 5, the present example successfully prepared L-rhamnose-OVA, and the L-rhamnose-OVA immunized mice had produced high titer of L-anti-rhamnose antibodies. Therefore, the L-rhamnose-OVA prepared by the embodiment can be used for injecting the crowd with low L-rhamnose natural antibody level so as to improve the L-rhamnose antibody level of the crowd with low L-rhamnose natural antibody level and further prevent various bacteria containing the L-rhamnose synthetic pathway from being infected in a broad spectrum.
Example 3 test of sterilizing Effect of humanized L-rhamnose Natural antibody
The specific method for extracting bacterial lipopolysaccharide and detecting SDS-PAGE/silver staining comprises the following steps:
(1) Extracting bacterial lipopolysaccharide: inoculating 5 μl of glycerol bacteria into 5mL of LB liquid medium, and culturing at 37deg.C for 220 r.t. overnight; centrifugally collecting the cultured thalli overnight, and extracting lipopolysaccharide by using a lipopolysaccharide extraction kit; the glycerinum is escherichia coli or pseudomonas aeruginosa;
(2) SDS-PAGE/silver staining detection: taking 5 mu L of extracted bacterial lipopolysaccharide, adding 1 mu L of 6 XSDS loading buffer solution, uniformly mixing, and boiling for 6 minutes; the resulting samples were subjected to 12% SDS-PAGE; after the completion of electrophoresis, the mixture was washed with a fixative solution (35 mL H 2 O, 15mL acetic acid, 50mL isopropanol) was slowly shaken for 2 hours, then twice with 200mL 7.5% glacial acetic acid gum for 10 minutes each; in oxidizing solution (139 mL H) 2 O, 11mL acetic acid, 1.059g periodic acid) for 3 minutes; then washing the gel with 200mL deionized water for three times, each time for 30 minutes; in silver staining solution (140 mL H) 2 O, 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 three times for 10 minutes each time; in the presence of a color development solution (250 mL H) 2 O, 12.5mg citric acid, 125. Mu.L formaldehyde) was slowly shaken until the strips were completeVisualization was then stopped with l% glacial acetic acid and the gel was scanned to record the silver staining results.
The in vitro killing experiment of the humanized L-rhamnose natural antibody comprises the following specific steps:
human L-rhamnose natural antibody gradient dilutions (1:20 to 1:320 fold diluted in 1 x PBS buffer, ph=7.4) were added to 96-well plates at 50 μl/well; a further 40. Mu.L of 1X 10 per well was added 4 E.coli or Pseudomonas aeruginosa (suspended in 1 XPBS buffer containing 1% (w/v) pancreatic protein powder, pH= 7.4,0.22 μm filter sterilized) and 10. Mu.L rabbit complement were mixed by shaking; the control group was not added with antibody, replaced with 1×pbs buffer (ph=7.4), and reacted at 37 ℃ for 1 hour; 10. Mu.L of CCK8 was added to each well and reacted at 37℃for 4 hours; OD determination with an ELISA apparatus 450 Is a light absorbance of (2); the kill rate was calculated according to the following formula: killing rate = 1- (OD) 450 antibody group /OD 450 control group )/100％。
SDS-PAGE/silver staining detection and Western blotting detection are carried out on lipopolysaccharides of Escherichia coli and Pseudomonas aeruginosa of various serotypes prepared and extracted in the embodiment, and detection results are shown in figures 6-11, wherein the humanized L-rhamnose natural antibody can be combined with Escherichia coli serotypes O1, O25b ST131 and O26 and Pseudomonas aeruginosa serotypes O4 and O10 with L-rhamnose in the lipopolysaccharide structure.
In vitro killing experiments are carried out on the humanized L-rhamnose natural antibody prepared in the embodiment, and the detection results are shown in figure 12, wherein the humanized 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 fig. 6 to 12, the human-derived L-rhamnose natural antibody prepared in this example can effectively bind and kill bacteria having L-rhamnose in lipopolysaccharide structure.
Experimental example: typical treatment cases
The effects of the present invention will be further described below in conjunction with three typical cases of clinical application of the L-rhamnose antibody prepared in the examples.
Case 1: the patient had urinary tract infection and even bacteremia symptoms, urine or blood was cultured by bacteria and 16s rRNA was identified as E.coli O25b ST131, and was infected with drug-resistant bacteria E.coli O25b ST131.
The application method of the L-rhamnose antibody comprises the following steps: the humanized L-rhamnose natural antibody prepared in example 1 was dissolved in physiological saline at a concentration of 10mg/mL, and then injected intravenously, 10mL each time, once every 3 days.
L-rhamnose antibody application effect: the urinary tract infection of the patient even the bacteremia symptoms are reduced or eliminated, and the escherichia coli O25b ST131 is not detected by urine or blood through bacteria culture or 16s rRNA.
Case 2: patients are hospitalized for a long time due to severe symptoms, and the patients have low immunity after long-term use of various antibiotics. The blood of the patient was withdrawn, and the L-rhamnose antibody level in the blood was found to be lower than the normal level, and was considered to be at risk of infection with a drug-resistant bacterium.
The application method of the L-rhamnose antibody comprises the following steps: the humanized L-rhamnose natural antibody prepared in example 1 was dissolved in physiological saline at a concentration of 5mg/mL and then injected intravenously, 10mL each time, once every 3 days.
L-rhamnose antibody application effect: the risk of a patient being free of or having a drug-resistant bacterial infection during hospitalization is reduced.
Case 3: the burnt skin surface of the burnt patient produces blue-green or yellow-green lesions, and samples of lesion parts are taken, cultivated by bacteria and identified by 16s rRNA to be pseudomonas aeruginosa, and the samples are infected by the pseudomonas aeruginosa skin.
The application method of the L-rhamnose antibody comprises the following steps: dissolving the murine L-mouse Li Tangshan clone antibody prepared in the example 1 in normal saline, adding rabbit complement, vaseline and glycerol, and preparing into ointment, wherein the final concentration of the murine L-mouse Li Tangshan clone antibody in the ointment is 5mg/mL; the prepared ointment is uniformly smeared on the skin infection part of the pseudomonas aeruginosa of a patient 3 times a day.
L-rhamnose antibody application effect: bluish green or yellowish green lesions on the surface of burned skin of a burn patient weaken or disappear, and pseudomonas aeruginosa is not detected by taking a lesion part sample and culturing the bacteria and 16s rRNA.
Claims (5)
- The use of an L-rhamnose antibody in the preparation of a medicament for the prevention and/or treatment of a drug-resistant bacterial infection, characterized in that the L-rhamnose antibody specifically recognizes L-rhamnose, which is a natural human L-rhamnose antibody; the drug-resistant bacteria are escherichia coli or pseudomonas aeruginosa;the preparation method of the humanized L-rhamnose natural antibody comprises the following steps:(1) Synthesis of L-rhamnose-NH 2The L-mouse Li Tangjing acetyl protection and thiophenol glycosylation reaction are carried out to obtain an intermediate product 1, the intermediate product 1 is connected with 3-azidopropanol to obtain an intermediate product 2, and the intermediate product 2 is subjected to deacetylation protection and reduction reaction to obtain L-rhamnose-NH 2 ；Intermediate 1 intermediate 2(2) Preparation of L-rhamnose affinity chromatography columnLoading the NHS activated agarose gel into a column, washing and balancing, and adding the L-rhamnose-NH prepared in the step (1) 2 Uniformly mixing, slowly shaking at room temperature for reaction for 3-5 hours, then adding a sealing solution, slowly shaking at room temperature for reaction for 0.5-1.5 hours, washing by a washing solution A and a washing solution B to obtain an L-rhamnose affinity chromatographic column, and storing in 20% ethanol at 4 ℃;the flushing liquid is precooled 1mM HCl, and the volume of the flushing liquid is 10-15 column volumes; the balance liquid is 0.2M NaHCO 3 0.5M NaCl solution, pH value of 8-9, and the volume of the balance liquid is 3-5 column volumes; the L-rhamnose-NH 2 The addition amount is 1-2 column volumes, and the concentration is 5-15 mg/mL; the blocking solution is 0.1M Tris-HCl solution, the pH 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 solutionpH is 8-9; the washing solution B is a solution of 0.1M HCOOH and 0.5M NaCl, and the pH value is 3-5; the washing liquid A and the washing liquid B are alternately washed for 3-6 times, 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 deionized water with 2-4 column volumes is used for washing 1-2 times;(3) Purification of L-rhamnose Natural antibodiesAnd (3) taking the L-rhamnose affinity chromatography column prepared in the step (2), adding serum after washing and balancing, incubating for 3-4 hours at room temperature after uniformly mixing, washing the L-rhamnose affinity chromatography column again, eluting, collecting eluent, adjusting the pH of the eluent to be neutral, and finally obtaining the humanized L-rhamnose natural antibody after ultrafiltration and concentration of the eluent.
- 2. The use according to claim 1, 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.
- 3. The use according to claim 1, wherein in step (3), the rinse solution is deionized water, and the volume of the rinse solution is 10 to 15 column volumes; the balanced liquid is 1 XPBS buffer solution, the pH value is 7.4, and the volume of the balanced liquid is 10-15 column volumes; the serum is healthy adult serum, and the addition amount of the serum is 10-15 column volumes; the eluted eluent is 0.2M glycine solution, and the pH value is 2-3; the ultrafiltration concentration is carried out by using an ultrafiltration tube with the molecular weight of 8-12 kDa.
- 4. The use according to claim 1, wherein the use is intravenous injection after dilution of a solution of a natural human L-rhamnose antibody to 5-15 mg/mL.
- 5. The application of claim 1, wherein the application is to dilute the natural human L-rhamnose antibody to a solution of 5-15 mg/mL, then add complement, vaseline and glycerol, and make ointment for direct application.
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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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| JPH06306092A (en) * | 1993-02-26 | 1994-11-01 | D D S Kenkyusho:Kk | Compound having specific bonding ability to adhesive molecule elam-1 |
| WO2009040726A2 (en) * | 2007-09-24 | 2009-04-02 | Universite De Geneve | Rhamnose antagonists and use thereof |
| CN103506080A (en) * | 2012-06-19 | 2014-01-15 | 汪志友 | Blood coagulation factor VIII separating and purifying medium and preparation method thereof |
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