WO2023153805A1 - Modèle de maladie infectieuse et procédé de criblage de médicament l'utilisant - Google Patents

Modèle de maladie infectieuse et procédé de criblage de médicament l'utilisant Download PDF

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WO2023153805A1
WO2023153805A1 PCT/KR2023/001859 KR2023001859W WO2023153805A1 WO 2023153805 A1 WO2023153805 A1 WO 2023153805A1 KR 2023001859 W KR2023001859 W KR 2023001859W WO 2023153805 A1 WO2023153805 A1 WO 2023153805A1
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mycoplasma
infectious
disease
recombinant expression
expression vector
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정의숙
성민지
오세경
김길수
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재단법인 대구경북첨단의료산업진흥재단
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/66—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • A—HUMAN NECESSITIES
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    • A01K2207/00—Modified animals
    • A01K2207/12—Animals modified by administration of exogenous cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0337—Animal models for infectious diseases
    • C—CHEMISTRY; METALLURGY
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    • C12Q2304/00—Chemical means of detecting microorganisms
    • C12Q2304/60—Chemiluminescent detection using ATP-luciferin-luciferase system
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y113/00—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13)
    • C12Y113/12—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13) with incorporation of one atom of oxygen (internal monooxygenases or internal mixed function oxidases)(1.13.12)
    • C12Y113/12007—Photinus-luciferin 4-monooxygenase (ATP-hydrolysing) (1.13.12.7), i.e. firefly-luciferase
    • 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

Definitions

  • the present invention relates to a transformant into which a luciferase gene has been introduced, an animal model infected therewith, and a method for screening a drug using the same.
  • Infectious diseases caused by bacterial infections of the gastrointestinal tract are very common. Specifically, an infectious gastrointestinal disease may occur as a cause such as bacteria or parasites is transmitted through contact with food or people. Common causative bacteria include E. coli , Salmonella , Shigella , Pseudomonas , and the like. Infectious diseases occurring in the gastrointestinal tract cause diarrhea and vomiting due to inflammation of the stomach and intestines. Diarrhea causes the anus to leak, and vomiting and diarrhea cause gas to build up in the abdomen, causing abdominal distension. In addition, there is no energy due to dehydration, severe pain in the abdomen, and headache due to high fever.
  • Bacteria that cause these infectious gastrointestinal diseases include Escherichia coli, Bacillus cereus, Campylobacter jejuni, Clostridium perfringens, and Salmonella species. species), Shigella species, Staphylococcus aureus, Vibrio parahaemolyticus, etc. are known, and recently, the existence of super bacteria has been confirmed, and the severity of diseases related to them is significantly increasing. .
  • infectious diseases caused by bacterial infections in the respiratory tract are also very common diseases, and depending on the degree of infection, severe or more severe diseases are caused.
  • toxin production caused during infection causes excessive inflammatory reactions in the bronchi and lungs, and also inhibits phagocytosis by white blood cells, producing leukotoxin as a by-product in the lower respiratory tract. It often causes infectious bronchopneumonia.
  • Symptoms of this infectious respiratory disease include sudden fever, initial high fever, dry cough accompanied by purulent sputum, severe chills, chest pain, and muscle pain.
  • Causative bacteria causing common infectious respiratory diseases are, for example, Acinetobacter baumannii , Pasteurella multocida , Haemophilus parasuis , Bordetella bronchiseptica ( Bordetella bronchiseptica ), Actinobacillus pleuropneumoniae , Streptococcus suis , and Mycoplasma spp.
  • Cefdinir Azithromycin, Clarithromycin, Dirithromycin, Erythromycin for the treatment of infectious diseases including gastric infectious gastrointestinal diseases and infectious respiratory diseases ), Roxithromycin, Telithromycin, Carbomicin A, Josamycin, Kitasamycin, Midecamycin/Midecamycin acetate, Antibiotic drugs such as Oleandomycin, Spiramycin, Troleandomycin, and Tylosin are used for treatment.
  • antibiotics as described above usually exhibits a sufficient effect in treating diseases, it causes an ironic situation in that the incidence of infectious diseases increases due to the frequent use of antibiotics. It is very common that the increase in the incidence of such an infectious disease occurs when a drug does not exhibit sufficient efficacy due to the development of drug resistance or the like.
  • a method of detecting the reporter gene by inserting a reporter gene into chromosomal DNA for the above screening can be considered as one method. Stable expression is possible when a reporter gene is inserted on the chromosome, but it has a low gene expression due to the number of copies of 1 copy, is more difficult to manufacture than using a plasmid, and takes a lot of time. In addition, it is very difficult to develop a screening method for the above method in that the phenotype can change when a gene is inserted into chromosomal DNA.
  • the present inventors prepared a transformant stably transfected with an optical reporter gene and an animal model infected with the transformant. Then, the present invention was completed by enabling non-invasive, real-time monitoring of drug screening along with tracking and quantification of in vitro and in vivo distribution of bacteria over time.
  • An object of the present invention is a recombinant expression vector comprising a lacI-deleted trc promoter and a Firefly luciferase gene operably linked thereto; Transformants transformed with the recombinant expression vector; And to provide an animal model infected with the above transformant.
  • Another object of the present invention is (a) processing a drug candidate to a transformant transformed with the recombinant expression vector;
  • Another object of the present invention is (a) infecting a transformant transformed with the recombinant expression vector to a disease-causing site of an animal other than human;
  • the T7 promoter and lac promoter which are generally known as high-efficiency gene expression systems, are IPTG inducible promoters and can be highly induced. However, the expression level is not sufficient in terms of overexpression due to the high price of IPTG and the suppression of gene expression by the lacI repressor.
  • the lacI-deleted trc promoter was used and firefly luciferase with high luminescence level was used to more strongly and specifically confirm the change in luminescence level.
  • the present invention provides a recombinant expression vector comprising a lacI-deleted trc promoter and a firefly luciferase gene operably linked thereto.
  • vector refers to a genetic construct comprising a nucleotide sequence of a gene operably linked to a suitable control sequence so as to express a target gene in a suitable host, and the control sequence is capable of initiating transcription.
  • promoters capable of controlling transcription, optional operator sequences for regulating such transcription, and sequences regulating termination of transcription and translation.
  • the term "recombinant expression vector” is a vector capable of expressing a target protein or target RNA in a suitable host cell, and refers to a genetic construct containing essential regulatory elements operably linked to express a gene insert.
  • operably linked refers to functional linkage between a nucleic acid expression control sequence and a nucleic acid sequence encoding a protein or RNA of interest so as to perform a general function.
  • a promoter and a nucleic acid sequence encoding a protein or RNA may be operably linked to affect expression of the encoding nucleic acid sequence.
  • Operational linkage with a recombinant vector can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cutting and linking uses enzymes generally known in the art.
  • the recombinant expression vector of the present invention contains the firefly luciferase gene of SEQ ID NO: 1.
  • the firefly luciferase gene of SEQ ID NO: 1 is a material used for visualization and is an enzyme that converts chemical energy into light energy by accelerating the oxidation of luciferin to emit light. It can be obtained directly from firefly or by expression or synthesis from a microorganism containing a recombinant DNA fragment encoding such an enzyme.
  • firefly luciferase (Luc) is used in the present invention to express strong luminescence intensity in vitro and/or in vivo . Accordingly, by using this, the time for measuring a similar detection amount is greatly reduced, and the level of luminescence change is increased so that luminescence change due to drug treatment can be more clearly and clearly identified.
  • firefly luciferase (Luc) emits light at a long wavelength of 600 nm, it can penetrate deeper tissues, which can show better action under in vivo imaging conditions.
  • the amino acid sequence of the firefly luciferase prepared from SEQ ID NO: 1 is shown in SEQ ID NO: 2. Any firefly luciferase gene sequence capable of synthesizing the sequence of SEQ ID NO: 2 is also included as a substantially homologous category of the firefly luciferase gene sequence of SEQ ID NO: 1 of the present invention.
  • promoter refers to a polynucleotide sequence that allows and regulates the transcription of a gene operably linked thereto.
  • the recombinant expression vector of the present invention also contains a trc promoter from which lacI has been removed.
  • the continuous expression of firefly luciferase is an object. Accordingly, constant expression of the firefly luciferase gene was ensured by using the trc promoter as the promoter sequence excluding the lac repressor (lacI) sequence. In particular, it shows an excellent effect in that it can bring the expression level to a high level with constant and high efficiency regardless of the presence or absence of IPTG (isopropyl- ⁇ -D-thiogalactoside, IPTG), an inducer for current induction.
  • IPTG isopropyl- ⁇ -D-thiogalactoside, IPTG
  • the trc promoter sequence is represented by SEQ ID NO: 3. That is, the trc promoter sequence may be the nucleotide sequence of SEQ ID NO: 3.
  • the recombinant expression vector according to the present invention further includes an origin of replication (oriV) and an origin of migration (oriT).
  • oriV origin of replication
  • oriT origin of migration
  • the recombinant expression vector according to the present invention is intended to be expressed in various bacteria through a broad host range. Accordingly, the recombinant expression vector according to the present invention includes an origin of replication (oriV) and an origin of migration (oriT), and thus has the advantage of being universally applicable to various pathogenic strains.
  • the replication origin (oriV) sequence is shown in SEQ ID NO: 4
  • the migration origin (oriT) sequence is shown in SEQ ID NO: 5.
  • pRO1600 oriV SEQ ID NO: 6
  • pRO1600 Rep SEQ ID NO: 7
  • the recombinant expression vector further comprises at least one antibiotic resistance gene selected from the group consisting of an ampicillin resistance gene, a kanamycin resistance gene and a chloramphenicol acetyl transferase gene. It may be a recombinant vector to.
  • "Antibiotic resistance gene” is a gene having resistance to antibiotics, and since cells having this gene survive in an environment treated with the antibiotic, it is usefully used as a selection marker in the process of obtaining plasmids in large quantities from E. coli.
  • antibiotic resistance genes commonly used as selection markers can be used without limitation.
  • resistance genes to ampicillin, tetracyclin, kanamycin, chloroamphenicol, streptomycin, or neomycin can be used, preferably It may be an ampicillin resistance gene.
  • the recombinant expression vector according to the present invention may preferably have a firefly luciferase (Luc) sequence of SEQ ID NO: 1 inserted at the insertion site of a vector known as pMF36.
  • Luc firefly luciferase
  • SEQ ID NO: 8 shows the full-length sequence of the recombinant vector into which the firefly luciferase (Luc) sequence of SEQ ID NO: 1 is inserted. That is, the recombinant expression vector may have the nucleotide sequence of SEQ ID NO: 8.
  • FIG. 1 A cleavage map of the recombinant expression vector according to the present invention is shown in FIG. 1 .
  • FIG. 1 shows the pMF36 vector, and the recombinant vector into which the firefly luciferase (Luc) sequence of SEQ ID NO: 1 according to the present invention is inserted is shown in B of FIG. 1 (pJS-1).
  • in vivo screening measures the luminescent signal present in the skin, it must have a certain level of expression or higher to be detected, and a high luminescent expression level detects a small amount of luminescent bacteria following drug treatment. make it possible
  • Xba I and Hind III of the recombinant vector The firefly luciferase gene represented by SEQ ID NO: 1 may be inserted in between.
  • the present invention also relates to a transformant (preferably a bacterium or the like) transformed with the recombinant expression vector described above.
  • a transformant preferably a bacterium or the like transformed with the recombinant expression vector described above.
  • transformation refers to the transfer of a nucleic acid fragment into the genome of a host cell to cause genetically stable inheritance.
  • the method of transforming the vector of the present invention into the cell includes any method of introducing a base into the cell, and can be performed by selecting a suitable standard technique as known in the art. Electroporation, calcium phosphate co-precipitation, retroviral infection, microinjection, DEAE-dextran, cationic liposome laws, etc., but are not limited thereto. Preferably, a heat shock and/or electroporation method may be used.
  • Transformant transformed with a recombinant vector refers to a cell transformed with a vector having a gene encoding one or more target proteins.
  • Transformants according to the present invention may be, for example, bacteria, viruses, parasites and the like.
  • Transformants according to the present invention are preferably bacteria.
  • Such bacteria may in particular be pathogens.
  • Salmonella enterica Escherichia coli, Citrobacter rodentium , Bordetella bronchiseptica , Acinetobacter baumannii . _ _ _ _ typhimurium ), Shigella dysenteriae, Yersinia enterocolitica, Acinetobacter calcoaceticus, Francisella tularensis , Legionella pneumophila Phila ( Legionella pneumophila ), Proteus vulgaris ( Proteus vulgaris ), Proteus mirabilis ( Proteus mirabilis ), Stenotrophomonas maltophilia ( Stenotrophomonas maltophilia ), Pasteurella multocida ( Pasteurella multocida ), Haemophilus parasu Is ( Haemophilus parasuis ), Actinobacillus pluronicumoniae ( Actinobacillus pleuropneumoniae ), Streptococcus suis
  • the present invention also provides animals infected with the transformants disclosed above.
  • Such animals may be mammals such as mice, rats, rabbits, beagles, goats, pigs, sheep, mice, and cows.
  • the animal infected with the transformant may be a mouse or a rat.
  • the amount of the transformant infected may vary depending on the type of organism, severity of infectious disease, age, sex, drug activity, administration time, administration route, and excretion rate.
  • mice and/or rats may be infected with 1 x 10 6 to 1 x 10 9 CFU, but it varies depending on the type of bacteria, route of administration, age and weight of the mouse, and so is not limited thereto.
  • the administration or infection may vary depending on the desired disease site.
  • oral administration, intragastric administration, intratracheal administration, inner ear administration, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intrauterine intrathecal administration, intracardiovascular administration, etc. may be administered differently depending on the target disease inducing site. .
  • it may be administration by intragastric or intratracheal administration or infection. This makes it possible to assess the level of disease following infection, for example in the gastrointestinal or respiratory tract.
  • the present invention also includes (a) processing a drug candidate into a transformant transformed with the recombinant expression vector;
  • Infectious diseases refer to diseases caused by disease-causing pathogens, such as viruses, bacteria, fungi, and parasites, spreading to or invading animals or humans, and for the purpose of the present invention, it means all infectious diseases caused by bacteria. do.
  • infections may be infections of the gastrointestinal tract, respiratory tract, skin or cardiovascular system.
  • the gastrointestinal tract refers to all organs of the digestive system from the mouth to the anus, including the esophagus, stomach, large intestine, small intestine, and the like.
  • the respiratory tract refers to all organs involved in the mechanical exchange of inhaling oxygen and exhaling carbon dioxide through the mouth, including the nose, pharynx, larynx, trachea, bronchi, lungs, and the like.
  • the cardiovascular system refers to related organs that play a major role in transporting blood in the body, including the aortic valve, pulmonary valve, mitral valve, tricuspid valve, heart muscle, epicardium, and endocardium.
  • the skin refers to an external tissue of the body including subcutaneous tissue, dermis, epidermis, hair follicles, and the like.
  • the infectious disease may be an infectious gastrointestinal disease, an infectious respiratory disease, an infectious skin disease, or an infectious cardiovascular disease caused by infection.
  • An infectious gastrointestinal disease according to the present invention is a disease caused by infection of the gastrointestinal tract, such as bacteria, viruses, and parasites. Symptoms may include anorexia, nausea, vomiting, flatulence and abdominal cramps. Also, diarrhea is the most common symptom and may be accompanied by blood and mucus. It can also cause fever, nausea, muscle pain and exhaustion. Infectious gastrointestinal diseases according to the present invention include, for example, infectious gastroenteritis, infectious colitis, infectious colitis, inflammatory bowel disease and the like.
  • Bacteria that cause gastric infectious diseases of the gastrointestinal tract can produce enterotoxins while remaining attached without invading the intestinal wall. These toxins cause the intestines to secrete water and electrolytes, which can lead to watery diarrhea. Some bacteria can invade the walls of the small intestine or colon and damage cells, forming small ulcers (ulceration) that cause bleeding and large leaks of fluids containing proteins, electrolytes, and water. Accordingly, diarrhea contains white blood cells and red blood cells, and sometimes blood is visible.
  • Strains that can cause these diseases include, for example , E. coli, C. rodentium, Campylobacter spp., Salmonella spp., Listeria spp., Shigella spp., Trichinella spp. etc. can be considered.
  • An infectious respiratory disease according to the present invention is a disease caused by infection of the respiratory tract by bacteria, viruses, parasites, and the like. Symptoms such as fever, initial high fever, dry cough with purulent sputum, severe chills, chest pain, and muscle pain may appear. Infectious respiratory diseases according to the present invention include, for example, infectious pneumonia, lung abscess, sepsis , upper respiratory tract infection, and the like.
  • Strains that can cause these diseases include, for example , Yersinia pestis, Streptococcus spp., Pateurella spp., B. bronchiseptica, A. baumannii, Mycoplasma spp. etc. can be considered.
  • An infectious skin disease according to the present invention is a disease caused by infection of the skin by bacteria, viruses, parasites, and the like. Symptoms accompanying skin inflammation are commonly present. Infectious skin diseases according to the present invention include, for example, impetigo, folliculitis, excision, erysipelas, cellulitis, and acute gastritis.
  • Strains that can cause these diseases include, for example, Staphylococcus spp., Micrococcus spp., Corynebacterium spp., Streptococcus spp. etc. can be considered.
  • Infectious cardiovascular diseases are diseases caused by infections of the cardiovascular system such as bacteria, viruses, parasites, and the like. Its main feature is inflammation that occurs in the cardiovascular system, and serious symptoms can occur when blood clots accumulate in the inflamed area. Infectious cardiovascular diseases according to the present invention include, for example, endocarditis, myocarditis, pericarditis, suppurative thrombophlebitis, endoarteritis and the like.
  • Streptococcus spp. and Stapylococcus spp. etc. can be considered as common causative bacteria, but is not limited to a specific strain.
  • a specific infectious disease according to the present invention is an infectious gastrointestinal disease or an infectious respiratory disease.
  • the infectious gastrointestinal disease drug screening method includes the step of treating a drug candidate to a transformant transformed with a recombinant expression vector.
  • the transformant according to the present invention is preferably a bacterium as mentioned above.
  • the drug candidate may be, for example, any one selected from the group consisting of antibodies, proteins, antigens, peptides, nucleic acids, enzymes, cells, bioactive polymers, bioactive inorganic substances, and drugs (eg, compounds). .
  • luciferin is a generic term for luminescent substrates of luminescence reaction that exhibit luciferin-luciferase reaction (LL reaction) in bioluminescence. , ratiaru cipherin, and coelenterazine. It is oxidized by oxygen molecules in the presence of a luminescent enzyme, and an oxidation product (oxyluciferin) is produced in an excited state, and when it becomes a ground state, visible light is generated. To summarize the process, luciferin + O 2 ⁇ oxyluciferin + light do.
  • the luciferin may be, but is not limited to, D-luciferin, native coelenterazine, or h-coelenterazine, which is a 2-deoxy derivative. More specifically, D-luciferin can be used.
  • Transformants transformed with the recombinant expression vector exhibit luminescent properties by luciferin.
  • Treatment of luciferin according to the present invention means placing a transformant transformed with a recombinant expression vector in the same reaction system, for example, adding luciferin to a container containing a transformant, adding a transformant to a container containing luciferin, or mixing the transformant and luciferin.
  • treatment of the above-mentioned drug candidates may change the luminous efficiency.
  • the infectious disease drug screening method according to the present invention includes (b) measuring the change in luminescence of the transformant after treatment with luciferin.
  • the measurement of the luminous properties refers to detecting the level of light generated in the step (a), quantitatively or qualitatively.
  • Luminescence can be detected by any means known in the art, for example, by luminescence microscopes, photometers, luminescence plate readers, multiplier tube detectors, and the like.
  • the luminescence level is reduced compared to the control group not treated with the drug candidate by the detection of the luminescence level, it can be considered as the drug candidate material.
  • the present invention may further include (c) selecting a drug candidate having a reduced emission level compared to a control group not treated with the drug candidate.
  • the present invention also provides (a) infecting a disease-causing site of an animal other than human with a transformant transformed with the recombinant expression vector;
  • the infectious disease drug screening method includes the step of (a) infecting a transformant transformed with the recombinant expression vector into a disease-causing site of an animal other than human.
  • the disease-causing site may be, for example, any site in the gastrointestinal tract, respiratory tract, skin, or cardiovascular system mentioned above.
  • Administration can produce an appropriate infectious disease model.
  • the transformant may preferably be a bacterium capable of causing an infectious disease, as described above.
  • Such animals may be mammals such as mice, rats, rabbits, beagles, goats, pigs, sheep, mice, and cows. Infectious diseases can be induced through infection of animals through the above transformants.
  • the number of bacteria administered or infected for causing such an infectious disease may vary depending on the severity of the desired disease, the disease-causing strain, the type of animal to be administered, and the like.
  • the administration or infection may be administration or infection by intragastric administration or intratracheal administration. Through this, it is possible to evaluate the level of the disease according to the infection at the disease-causing site.
  • the disease-causing site may be the gastrointestinal system. More specifically, it may be the stomach, small intestine, large intestine or esophagus.
  • the disease-causing site may be the respiratory tract. More specifically, it may be the nose, trachea, bronchi or lungs.
  • the drug screening method for infectious diseases according to the present invention also includes the step of treating an infected animal with a drug candidate.
  • Treatment of the drug candidate to the infected animal may proceed through various administration routes.
  • administration routes such as oral administration, intragastric administration, intratracheal administration, inner ear administration, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intrauterine intrathecal administration, and intracardiovascular administration. It can be administered in an appropriate amount.
  • administration and treatment of the drug candidate may vary depending on the severity of the target disease, the disease-causing strain, and the type of animal to be administered.
  • the infectious disease drug screening method according to the present invention also includes the step of (c) treating luciferin and measuring the change in luminescence at the site of infection of the animal.
  • Treatment of luciferin means placing a transformant transformed with a recombinant expression vector in the same reaction system, and accordingly, luciferin is administered orally, intragastrically, intratracheally, inner ear, or intraperitoneally. It may include administration through administration, intravenous administration, intramuscular administration, subcutaneous administration, intrauterine intrathecal administration, intracardiovascular administration, and the like.
  • Such treatment with luciferin may be considered differently depending on the number of transformants administered , the body weight of the animal, and the like.
  • the method according to the present invention can be used as a practical means for screening drug efficacy by confirming the efficacy of drug candidates in vivo without sacrificing animals or fixing biological specimens.
  • an animal model for infection is prepared in vivo , then the drug candidate is treated, and then the animal is sacrificed over time to perform various experiments (using molecular biological methods, histopathological methods, or target organs). Bacteria count, etc.) is required to confirm the potential as a drug candidate.
  • the method according to the present invention has a great advantage in evaluating the possibility of a drug candidate because it can non-invasively evaluate the in vivo response in real time.
  • the superiority is also recognized in that drug changes can be confirmed over a long period of time through high expression levels.
  • the luminescence level is reduced compared to the control group not treated with the drug candidate by the detection of the luminescence level, it can be considered as the drug candidate material.
  • the present invention may further include (d) selecting a drug candidate having a reduced luminescence level compared to a control group not treated with the drug candidate.
  • the recombinant vector according to the present invention can provide universal, non-invasive and real-time evaluation of the efficacy of drug candidates for the treatment of novel infectious diseases in vitro and in vivo. It can be usefully used for drug screening in that it exists.
  • Figure 1 shows the vector structure of pMF36 and the structure of the recombinant vector pJS-1 prepared in the present invention.
  • Figure 2 shows the result of confirming stable luminescence in vitro of P. aeruginosa transformed with a recombinant vector containing a FireFly luciferase gene in a solid medium.
  • Figure 3 shows the result of confirming the stable light emission of P. aeruginosa transformed with the recombinant vector containing the FireFly luciferase gene in a liquid medium in vitro .
  • Figure 4 shows the result of confirming the stable luminescence of B. bronchiseptica transformed with the recombinant vector containing the FireFly luciferase gene in a solid medium in vitro .
  • FIG. 5 shows the result of confirming the stable luminescence efficacy of P. aeruginosa transformed with the recombinant vector containing the FireFly luciferase gene in an experimental animal (or mouse) in vivo .
  • Figure 6 is a comparative example of a recombinant vector (pJS-2) in which the FireFly luciferase gene was inserted into pLI50, a recombinant vector (pJS-3) in which the FireFly luciferase gene was inserted in pCN60, and a pAKlux2 vector (pJS-4). , and a schematic diagram of a recombinant vector (pJS-5) in which the FireFly luciferase gene is inserted into pRMC2.
  • FIG. 7 shows the results of drug efficacy evaluation by treating P. aeruginosa transformed with a recombinant vector containing a FireFly luciferase gene in vitro with an antibiotic.
  • FIG. 8 shows the results of drug efficacy evaluation by treating B. bronchiseptica transformed with a recombinant vector containing a FireFly luciferase gene in vitro with an antibiotic.
  • Figure 9 The results of drug efficacy evaluation according to antibiotic treatment on an animal model of an infectious gastrointestinal disease are shown.
  • Figure 10 shows the result of confirming the difference in luminescence efficacy according to the type of recombinant vector (left: S. Typhimurium/pJS-2), right: S. Typhimurium/pJS-1).
  • Figure 11 is The results of drug efficacy evaluation according to antibiotic treatment on an animal model of infectious respiratory disease are shown.
  • Figure 12 shows the difference in luminescence efficacy according to the type of recombinant vector containing the FireFly luciferase gene. The results confirmed in vitro are shown ((a): negative control, (b): pJS-3, (c): pJS-4, (d): pJS-2, (e): pJS-5, (f) :pJS-1).
  • Figure 15 shows the results of in vitro growth experiments monitoring changes in OD (A), luminescence value (B), and bacterial count (C) of the transformed P. aeruginosa strain and the wild type.
  • FIG. 16 shows the results of in vitro growth experiments monitoring changes in OD (A), luminescence values (B), and bacterial counts (C) of transformed S. Typhimurium strains and wild type.
  • Figure 21 is The results of drug efficacy evaluation according to antibiotic treatment on an animal model of an infectious gastrointestinal disease are shown.
  • Example 1 Construction of a recombinant vector containing the FireFly luciferase gene
  • pMF36 a broad host range plasmid
  • pMF36 contains an ampicillin sequence and a trc promoter, which are selectable factors, but does not contain a LacI sequence.
  • luciferase gene In order to insert the luciferase gene, Xba I, Hind III restriction enzymes present in MCS (Multiple Cloning Site) were selected, and a primer set containing Xba I and Hind III was used with the luciferase gene (SEQ ID NO: 1) as a template. PCR was performed using
  • PCR was performed under the following conditions; Initial denaturation process at 98 °C for 2 minutes; Denaturation at 95 °C for 10 seconds, Primer Annealing at 58 °C for 10 seconds, Extension at 72 °C for 35 cycles; And the final extension process was carried out at 72 °C for 5 minutes.
  • the amplification products were digested with Xba I and Hind III restriction enzymes and then purified using a Purification Kit.
  • a sequence to be inserted into the vector is shown in SEQ ID NO: 11.
  • pMF36 was also digested with Xba I and Hind III restriction enzymes in the same way, and pMF36 and luciferase genes were reacted overnight at 16 ° C with T4 ligase (Takara) to construct a recombinant vector, which was named pJS-1.
  • the constructed luciferase labeled plasmid was transformed into E. coli DH5a strain using heat shock.
  • a recombinant vector was purified from the selected strain and used in the experiment.
  • the DNA is competent for transformation. made of cells.
  • E. coli DH5a , S. Typhimurium, and P. aeruginosa were cultured in Luria Bertani (LB) while A. baumannii , C. redentium, and B. bronchiseptica were cultured in Brain Heart Infusion (BHI) liquid medium with shaking at 200 rpm or 1.5 % (w/v) was cultured at 37 °C using a solid medium supplemented with agar.
  • BHI Brain Heart Infusion
  • the antibiotic ampicillin was added at a concentration of 100 ⁇ g/ml.
  • the overnight bacterial solution was inoculated into this culture medium and O.D. It was cultured to about 0.5. The cultured bacterial solution was left cold on ice for 30 minutes, and then centrifuged at 2,500 g at 4 ° C. for 10 minutes, and the supernatant was removed. The bacterial cell pellet was washed several times with 10% glycerol, and finally suspended in 1/50 of the culture medium in 10% glycerol and stored at -80 °C until transformation.
  • ampicillin medium was used to select bacteria into which the recombinant plasmid was introduced, and the substrate D-luciferin Potassium Salt Bioluminescent Substrate (XenoLight) was added to a final concentration of 0.3 mg/ml using a microplate reader, and the luminescence value was measured. Confirmed.
  • Figure 2 shows the result of comparing the luminescence change of the transformed P. aeruginosa strain and Wild type . As can be seen from Figure 2, it was confirmed that the transformed P. aeruginosa strain stably emits light.
  • 3 and 13 show the result of confirming the same stable expression level on the E-tube.
  • 3 A shows a high concentration of P. aeruginosa
  • B shows a low concentration of P. aeruginosa
  • C shows a Wild type.
  • Figure 13 shows the remarkable luminescence efficacy of transformed S. Typhimurium and P. aeruginosa .
  • the luminescent efficacy was confirmed for B. bronchiseptica and is shown in FIG. 4 .
  • the strain transformed according to the present invention showed stable luminescence efficacy.
  • Example 2 The transformed bacteria prepared in Example 2 and the negative control bacteria were cultured in a suitable medium to an appropriate O.D, centrifuged to remove the supernatant, and washed with PBS to prepare.
  • E. coli DH5a, C. rodentium , B. bronchiseptica , P. aeruginosa, S. Typhimurium strains or negative control bacteria prepared in Example 2 were prepared using 7- to 8-week-old ICR or C57BL/6 mice. Depending on the species, 200 ⁇ l of 1 x 10 8 CFU or 1 x 10 9 CFU was intragastricly administered. Prior to imaging, mice were inoculated with 100 ⁇ l of 0.3 mg/ml D-luciferin Potassium Salt Bioluminescent Substrate (XenoLight) by intragastric administration. Mice were anesthetized with isoflurane/Oxygen and luminescence imaging images were taken using IVIS® Lumina K (Perkin Elmer). The intensity of the luminescence signal was quantified as photon counts per second (p/s).
  • a pJS-2 vector was prepared in the same manner as in the pJS-1 production method.
  • pLI50 contains the ampicillin sequence, which is a selective factor, and was purchased from Addgene.
  • the amplification product was digested with Bam HI and Hind III restriction enzymes and then purified using a Purification Kit.
  • the vector was also digested with Bam HI and Hind III restriction enzymes in the same way, and pLI50 and luciferase genes were reacted overnight at 16 ° C with T4 ligase (Takara) to construct a recombinant vector, which was named pJS-2.
  • the constructed luciferase labeled plasmid was transformed into E. coli DH5a strain by electroporation.
  • a recombinant vector was isolated from the selected strain, and after confirmation of plasmid insertion, the vector was purified from an E. coli strain containing the recombinant vector and used for the experiment.
  • pJS-2 recombinant vector to S. Typhimurium In order to insert the strain, competent cells for transformation were prepared similarly to Example 2 so that the DNA could enter well. Then, after adding 1 ⁇ g of pJS-2 DNA to the prepared competent cells, it was introduced into the cells through electroporation.
  • ampicillin medium was used to screen strains into which the recombinant plasmid was introduced, and the substrate D-luciferin Potassium Salt Bioluminescent Substrate (XenoLight) was added to a final concentration of 0.3 mg/ml using a microplate reader, and the luminescence value was measured. Confirmed.
  • the transformed bacteria prepared in Comparative Example 1.2 were cultured in a suitable medium to an appropriate O.D., centrifuged to remove the supernatant, and washed with PBS to prepare.
  • mice Prior to imaging, mice were inoculated with 100 ⁇ l of 0.3 mg/ml D-luciferin Potassium Salt Bioluminescent Substrate (XenoLight) by intragastric administration. Mice were anesthetized with isoflurane/Oxygen and luminescence imaging images were taken using IVIS® Lumina K (Perkin Elmer). The intensity of the luminescence signal was quantified as photon counts per second (p/s).
  • the pJS-3 vector was prepared by inserting the luciferase gene into pCN60, a commercial vector, in the same manner as the pJS-1 production method ( Bam HI, Hind III restriction enzyme selection).
  • Transformants into which pJS-3 was introduced were prepared in the same manner as in Example 2.
  • Transformants into which pJS-4 was introduced were prepared in the same manner as in Example 2.
  • the pJS-5 vector was prepared by inserting the luciferase gene into pRMC2, a commercial vector, in the same manner as the pJS-1 production method ( Bam HI, Xba I restriction enzyme selection).
  • Transformants into which pJS-5 was introduced were prepared in the same manner as in Example 2.
  • Example 4 Infectious animal model (infectious respiratory disease model) production
  • Transformed B. bronchiseptica , A. baumannii , and P. aeruginosa strains are shown in FIG. 17 to confirm the stable luminescence 2 hours after strain infection in an animal model infected, and the transformed B. bronchiseptica , A. baumannii, and The result of confirming the luminescence of the lung isolated from the animal model infected with the P. aeruginosa strain is shown in FIG. 18 .
  • the reaction was performed by diluting each antibiotic and bacterial solution by concentration.
  • antibiotics were intragastricly administered to the infected animal models prepared in Example 3, and changes in the in vivo drug over time were confirmed.
  • Example 3 the experiment was performed using the infected animal model prepared in Example 3 above.
  • the antibiotic clarithromycin was administered to an animal model of pneumonia by intranasal administration.
  • images were taken using the IVIS imaging system (PerkinElmer) after anesthesia with isoflurane/oxygen. The intensity of the luminescence signal was quantified as photon counts per second (p/s).
  • Figure 12 shows the result of confirming the significantly superior expression level of the pJS-1 recombinant vector (Example 1) compared to the pJS-2 recombinant vector (Comparative Example 1) on the E-tube.
  • 12 (a) is a negative control
  • (b) to (e) are pJS-2, pJS-3, pJS-4 and pJS-5 recombinant vectors
  • (f) is a pJS-1 recombinant vector indicate
  • the pJS-1 recombinant vector prepared according to the present invention exhibited significantly higher luminescent efficiency compared to other recombinant vectors.
  • the transformed bacteria according to the present invention can maximize the expression (luminescence) of the target gene without affecting the basic characteristics or functionality of the pathogen, such as interference. It was confirmed that the understanding of and the evaluation of the efficacy and stability of treatment candidates are possible in real time and noninvasively.

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Abstract

La présente invention concerne un transformant dans lequel est introduit un gène de la luciférase, un modèle animal infecté par ce transformant, ainsi qu'un procédé de criblage d'un médicament utilisant ce transformant. Un vecteur recombiné, un transformant de celui-ci et un modèle animal infecté par le transformant, selon la présente invention, peuvent être utilisés efficacement dans le criblage de médicaments et autres en ce sens qu'ils peuvent fournir une évaluation polyvalente in vitro et in vivo de l'efficacité de nouveaux médicaments candidats pour le traitement de maladies infectieuses, d'une manière non invasive et en temps réel.
PCT/KR2023/001859 2022-02-09 2023-02-08 Modèle de maladie infectieuse et procédé de criblage de médicament l'utilisant WO2023153805A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5229285A (en) * 1991-06-27 1993-07-20 Kikkoman Corporation Thermostable luciferase of firefly, thermostable luciferase gene of firefly, novel recombinant dna, and process for the preparation of thermostable luciferase of firefly
US20030108483A1 (en) * 1994-07-01 2003-06-12 The Board Of Trustees Of The Leland Stanford Junior University Non-invasive localization of a light-emitting conjugate in a mammal
CN102876702A (zh) * 2012-10-15 2013-01-16 中国科学院微生物研究所 一种具有广泛宿主的穿梭表达载体
US20200063146A1 (en) * 2014-06-18 2020-02-27 Calysta, Inc. Nucleic acids and vectors for use with methanotrophic bacteria
KR20210034743A (ko) * 2019-09-20 2021-03-31 성균관대학교산학협력단 변형된 시아노박테리아 유전자 발현 시스템

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5229285A (en) * 1991-06-27 1993-07-20 Kikkoman Corporation Thermostable luciferase of firefly, thermostable luciferase gene of firefly, novel recombinant dna, and process for the preparation of thermostable luciferase of firefly
US20030108483A1 (en) * 1994-07-01 2003-06-12 The Board Of Trustees Of The Leland Stanford Junior University Non-invasive localization of a light-emitting conjugate in a mammal
CN102876702A (zh) * 2012-10-15 2013-01-16 中国科学院微生物研究所 一种具有广泛宿主的穿梭表达载体
US20200063146A1 (en) * 2014-06-18 2020-02-27 Calysta, Inc. Nucleic acids and vectors for use with methanotrophic bacteria
KR20210034743A (ko) * 2019-09-20 2021-03-31 성균관대학교산학협력단 변형된 시아노박테리아 유전자 발현 시스템

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