TW201028163A - Detection and therapy of bacterial infection caused by Enterobacteriaceae - Google Patents

Abstract

The present invention provides a method for the treatment and/or prevention of bacterial infection caused by Enterobacteriaceae in central nervous system and/or peripheral blood circulation in a mammal by administering effective amount of outer membrane protein A (OmpA) or its derivatives to a mammal. Also provided are a method for vaccinating a mammal to produce an antibody against bacterial infection caused by Enterobacteriaceae in central nervous system and/or peripheral blood circulation and a method of detecting or diagnosing bacterial infections caused by Enterobacteriaceae in central nervous system and/or peripheral blood circulation in a mammal. An antibody and a kit on the basis of the vaccinating method and detecting/diagnosing method are also provided.

Description

201028163 VI. Description of the Invention: [Technical Field] The present invention relates to a method for treating and/or preventing and/or diagnosing a bacterial infection of the central nervous system and/or peripheral blood circulation caused by Enterobacteriaceae in a mammal, It is administered to a mammal by administering an effective amount of outer membrane protein A or a derivative thereof. Based on this method, the invention further provides antibodies against bacterial infections and reagents and/or kits for diagnosing and/or treating bacterial infections. [Prior Art] The body of the f-zone (CNS) is well protected against infection. The spine and brain are coated in a hard, protective membrane. The outermost membrane, the dura mater, and the underlying membrane, the arachnoid membrane, completely encircle the brain and ridge. These protections are not absolute. In some cases, the bacteria are close to the area within the CNS. Bacterial infections can be purulent infections (such as meningitis, cerebral abdominal swelling, subdural abscess and epidural abscess), tuberculosis, neurosyphilis or leprosy. Typically, bacterial invasion is caused by the spread of adjacent infections; for example, chronic sinusitis or middle ear infections can expand away from their starting position. Bacteria can also be passed to the CNS by blood flow from the distal infection site. In rare cases, bacteria can be introduced directly into the CNS during head trauma or surgery. However, sources of infection are often not identified. The purpose of treatment for bacterial infections is to stop the infection, alleviate the symptoms, prevent complications and, if necessary, maintain life. A two-track approach was adopted to treat bacterial infections. First, antibiotics were used; and Taiwanese treatments were confronted—(iv) possible infections. The second phase involves surgery to clear the position of the money. Once the bacteria are used, the drug treatment can be changed to a specific antibiotic. However, surgery is not a good choice in some situations, such as a large number of infections or infections that are inaccessible to the brain. Outer membrane protein A (OmpA) was originally described by Henning and colleagues in 1975. It has 325 amino acid residues and exhibits heat-modifiable electrophoretic movement in SDS-PAGE. . The N-terminal domain of OmpA consists of 177 amino acids and traverses the outer membrane 8 times. OmpA is involved in the maintenance of bacterial shape as a phage receptor and F-mediated conjugation, and has limited pore formation properties. OmpA enhances LPS uptake into macrophages and has been reported to be associated with the invasion of the sacral nervous system in E. coli. WO 9201001 provides a method for producing purely selected outer membrane proteins and provides a method for renaturation thereof to obtain a biologically or immunologically active antigenic epitope which can be Animals are induced to produce antibodies. Pascale Jeannin et al. reported that outer membrane protein A (OmpA) is a highly retained protein in the evolution of Enterobacteriaceae and OmpA is a novel pathogen-associated molecular pattern (PAMP). Inducing CTL as a carrier in infectious and therapeutic anti-tumor vaccines (Vaccine, Volume 20, Supplement 4, 19

December 2002, pages A23-A27) 0 However, there are no reports of new uses of outer membrane protein A and its derivatives for the treatment and/or prevention and/or diagnosis of sacral nervous system and/or peripheral blood circulation bacterial infections. . SUMMARY OF THE INVENTION 144249.doc 201028163 The purpose of this month is to provide an enterobacteriaceae (Enterobacteriaceae) outer membrane protein A (OmpA) for the treatment and/or prevention of the central nervous system caused by Enterobacteriaceae in mammals and / Or the use of bacterial infections in the peripheral blood circulation. Another object of the present invention is to provide an enterobacteriaceae outer membrane protein A (OmpA) for use in immunizing a mammal to produce an antibody against a bacterial infection of the central nervous system and/or peripheral blood circulation caused by Enterobacteriaceae. Another object of the present invention is to provide an isolated antibody obtained from the immunization method of the present invention and a therapeutic or diagnostic agent comprising the antibody of the present invention. A further object of the present invention is to provide a method for detecting or diagnosing a bacterial infection of the middle sacral nervous system and/or peripheral blood circulation caused by Enterobacteriaceae in a mammal, comprising coating a first specific anti-〇 mpA antibody a substrate surface that immunospecifically binds to 〇mpA molecules in the blood or OmpA on the bacterial cell membrane; a sample taken from the peripheral blood circulation and/or the central nervous system is added to the matrix; a second label is added Anti-OmpA antibody; and detecting binding of an anti-〇mpA antibody to OmpA on the mpA molecule or bacterial cell membrane, wherein the binding result indicates that the mammalian central nervous system and/or peripheral blood circulation is subjected to bacterial infection, and OmpA is from the family Enterobacteriaceae. A further object of the present invention is to provide a kit for diagnosing bacterial infections of the central nervous system and/or peripheral blood circulation caused by Enterobacteriaceae bacteria. [Embodiment] The present invention unexpectedly finds that outer membrane protein A (OmpA) can be used for the treatment and/or prevention and/or diagnosis of bacteria in the central nervous system and/or peripheral blood circulation 144249.doc -6- 201028163 infection. In addition, antibodies that bind to 0mpA have been developed to analyze the amount of OmpA in biological samples and to detect or diagnose bacterial infections in the central nervous system and/or peripheral blood. Such antibodies can also be used to rapidly detect whether bacterial infections (such as sepsis) are caused by Enterobacteriaceae, allowing physicians to quickly and correctly select therapeutic antibiotics to reduce mortality and improve prognosis. The present invention provides a method for treating and/or preventing a bacterial infection of the central nervous system and/or peripheral blood circulation caused by Enterobacteriaceae in a mammal, comprising administering an effective amount of Enterobacteriaceae outer membrane protein A or Derivatives to the mammal ^ In other words, the present invention provides an enterobacteriaceae outer membrane protein A or a derivative thereof for treating and/or preventing a central nervous system and/or peripheral blood circulation bacterial infection caused by Enterobacteriaceae in a mammal Use. As used herein, "central nervous system" means a portion of the nervous system consisting of the brain and the spinal cord. "Peripheral blood circulation" means blood in the systemic circulation. As used herein, "bacterial infection" means φ infection caused by Gram-negative bacteria. Preferably, the bacterial infection is caused by Enterobacteriaceae or other Gram-negative bacteria. More preferably, the 5 mycelial infection is caused by Shigella (from ^, Salmonella * (Sa丨monella), Klebsiella, Escherichia (heart, Citrobacter) (c), enterobacteria (five or Serratia genus such as. Caused by the best, S Haicheng infection by the large intestine function (five, Klebsiella pneumoniae (feet pneumoniae)' Salmonella typhimurium (iSiz/wowe/Za 7yp/n·) or aerobacteria (five 144249.doc 201028163). As used herein, "outer membrane protein A (OmpA)" means any derived from Gram. Negative bacteria OmpA and any recombinant OmpA. OmpA is a structural protein rich in the outer membrane of Gram-negative bacteria. The "OmpA derivative" means a protein derived from OmpA. For example, it has the same function as OmpA. The OmpA recombinant is an OmpA derivative. Preferably, OmpA or a derivative thereof is obtained from Enterobacteriaceae or other Gram-negative bacteria. Outer membrane protein A (OmpA) is a class which is highly retained in the evolution of Enterobacteriaceae. Protein (Vaccine, Volume 20, Supplement 4, 19

December 2002, pages A23-A27), therefore, the skilled person agrees that OmpA from various Enterobacteriaceae bacteria has the same function. Nguyen TN et al. found that OmpA of 'Altobacillus, Klebsiella pneumoniae, Salmonella typhimurium or Aerobacter aerogenes was highly similar after sequence alignment (Gene 1998 210: 93). In a specific embodiment of the invention, OmpA or a derivative thereof is obtained from an Enterobacteriaceae bacterium. Preferably, the Enterobacteriaceae bacterium is selected from the group consisting of Escherichia (Fructus / ΓΖϋβΓΖϋ / π'α), Shigella, Salmonella (Sa/mowe/Za), gram Rhodobacter, Citrobacter, Ewieroftacior or Serraiia. Optimally, the Enterobacteriaceae bacterium is selected from the group consisting of Escherichia coli (five κΛβγζ'ο/π'ύ! co/z·), Shigella sojae (iS/2z'ge//a s(10) Ez·), Shigella flexneri//exwerz·), Salmonella typhimurium, group A Salmonella group A), group B Salmonella (Sa/wowe/Za group B), group D Salmonella 144249.doc 201028163 Group D), Citrobacter freundii/rw»Shan·ζ·), C. sinensis (C/iroZ^c/er Aroserz·), Klebsiella pneumoniae/mew/wom'ae), Klebsiella oxytosus (ash 7eh/e//a 〇j£:_yic>ca), EwierMacier aerogewe·?, Enterobacter cloacae (_£«iero6izcier • c'oac<3e) , Serratia marcescens ("Serra/^a wareescew·?"). More preferably, the bacterium of the genus Enterobacter is Escherichia coli (£^c/zerz'c/zz_a co/〇, Klebsiella pneumoniae (especially / eh / / α / 7 like please <?« / (mail), Salmonella typhimurium ❹ (Heart 'speak (10) pan " 0 noisy /? 〇 or aerogenic bacillus (_£Wer<90<3cier 〇 optimally, Enterobacteriaceae bacteria for E. coli (any yc /^Wc/„.ae〇//) or Klebsiella pneumoniae (_/^7eZ?j/e//ap/2g2//72c>w/ae). According to one embodiment of the present invention Recombinant E·c〇u 〇mpA can be effectively treated by Escherichia coli (£^c/;erk;H'a co/z·), Khbsiella P"eumoriiae" Salmonella typhimurium An infection caused by (SaIm〇neUa?>ρ/π·) or aerogenic bacillus (five „ierc^cier敝〇#胆). According to the present invention, the therapeutically effective amount of 〇mpA or a derivative thereof of the present invention It can be administered to mammals (including human or non-human mammals) suffering from bacterial infections of the central nervous system and/or peripheral blood circulation system. According to the present invention, the administration of OmpA or its derivatives of the present invention can be carried out by various general parties. get on. Administration forms suitable for oral administration are those which act according to the state of the prior invention and which deliver the 〇mpA or its derivative 'for example' a lozenge (uncoated or coated lozenge, for example against gastric acid) in a rapid and/or modified manner. Or a slow-dissolving or insoluble coating or a coating that controls the release of the compound of the invention), a rapid tablet in the h's or a membranous drug/flake, a membranous drug/bed dry tablet, a capsule (eg a hard or soft capsule) , sugar-coated keys, granules, pills, powder 144249.doc 201028163 End, ritual, suspension, aerosol or solution. Non-witch remedies include intravenous drip or perfusion, subcutaneous injection, intraperitoneal, injection or intramuscular Main injection, pulmonary administration (eg by inhalation or insufflation) or intraspinal or intraventricular administration. Suitable for parenteral administration is solution, u liquid n #丨,; Donggan agent or _ powder Forms of injection and perfusion preparations. "Oral or parenteral administration is preferred, especially oral and intravenous administration. Particularly preferred are intravenous dosage forms, for example for the treatment of acute central nervous system infections. 0mpA according to the invention or Its derivatives can be transferred Suitable pharmaceutical compositions can be achieved in a known manner by mixing inert, non-toxic, pharmaceutically suitable excipients, including such carriers (eg, microcrystalline cellulose, lactose, mannitol) ), a solvent (such as liquid polyethylene glycol), an emulsion and a dispersing agent or a wetting agent (for example, s(dium) sulphate (8) sulfate, P〇lyoxysorbitan 〇leate), a binder (for example, polyethylene) Ketones, synthetic and natural polymers (such as albumin), stabilizers (such as antioxidants such as ascorbic acid), colorants (such as inorganic pigments such as iron oxide), and masking agents and/or fragrances. The dosage form of the present invention includes 〇. %% to 〇% by weight of 〇mPA or a derivative thereof, preferably 〇.5% to 5% by weight of 〇_ or a derivative thereof . The time of treatment with the pharmaceutical composition of the present invention is determined based on the severity of the condition to be treated and the condition of the individual patient. The physician should decide on a sufficient time to treat with the pharmaceutical composition of the invention. The present invention also provides a method for immunizing a mammal to produce an antibody against a bacterial infection of the central nervous system 144249.doc •10·201028163 and/or peripheral blood circulation, which comprises an outer membrane protein A or a biologically active substance thereof; To the mammal. In other words, the present invention provides the use of an antibody having an outer membrane protein A or a derivative thereof for immunizing a mammal to produce a bacterial infection against the central nervous system and/or peripheral blood circulation. Based on the discovery that 〇mpA can treat and/or prevent and/or diagnose • bacterial infections of the central nervous system and/or peripheral blood circulation, OmpA is used for introduction into animals for immunization. The present inventors have found that 〇mpA can induce the production of specific antibodies. The present invention also provides an isolated antibody obtained by the above method, which specifically binds OmpA. In a specific embodiment, the antibody is a monoclonal antibody, a plurality of antibodies or a synthetic antibody or an antibody fragment thereof. Preferably, the antibody is an anti-〇ηιρΑ IgY antibody. More preferably, the antibody is a chicken anti-〇mpA IgY antibody. In another preferred embodiment, the antibody of the present invention can treat and/or prevent and/or detect (diagnose) the central nervous system caused by Enterobacteriaceae bacteria. And / or peripheral blood circulation bacterial infection. Preferably, the Enterobacteriaceae bacterium is selected from the group consisting of the following species: Escherichia, Shigella, Salmonella, Klebsiella (feet/eh/e) /Za), Phytophthora, Enterobacter (five "ierofeacior" or Serrim'a). Most preferably, the Enterobacteriaceae bacterium is selected from the group consisting of Escherichia coli (five sc/jer/c/n'a co/z·), Shigella sojae, Shigella flexneri (57n> e//a //exweW), Salmonella typhimurium (5^/ 〇 / / / « 7 ^ /? / π ·), group A Salmonella group A), group B Salmonella (Sa / moneZ / ci group B), group D Salmonella (*Sa/wo«e//a group D), Citrobacter freundii (C7iro0acier 144249.doc -11 - 201028163 freundii) ' ^ {Citrobacter koseri) ' ^ ^ Klebsiella (feet, Klebsiella pneumoniae (Α7βΖ)·5ζ·β//α ox_yioca), aerobacteria (five “ierofeacier aerogewe·?”, Enterobacter cloacae c/oacae), Serratia marcescens (•Serrai/ a marcescews). More preferably, the Enterobacteriaceae bacterium is Escherichia coli (five jcAerz'c/na co/z·), Klebsiella pneumoniae (feet/βί&//α pneumoniae) 'r}' H ^ ® {Salmonella Typhi ) ^ Bacillus (five aeroge / iej). Most preferably, the Enterobacteriaceae bacterium is Escherichia coli (five co/f) or Klebsiella pneumoniae (feet/ewe//a pneumoniae). The invention also provides a therapeutic comprising an antibody of the invention. Antibodies of the invention can be produced by methods known in the art. Multiple antibodies and monoclonal antibodies against mp A can be produced by various processes known in the art. For example, OmpA can be administered to a variety of host animals (including, but not limited to, chickens, rabbits, mice, rats, etc.) to induce production of polyclonal antibodies comprising OmpA specificity. Preferably, the multi-strain antibody is an anti-OmpA IgY antibody. Preferably, the multi-strain antibody is a chicken anti-OmpA IgY antibody. Depending on the host species, different adjuvants may be used to increase the immune response; such adjuvants include, but are not limited to, Freund's adjuvant (complete and incomplete), mineral gels (such as aluminum hydroxide) ), interface active substances (such as lysolecithin, pluronic p〇iy〇ls, polyanions), peptides, oil emulsions, hemocyanin (keyhole limpet hemocyanin), dinitrophenol and possibly Human adjuvants (such as BCG (bacilli Calmette-Guerin)) and short rods @1 (Cor_y«e& acier/wm. These 144249.doc -12-201028163 adjuvants are known in the art. Antibodies can be used to analyze the presence of OmpA or the amount of OmpA in a biological sample, using serological and immunohistological methods as described herein or using methods known in the art. Other antibody-based methods for detecting protein gene expression Including immunoassays, such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). Suitable antibody assay tags are techniques Known and include enzyme labels such as glucose oxidase, radioisotopes (such as iodine (1251, 1311), carbon (14C), sulfur (35S), strontium (3H), indium (1211η) and chromium (99Tc)), Cold light labels (such as luminol) and fluorescent labels (such as fluorescein and rhodamine) and biotin. The antibodies or therapeutic agents of the invention may be administered in any conventional or desired manner, for example, orally or parenterally. In general, the pharmaceutical compositions can be administered parenterally; i.e., subcutaneously, intramuscularly or intravenously. For such administration, the carrier is preferably an aqueous carrier. A variety of aqueous carriers can be used, such as water, buffered φ solution, 0.4% physiological saline, 0.3% glycerol, and the like. These solutions are sterile and generally free of specific materials. The reagent may contain pharmaceutically acceptable adjuvants suitable for physiological conditions, such as pH adjustment and buffering agents, tonicity adjusting agents, and the like, for example, sodium acetate, gasification, chlorination, gas Huama, lactate and so on. The concentration of the antibody in such formulations can vary widely, such as 'from less than 0.5% (typically 1%) up to 15 or 20% by weight, and can be selected based on liquid volume, viscosity, etc.; preferably based on the particular mode of administration . The invention also provides a diagnostic agent comprising an antibody of the invention. The present invention - 13-144249.doc 201028163 A diagnostic or therapeutic agent can diagnose or treat a mid-framed nervous system and/or peripheral blood circulation bacterial infection caused by Enterobacteriaceae bacteria. The present invention also provides a kit for collecting central nervous system and/or peripheral blood circulation bacterial infection caused by bacteria of the bacterium, comprising the antibody of the present invention, a reagent suitable for constituting a medium for performing 〇mpA•antibody reaction, and An agent that allows detection of the complex formed. The invention further provides a method of detecting or diagnosing a bacterial infection in the central nervous system and/or peripheral blood circulation of a mammal. In a specific embodiment, the detecting or diagnosing comprises: coating - the first specific anti-OmpA antibody on a substrate surface (such as an ELISA disk or a magnetic nanoparticle), the antibody can be immunologically specific to the 〇mpA molecule in the blood Or binding on the bacterial cell membrane. Adding a sample taken from the peripheral blood circulation and/or the central nervous system to the matrix; adding a labeled second anti-〇 mpA antibody; and detecting the anti-〇 mpA antibody and the OmpA molecule or bacterial cell membrane A combination of 〇mpA, wherein the binding result indicates that the mammalian central nervous system and/or peripheral blood circulation is subjected to a bacterial infection. The first specific anti-OmpA anti-system according to the present invention is applied to the surface of the substrate using methods known in the art and commercial coating buffers, and any suitable substrate can be used in the method of the present invention. The substrate is preferably an ELIS a disk or a magnetic nanoparticle. The anti-〇 mpA antibody specifically binds to 〇ιηρΑ molecules in the blood or 〇mpA on the bacterial membrane. To allow binding to be detected, the anti-〇mpA antibody specific for binding to the OmpA molecule is detected by using a labeled second anti-OmpA antibody. According to the invention, the term "tag" is a molecule or group having detectable properties or properties. A label can be directly detected 144249.doc 201028163 (such as radioisotopes, fluorophores, or chemical luminescent substances (Chemilumiph〇res); or a label can be detected directly (such as hapten or poly Nucleotides). When not directly labeled for detection or signal purposes, they are used to bind to a signal body complex. The "signal body" is a molecule or group that provides detectable properties or properties. Can be direct • ground (such as colloidal particles (such as colloidal gold or selenium)); or may be not directly (such as enzymes (such as alkaline phosphatase, β-galactosidase or horseradish peroxidase)). Direct _ 汛 体 may require additional components known in the art (such as a substrate). 忒 "Signal complex" includes a signal body that binds to a specific binding partner (such as an antibody or polynucleotide). These combinations may be prepared according to any known binding method. According to the present invention, the kit of the present invention preferably includes a reagent for carrying out the method of the present invention, a container for carrying out the reaction, a substance for identifying the reaction product, instructions for use, and a package. The reagent may include a washing buffer, a reaction mixture, an enzyme, and a label. The kit may include a group of the above-listed components as needed, but includes the guanidine antibody of the present invention. The kit also includes a medium constituting the OmpA-antibody reaction. Reagents required and reagents permitting detection of the complex formed. According to the present invention, binding of an anti-〇mpA antibody to peripheral blood or an Omp A knife in the central nervous system can be used to detect the presence of a 〇mpa molecule, such as the OmPA molecule. Exist in the peripheral blood or central nervous system of a body, which means that the individual is infected with bacteria with 〇mp A molecules, such as Enterobacteriaceae. Infections caused by Enterobacteriaceae can be detected quickly and correctly, even in one day. Therefore, the physician can quickly and correctly use antibiotics to treat bacterial infections caused by Enterobacteriaceae (such as sepsis) 144249.doc -15- 201028163 to reduce the mortality rate and improve the prognosis. Example 1 Adhesion And Adhesion and Invasion Assay for C6 glioma cell-associated bacteria, fusion cells The layers were cultured at 37 ° C for a set time interval with the bacterial strains 5.co/ί E44, E91 and MG1655. V·E44 is the K1 strain 5..〇"118218(018:1<:1:117 And it is derived from the cerebrospinal fluid of infants with meningitis. £. co" E91 is a mutant strain derived from strain E44 and lacking the complete OmpA gene. 5.co/ί MG1655 is a non-pathological strain and is not a blood-brain barrier Invasive, it was used as a control group. In the adhesion analysis, C6 glioma cells (MOI (bacteria-to-cell) = 10) were infected with the above bacterial strain at set time intervals. The C6 glioma cell monolayer was then washed 3 times with the culture medium and lysed with 0.5% Triton X-100. The released bacteria were counted by applying to a sheep blood agar plate. As shown by the results of the adhesion analysis (Fig. 1), the number of five. co/z·E44 was greatly increased with time. However, the number of five co/ί E91 and MG1655 is less than five.co" E44. In the invasive assay (gentamicin protection assay), for the intracellular bacterial study, the glioma C6 fusion cell monolayer (MOI (bacteria-to) was infected at the time interval set by the above bacterial strain at 37 °C. -cell)=10). The C6 glioma cell monolayer was washed 3 times with medium and further cultured for 2 hours in a medium containing gentamicin (100 pg/ml) to kill extracellular bacteria. The monolayer was washed again 3 times and the cells were lysed with 0.5% Triton X-100. The released bacteria were applied to the sheep blood lipid agar plate by 144249.doc -16- 201028163. As shown by the invasive analysis in Figure 2, the number of co/ί E44 is much larger than the number of five.co/ί Ε91 and MG1655. In the invasive inhibition assay, C6 stromal cell monolayers were infected for 2 hours with the simultaneous addition of 4 pg or 40 pg of OmpA and penta·co//Ε44. The cell monolayer was then washed 3 times with medium and further cultured for 2 hours in a medium containing gentamicin (100 pg/ml) to kill extracellular bacteria. The released bacteria were counted by applying to a sheep blood agar plate. As shown in Figure 3, brother E44 and

The mixture of OmpA does inhibit the invasion of E44. OmpA inhibits the attack of 5.α/ζ· E44 greater than 55% (4 pg) and 80% (40 pg). Example 2 Animal experiments C57BL/6 mice were obtained from the National Laboratory Animal Center and kept in a pathogen free state. The animal's treatment procedures are based on the approved trial plan of the Taipei Medical University. C57BL/6 mice, 8 to 12 weeks old, were randomly divided into arrays, each containing 5 to 10 mice. The mice were anesthetized with intraperitoneal injection of barbituric sodium salt (5 Omg/kg), followed by intracerebral injection of 5χ1055.co"strain E44, E91 or MG1655 in 20 μΐ PBS or 5 pg LPS in 20 μΐ PBS The brain of each mouse. 20 μΐ PBS was used as the control group. 5. Co-quot; Survival of C57BL/6 mice continued to be evaluated until 8 days after administration. As shown in Figure 4, all mice infected with 5.6 co/z·Ε44 died after 2 days of administration. . To investigate the role of recombinant OmpA in the survival of C57BL/6 mice administered intracranial sputum 44, anesthetize with intraperitoneal injection of barbituric sodium salt (50 mg/kg) 8-12 144249.doc •17· 201028163 C5 7BL/6 mice. The brain of each mouse was then infected by intracerebral injection of 5xl055.co/f strain E44 in 20 μΐ PBS with or without premixed 8 pg or 20 pg of recombinant pentaco.co/z·OmpA. 30 μΐ PBS was used as the control group. The survival of the five co-quot; strains infected with C57BL/6 mice continued to be evaluated until 8 days after administration. Figure 5 shows that OmpA prolongs the survival of mice injected with pentaco" E44 in the brain. The mice treated with 8 pg OmpA·5· co" E44 and the 5 cog/E44 containing 20 pg OmpA had a survival rate of about 40% and 80%, respectively. The above mouse experiments were carried out on infections caused by Klebsiella pneumoniae or Salmonella typhimurium, and the experimental conditions and procedures used were used in addition to only 20 pg of recombinant pentaco/i OmpA. The experiment of the above five. co/z· is the same. Figures 6 and 7 show that OmpA prolongs the survival of C57BL/6 mice infected with Klebsiella pneumoniae or Salmonella typhimurium. When mice infected with Klebsiella pneumoniae were treated with 20 pg of co/z·OmpA, about 80% of the mice survived. Similarly, when mice infected with Salmonella typhimurium were treated with 20 pg of 5.OmpA, the survival of the mice was significantly prolonged. Example 3 Preparation and Binding Analysis of Chicken Anti-OmpA Antibody of the Invention Selection of OmpA Gene Fragments To amplify various ompA gene fragments, primers were designed based on the published nucleotide sequence of the E. coli genome (Johns Hopkins University, USA). Polymerase chain reaction (PCR) conditions include the following: 95 ° C, 5 minutes; 94 ° C, 30 seconds, 30 cycles; 58 ° C, 30 seconds; . , 90 seconds; then 72 ° C, 10 minutes. The amplified fragment was purified and treated with Sac I and Xho I (Bio-Labs, 144249. doc -18-201028163 USA), respectively, and ligated to the ρΕΤ-2 1 expression vector. Recombinant plastid DNA was transformed into a competent five.co/z·BL-21 by heat shock. Screened with a selective LB agar extract containing 50 gg/ml ampicillin. Next, randomly selected plastid DNA was isolated, treated with Xho I and Sac I, and analyzed for gene insertion by electrophoresis.

Performance and Purification of His-Fused OmpA Protein The overnight culture of the bacterial culture was diluted 100-fold in LB medium at 37. (: growth for 1.5 to 2 hours until OD6〇q reaches 0.4-0.8. To induce the expression of His-fused OmpA fragment protein, isopropyl-β-D-thiogalactoside (IPTG) was added to the culture to The final concentration was 0.5 mM and further cultured for 4-6 hours at 37° C. After the culture, the cells were collected, and the cell pellet was resuspended in histidine (Hisidine binding buffer) and sonicated. (sonication) breaking the cells. After centrifugation, the total protein containing His-fusion protein was mixed with 50% Ni Sepharose High Performance solution (Amersham Biosciences, Sweden) and incubated at room temperature for 0.5 to 1 hour. The mixture was centrifuged at 500 g 5 Minutes, and wash the pellet with 5 volumes of His Wash Buffer for 5 minutes. Repeat at least 2 wash steps to increase the purity of His fusion protein. Finally, add His rushing buffer and incubate for 15-20 minutes. Under 500 g The supernatant was collected by centrifugation for 5 minutes. Immunization with purified His-fused OmpA protein was used to immunize the mature white Leghorn hen 4 times using the antigen mixture at intervals of 7 days. The preparation of the first immunizing antigen was mixed at 500 μΐ. Phosphate buffer 50 pg purified His-fusion protein in water and an equal volume of Freund's adjuvant. For subsequent immunization, anti-144249.doc -19- 201028163 original and incomplete Freund's adjuvant (Freund's Admixture. ° Eggs from pre-immune and post-immunization hens were collected and stored at 4 ° C. Purification of total IgY antibodies in eggs The egg yolk was separated from the protein and diluted with 4 volumes of TBS. At 25 ° C ' Centrifuge at 2000-3000 g for 20 minutes, add 120 μM dextran sulfate solution per ml of supernatant, mix well and incubate at 25 ° C for 30 minutes, then add 50 μM calcium chloride per ml of supernatant and proceed The above procedure was carried out. The mixture was centrifuged at 2000-3000 g for 20 minutes at 25 ° C, and the supernatant volume was adjusted to 100 ml with TBS. The solution was stirred while slowly adding 20 g of anhydrous sodium sulfate until completely dissolved, and then at 25 The solution was allowed to stand for 30 minutes at ° C. After centrifugation as described above, the precipitate was redissolved in 10 ml of TBS and centrifuged again to collect the supernatant. The total IgY antibody was repeatedly precipitated by slowly adding 8 ml of a 36% sodium sulfate solution. Redissolved in 5 ml TBS containing 0.05% sodium azide and stored at 4 ° C. Western blots will be obtained from wild-type and mutant E. coli strains of purified IgY antibodies, different His-fused OmpA proteins and total cell lysis The split was subjected to SDS-PAGE and transferred to a nitrocellulose membrane. The nitrocellulose membrane containing the protein was blocked with 5% skim milk. A 1:5,000 dilution of chrome mouse anti-His antibody (Amersham Biosciences, UK) and the resulting anti-OmpA IgY antibody (1: 10,000) were added as primary antibodies at room temperature for 1 hour. The membrane was washed 3 times with PBST (containing 0.05% T-20) and incubated with HRP-conjugated sputum anti-chicken IgY Ab (1: 10,000 dilution) (Jackson ImmunoResearch, USA) for 1 hour at room temperature. After 3 washes, the protein band was spread with DAB until the desired intensity of 144249.doc -20- 201028163 was reached. In Fig. 8, (A) is SDS-PAGE, showing that purified OmpA and (B) are Western blots, showing that the anti-OmpA IgY antibody specifically binds to OmpA. In Fig. 9, (A) is SDS-PAGE, showing purified OmpA, and (B) to (F) are Western blots, showing that OmpA binds to purification from the first, first, second, and third times. And IgY antibodies in eggs produced by the fourth immunized chicken, wherein HRP-conjugated donkey anti-chicken IgY was used as a secondary antibody in Western blot analysis. Enzyme-Link Immunosorbent Assay (ELISA) was coated in a 96-well ELISA plate (Becton Dickinson and company, USA) at 0.5 °C with 0.5 pg of His-fused OmpA protein and BSA at 37 °C. hour. At 37 ° C, at 5 ° /. The skim milk was sealed for 1 hour. Multiple IgY antibodies from chickens from the first to the fourth immunization were diluted and incubated with the coated antigen for 1 hour at 37 °C. The BSA acts as a negative control group. After the incubation, the well plate was washed with PBST at least 3 times. HRP-conjugated donkey-chicken IgY Ab (l: 10,000 dilution) was used as a secondary antibody and incubated for an additional hour at 37 °C. After washing as above, 25 μM of TMB was added to each well to observe binding. Finally, 25 μΐ ' 1 N HCL· was added to terminate color development and the OD (optical density) value at 450 nm was determined by ELISA plate reader. Figure 10 shows the specific binding of multiple anti-OmpA IgY antibodies of different dilutions to His-fused OmpA in ELISA. BSA-containing IgY was used as a non-specific control group. The results indicated that the multi-strain anti-OmpA IgY antibody diluted from 8,000X to 16,000X was the most suitable concentration for binding. Immunochemistry 144249.doc -21- 201028163 A monolayer of C6 glioma cells infected with £.co/z· was immobilized with 4% triformaldehyde. The resulting C6 cell sample was blocked with 1% BSA and incubated with a solution containing chicken anti-OmpA IgY polyclonal antibody (diluted 1:100 and 1:1 〇〇〇). Multiple IgY antibodies from non-immune chickens were used as negative control groups. Finally, samples were incubated with FITC-labeled anti-IgY secondary antibody (diluted 1:500). Slides were fixed in 50% glycerol-PBS and then detected by the TCS SP5 Confocal Spectral Microscope Imaging System (Leica). Figure 11 shows that the anti-OmpA antibody produced binds to E44, whereas IgY from non-immune chickens does not show any binding activity. Example 4 Detection of Enterobacteriaceae bacteria by Western blotting. Co/ί E44, five co/z·E91, clinical £.co/i isolate, sonnei, S. flexneri, Salmonella group A, Salmonella group B ' Salmonella group D ' C. freundii ' C. koseri ' K. oxytoca, E. aerogenes, E. cloacae AS marcescens ^. Centrifuge at 3,500 rpm for 10 minutes and discard the supernatant to collect bacterial proteins. The resulting bacterial protein is appropriately diluted and added with protein loading dye containing beta-mercaptoethanol. The protein of *14 was separated by SDS-PAGE electrophoresis. Coomassie blue staining was performed on one of the obtained SDS-PAGE gels. Western ink dots as described in Example 4 were applied to other gels. Figure 12 shows co//, S. sowwei, flexneri, Salmonella group A, Salmonella group B, Salmonella group D 'C. freundii ' C. koseri ~ K. oxytoca ' E. aerogenes, E. cloacae A S. marcescens H ^ £. coli 144249.doc -22· 201028163

The anti-OmpA IgY antibody of Example 4 obtained by OmpA immunization was detected. (Genus: , "S1. iSowwez. (Genus: < S7n'ge//a) and flexneri (Genus: Shigella) are representative species of the Escherichieae family; <5^// »〇状//&&;3 group A (Genus: Salmonella) ' Salmonella group B (Genus: Salmonella) 3l Salmonella group D (Genus: like //<a) is a Salmonella family ("Sa/;wo«e/ Representative species of /eae); C (Genus: and C. bierz. (Genus: representative strain of Cz'iroZmciereae; and especially /meMwow/ae (Genus: A7e6sie//a) ) (see the results of Example 2), · oxytoca (Genus: Klebsiella) ' E. aerogenes, (Genus: Enterobacter) ' E. cloacae (Genus: Enterobacter) S^ S. warceae (Genus: Serra/z'a ) is a representative species of Klebsiella (the representative species of the ruler, so the detection of the above bacteria supports the infection of Enterobacteriaceae bacteria can be detected by anti-Omp A antibody and can be treated by OmpA, due to Escherichia, Salmonella Family (<Sa/wi?«e//efle), Cz7ro6aciereae and Klebsiella are the most relevant representatives of Enterobacteriaceae and clinical infections. Example 5 of the present invention Invasive inhibition assay of Omp A IgY antibody The C6 glioma cell monolayer was infected with the above bacterial strain (MOI=10) at a set time interval at 37 ° C. The monolayer was then washed 3 times with medium and further containing Qingda The medium of gentamicin (100 pg/ml) was cultured for 2 hours to kill extracellular bacteria. The monolayer was washed again 3 times and lysed with 0.5% Triton X-100. The released bacteria were coated on sheep. Blood agar 144249.doc • 23· 201028163 Shear blood agar plate count. Invasive analysis of chicken OmpA IgY antibody was performed according to the materials and procedures described in Example 1. The blank group used for this analysis was without any C6 cells of the reagent (see MOCK of Figure 13). The two positive control groups used in the analysis were (1) infected C6 cells with 5.4 co/z·E44 (see E44 of Figure 13) and (2) .co/ί E44 simultaneously added OmpA to infect C6 cells (see E44+OmpA in Figure 13). The four negative control groups used in the analysis were (1) C6 cells were infected with enolase at the same time as E.E44 (see E44+enolase in Figure 13); (2) OmpA to C6 cells were added (see Figure 13 for OmpA); (3) Adding enolase to C6 cells (see enolase of Figure 13); and (4) adding anti-OmpA IgY antibody to C6 cells (see aOmpA Ab of Figure 13). The experimental group in this analysis was to infect C6 cells with the addition of anti-OmpA IgY antibody at five co-quot; E44 (see E44+aOmpA Ab of Figure 13). Next, immunoblotting analysis was carried out in which β-actin was used as a control group. GFAP is an important protein found in brain stellate cells such as C6 cells. When £6?// infected C6 cells, GFAP performance increased. Figure 13 shows that OmpA IgY antibody specifically inhibits the expression of GFAP caused by invasion of Co., E44. In particular, in the E44+aOmpA Ab group, GFAP performance was significantly reduced. [Simple description of the diagram] Figure 1 shows the time course of the attachment of the strain:. Figure 2 shows the time course of co"strain invasion. Figure 3 shows that OmpA inhibits the invasion of C6 glioma cells by penta-co/ί E44. Figure 4 shows the survival rate of C57BL/6 mice injected with the £. co// strain in the brain. Figure 5 shows that OmpA prolongs the survival of C57BL/6 mice injected with co/z·E44 in the brain 144249.doc •24· 201028163. Figure 6 shows that OmpA prolongs the survival of brain-injected C57BL/6 mice. Figure 7 shows that OmpA prolongs the survival of mice injected with brain. Figure 8 shows OmpA protein purified using SDS-PAGE and Western blot analysis. Figure A shows SDS-PAGE; Figure B shows Western blots; Μ marks; and approximately 40 kDa of protein is OmpA ° ❿ Figure 9 shows the binding of multiple anti-OmpA IgY antibodies to OmpA in Western blots. (A): Soma-staining of Coomassie blue staining. (B) to (F): Western blotting, showing that ImpA binds to IgY antibodies in eggs produced from chickens that have been purified from the first, second, third, and fourth immunizations, and then HRP-conjugated donkey-chicken IgY (l: 10,000) was used to detect the humoral response of chickens. Lanes 1 and 2 in (A) to (F) are OmpA protein and BSA. Figure 10 shows the binding of multiple anti-OmpA IgY antibodies to OmpA in ELISA. Figure 11 shows the combination of anti-〇1^8 1 丫 丫 antibody shown in the Confocal Spectral Microscope Imaging System with 5.00. Figure 12 shows the detection of the OmpA protein of the Enterobacteriaceae bacterial strain by Western blotting. The first to the 14th are respectively 5.α/ί E44, five.co/ί E91, clinical five. co"isolated, "S., iS.," S^/mowd/a group A,

Salmonella group B ' Salmonella group D ' C. freundii, C. koseri, K. oxytoca, E. aerogenes, E. cloacae anti S.

The OmpA protein was detected using a plurality of anti-OmpA 144249.doc -25- 201028163 antibodies produced by chickens. Figure 13 shows that OmpA IgY antibody specifically inhibits the invasiveness of c. co/ί E44 and activates C 6 cells. 0 144249.doc 26-

Claims (1)

201028163 VII. Scope of application: 1. An enterobacteriaceae family outer membrane protein A (OmpA) for the treatment and/or prevention of bacteria in the central nervous system and/or peripheral blood circulation caused by Enterobacteriaceae in mammals The purpose of infection. 2. The use according to claim 1, wherein the mammal is a human. 3. According to the use of claim 1, wherein the OmpA is a recombinant OmpA. 4. According to the use of claim 1, wherein the OmpA is derived from Shigella (iS/i/ge/Za), Salmonella, Klebsiella, Escherichia (V., Citrobacter) Is a genus of the genus Enterobacteria or Serratic T. 5. According to the use of claim 1, the OmpA line is obtained from Escherichia coli, Shigella sonnei, Shigella (*S7^ge//a, Salmonella typhimurium (&lt;Sa/mo«e//a 7) φ/π·), Group A Salmonella (Sa/woneZ/a group A) , Group B Salmonella (heart /»2〇«6//« group B), Group D Salmonella (&lt;Sa/mo«e//&lt;3 group D), Citrobacter freundii (C7iro6acier, K. 1 citric acid bacteria (Cz'iroZ&gt; acier Arc^erz·), Klebsiella pneumoniae pwewwowz'ae), Klebsiella pneumoniae (A7e65k//&lt;3, aerobacteria (jEwieroftacier aerogewes), Enterobacter cloacae (five c/oacrae) or Serratia marcescens {Serratia marcescens, 6. According to the use of claim 1, the OmpA is obtained from E. coli (f. sc/zer/c/z/ Iz co/z·), Kreb's pneumonia Bacteria (A7eZ^/e//a 144249.doc 201028163 /?«ew/wowz'ae), Salmonella typhimurium (Sa/wowe/Za 7) 7?/»·) or aerogenic bacteria (5. 7) According to the use of claim 1, wherein the bacterial infection is caused by Shigella (&lt;S7n'ge//a), Salmonella (5Wwo«e//a), Klebsiella (Χ/ βΖ&gt;5·ζ·β//&lt;3), Escherichia, Chromosome (C&quot;r&lt;?0acier), Enterobacter (£«iero6acior) or Serratia ( (Serrai/α) caused by 8. According to the use of claim 1, wherein the bacterial infection is caused by Escherichia coif, Shigella sonnel, Shigella per Shigella (57π' hair) e//ar //awerz·), Salmonella typhimurium (&lt;Sa/mo«e//a/////), group A Salmonella (5·α/»ιο«β//α group A), Group B Salmonella (Sa/mcmeZ/a group B), Group D Salmonella {Salmonella group D), Acidophilus fuliginea (Cz'/ro6acier, K. oxysporum (Cz7ro0&lt; 3cier Aoserz· ), Klebsiella pneumoniae, K. oxysporum coc_yioca, aerogens (five aero e«ei), Enterobacter cloacae c/oacize or Serratia marcescens ^ ^ 0 9. According to the use of claim 1, the bacterial infection is caused by E. coli (jBsc/zeric/π' ϊ cohh), Klebsiella pneumoniae (A7e6sieZ/a pwewwom'ae), Salmonella typhimurium (5αΖ»ί0«β//α Γ&gt;&gt; ρ/π·) or aerogens (£« Caused by iero6&lt;3cier aerogewes). 10. The use according to claim 1, wherein the OmpA is administered orally or intravenously. 144249.doc 201028163 11. The use according to claim 1, wherein the dosage of the OmpA is 0.0001% to 10% by weight of OmpA. 12. The use according to claim 1, wherein the dosage of the OmpA is from 5% to 5% by weight of OmpA. 13. Use of an enterobacteriaceae outer membrane protein A (OmpA) to immunize a mammal. The use of an antibody against a bacterial infection of the central nervous system and/or peripheral blood circulation caused by Enterobacteriaceae. 14. The use according to claim 13, wherein the antibody is a polyclonal antibody. 15. The use according to claim 13, wherein the mammal is a human. 16. According to the use of claim 13, wherein the OmpA is a recombinant ΟιηρΑ. 17. The use according to claim 13, wherein the OmpA is derived from Shigella (*S7n_gd/a), Salmonella, Klebsiella, Escherichia, and Oak Acidic genus Enterobacter (Five or Serra genus φ 18. Use according to claim 13, wherein the OmpA line is obtained from Escherichia coli, Sogella sonnef Shigella Shigella (*S7n'ge//i2, Salmonella typhimurium accountant/^), Group A Salmonella group A), Group B Salmonella (iSa/mcme/Za group B), Group D Salmonella (&lt;S*a/mo«e//a group D), C. faecalis (C7iro6acer / rew7?&lt;iz_z·), Aojerz·K., Klebsiella pneumoniae (幻^We//a, Klebsiella oxytoca) 'IL # S (Enterobacter aerogenes) ' 144249.doc 201028163 Enterobacter cloacae (five c/oacae) or Serratia marcescens (JSerratia) Marcescens). 19. The use according to claim 13, wherein the OmpA is obtained from Escherichia coli co/z·), Klebsiella pneumoniae préumoniae 'r}' Η {Salmonella Typhi) or A. faecalis (V. 20. According to The use of claim 13, wherein the dosage of the OmpA is 0.0001% to 10% by weight of OmpA. 21. According to the use of claim 13, wherein the dosage of the OmpA is 0.5% to 5% by weight of OmpA 22. An isolated antibody which is the antibody obtained according to the use of claim 13. 23. The antibody of claim 22 which is a monoclonal antibody, a polyclonal antibody or a synthetic antibody or a fragment thereof. The antibody of claim 22, which is an anti-OmpA IgY antibody. 25. The antibody of claim 22 which is an anti-OmpA IgY antibody of mammalian or avian. 26. The antibody of claim 22 which is a pig, a rabbit, An antibody against a mouse, a monkey, a cow, a sheep, a chicken, or a duck. 27. The antibody of claim 22 which is an anti-OmpA IgY antibody of chicken or duck. 28. The antibody of claim 22, which is Chicken anti-OmpA IgY antibody. 29. The antibody of claim 22, for its treatment, prevention, detection and/or diagnosis A bacterial infection of the central nervous system and/or peripheral blood circulation caused by Enterobacteriaceae. 30. The antibody of claim 29, wherein the Enterobacteriaceae bacterium is selected from the group 144249.doc 201028163 Group: Shigella ("S7z!'ge//a), Salmonella (Sa/moweHa), lej, Klebsiella, Escherichia, Phytophthora, Enterobacter ( 5. Serratia genus (☆ ΓΓίϊΗβ). 3 1. The antibody of claim 29, wherein the Enterobacteriaceae bacterium is selected from the group consisting of Escherichia coii, Shig Ua son «ei·), Shigella flexneri (*S7n'ge//a //exnerij, Salmonella typhimurium (☆/wowe/Za 7&gt;/?/η·), group A Salmonella 〇group A) , Group B Salmonella (5^/wcme//a group B), Group D Salmonella group D), Citrobacter freundii (C7ir〇kcier / rew «A7), C. citrea (C^rohcier Ατοκη· ), Klebsiella pneumoniae (illusion ehie/M /? «ewwom'ae), Krebs oxyioca), aerogens (five "iero6acier aeroge" E5), Enterobacter cloacae (five "iero0&lt;3i; ier c/oacae" and Serratia marcescens {Serratia marcescens,. Φ 32. The antibody according to claim 29, wherein the Enterobacteriaceae bacterium is Escherichia coli {Escherichia co/〇, Klebsiella pneumoniae. pwewwowz'ae), Salmonella typhimurium (仏/molike//ίζ or Aerogenes (five "ie wide oftacier aerogenes". 33. The antibody of claim 29, wherein the bacterium of the genus Bacillus is Escherichia coli (where sc/^rz_c/n'a co/z·) or Klebsiella pneumoniae A therapeutic or diagnostic agent comprising the antibody of claim 22. 3 5. A central nervous system caused by Enterobacteriaceae in the detection or diagnosis of mammals 144249.doc 201028163 System and / Or a peripheral blood circulation bacterial infection method comprising: coating a first specific anti-OmpA antibody on a surface of a substrate, the antibody immunospecifically binding to OmpA on a blood_OmpA molecule or a bacterial cell membrane; the addition is taken from the periphery a sample of blood circulation and/or central nervous system in the matrix; adding a labeled second anti-OmpA antibody; and detecting binding of the anti-OmpA antibody to OmpA molecule or OmpA on the bacterial cell membrane, wherein the binding result indicates the lactation The central nervous system and/or the peripheral blood circulation is subjected to a bacterial infection, and wherein the OmpA line is from Enterobacteriaceae. 36. The method of claim 35, wherein the mammal is a human. 37. According to the method of claim 35, Wherein the OmpA is derived from Shigella (other/^^//"), Salmonella (*SWmo«e//a), Klebsiella, and Escherichia (Esc/zeWc/zia) </ RTI> </ RTI> </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; Shigella sonnei, Shigella sonnei, *S7n'gW/&lt;3 //exnerp, Salmonella typhimurium 7^/?/π·, Group A Salmonella (Sa/zwowe/ Za group A), Group B Salmonella group B), Group D Salmonella group D), C. faecalis (C7iroi > acier, C/iroZmcier Aroserz), Klebsiella pneumoniae (AT/ekiW/a /?«ewwom_ae), K. oxysporum oxjyioca), aerobacteria (five wierofeacier aerogewes), 144249.doc 2 01028163 Enterobacter cloacae (five c/oflcae) and Serratia marcescens °. 39. The method according to claim 35, wherein the OmpA is obtained from Escherichia coli (five "sc/ieric/z/a eo/z.", Klebsiella pneumoniae (foot/eZ^k/Za pneumoniae)" y}' Π # ϋ {Salmonella 7^/?/π·) or aerogenic bacillus (five aerogewe·?) ° 40. The method according to claim 35, wherein the substrate is an ELISA disk or a magnetic nanoparticle. 41. The method of claim 35, wherein the label is a radioisotope, a fluorescent substance, or a chemically luminescent substance. 42. A kit for diagnosing a bacterial infection of the central nervous system and/or peripheral blood circulation caused by Enterobacteriaceae, comprising an antibody as claimed in claim 22, a reagent required to constitute a medium for performing an OmpA-antibody reaction, and allowing An agent that detects the complex formed. 144249.doc