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WO2007122760A1 - Réactif de détection et procédé de détection de l'infection par l'influenza - Google Patents

Réactif de détection et procédé de détection de l'infection par l'influenza Download PDF

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WO2007122760A1
WO2007122760A1 PCT/JP2006/321891 JP2006321891W WO2007122760A1 WO 2007122760 A1 WO2007122760 A1 WO 2007122760A1 JP 2006321891 W JP2006321891 W JP 2006321891W WO 2007122760 A1 WO2007122760 A1 WO 2007122760A1
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aiv
influenza
antigen
nucleoprotein
test
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PCT/JP2006/321891
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Japanese (ja)
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Kazuaki Takehara
Masayuki Nakamura
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School Juridical Person Kitasato Gakuen
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    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6875—Nucleoproteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/14011—Baculoviridae
    • C12N2710/14111—Nucleopolyhedrovirus, e.g. autographa californica nucleopolyhedrovirus
    • C12N2710/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/11—Orthomyxoviridae, e.g. influenza virus

Definitions

  • the present invention relates to an influenza infection screening agent and a screening method. More specifically, the present invention relates to an influenza infection test agent and a test method using a common influenza A antigen capable of rapid, high sensitivity and multi-sample processing. Background art
  • Avian influenza (Avian Influenza, hereinafter abbreviated as AI) is an important viral disease in poultry and is highly pathogenic in livestock epidemic prevention methods.
  • avian influenza (hereinafter abbreviated as HPAI) is designated as a legal infectious disease
  • LPAI low pathogenic avian influenza
  • AIV An avian influenza virus (hereinafter abbreviated as AIV) belonging to the genus Orthomyxomyces influenza A virus has an envelope, a diameter of about 80 to 120 nm, and a negative single-stranded segmental RNA.
  • This virus is antigenically divided into three types, A, B, and C, by nucleoprotein (hereinafter abbreviated as NP) and matrix protein (hereinafter abbreviated as M).
  • NP nucleoprotein
  • M matrix protein
  • HA hemagglutinating protein
  • NA neuraminidase
  • M2 matrix 2 protein
  • Internal proteins include polymerase proteins (PA, PB1, PB2), NP, matrix 1 protein (hereinafter abbreviated as Ml), nonstructural protein 2 (named nonstructural, but within the virus particle) Incorporated: hereinafter abbreviated as NS2.
  • Nonstructural protein 1 (hereinafter abbreviated as NS 1) is produced in large quantities in infected cells. It is the only protein that cannot be turned over. According to the surface proteins HA and NA, serotypes (subtypes) are classified into 15 types (H1 to H15) for HA and 9 types (N1 to N9) for NA. Recently, a new H16, which is relatively closely related to H13, has been reported. Due to the various combinations of HA and NA, there are many subtypes of influenza virus. AI is also classified into HPAI and LPAI by pathogenicity. HPAI is an acute infectious disease that causes systemic symptoms in various birds, including chickens. The symptoms vary, but the fatality rate and transmission power are extremely strong.
  • HA and NA subtypes of AIV use antiserum prepared from 15 types of HA and 9 types of NA, respectively, and hemagglutination inhibition (hereinafter abbreviated as HI) test and It has been determined by neuraminidase inhibition (hereinafter abbreviated as NI) test.
  • HI hemagglutination inhibition
  • NI neuraminidase inhibition
  • FRT-PCR reverse transcriptase polymerase chain reaction
  • a method for diagnosing AIV using primers H1 to H15 was reported by Lee et al. (Journal of Virological Methods. 97: 13-22.2001: Non-Patent Document 1) and adopted in Japan. PCR is said to serve as an effective and rapid method for detecting influenza A virus, which requires rapid and antisera.
  • AIV has 16 HA subtypes, and its serological diagnosis requires a large number of antisera (16 types and amounts) and antigens, which complicates the diagnostic method.
  • antisera (16 types and amounts) and antigens
  • AGP in-gel sedimentation
  • HI test is widely used for serodiagnosis techniques for detecting AIV antibodies.
  • the AGP test uses both antigen and antibody to form sedimentation lines. Need at least 24 hours.
  • the HI test is very laborious and costly per specimen due to the 16 HA subtype that is more sensitive and quicker than the AGP test.
  • Non-patent document 2 shows that the sensitivity and specificity are higher than those of the AGP test. It is considered that there are many nonspecific reactions, and relatively specific antigens for colorimetric determination and species-specificity for each test animal. Enzyme-linked antibodies are required.
  • Competitive ELISA uses recombinant antigens and monoclonal antibodies (hereinafter abbreviated as MAb) to suppress non-specific reactions and increase sensitivity and specificity. Its usefulness has been proved by Zhou et al. (Avian Dis.42: 517_22.1998: Non-patent document 3) and Shafer et al. (Avian Dis. 42: 28-34.1998: Non-patent document 4). Since it does not require a bound antibody, it can be diagnosed in various animal species.
  • Non-Patent Document 4 also compared the competitive ELISA and AGP tests using chicken, turkey, running bird, quail, pheasant, and penguin sera. Re, correlation and competition showed high specificity and specificity of ELISA. In the competitive ELISA of Shafer et al. (Non-Patent Document 4), antigen purification is performed.
  • Non-Patent Document 1 Journal of Virological Methods. 97: 13-22.2001
  • Non-Patent Document 2 Avian Dis. 29: 136-44.1985
  • Non-Patent Document 3 Avian Dis. 42: 517-22.1998
  • Non-Patent Document 4 Avian Dis. 42: 28-34.1998
  • Non-Patent Document 5 ⁇ Med Virol. 27: 25-30.1989
  • Non-Patent Document 7 J Immunol. L26: 1814-9.1981
  • Non-Patent Document 8 Arch Virol. 115: 47-61.1990
  • the NP of AIV that the present inventors tried to express is a protein consisting of 498 amino acid residues having several different functions throughout the life cycle of the virus. It functions mainly as a negative single-stranded RNA-binding protein and is a structural protein in ribonucleoprotein particles (hereinafter abbreviated as RNPs).
  • RNPs ribonucleoprotein particles
  • NP plays an important role in the transport and transcription of RNPs between the cytoplasm and nucleus.
  • the NP gene is l, 565 bp and is encoded in segment 5.
  • NPs are highly conserved among viruses and have more than 95% amino acid sequence identity with other avian subtypes. Furthermore, because it exists on a different segment from the gene segment encoding HA or NA that defines the subtype, it is not affected by antigenic mutations in HA and NA. Because it is common to influenza A, it is an important protein used for diagnostic tests
  • the present inventors used baculovirus-insect cells as an expression system.
  • baculovirus Autographa California nuclear polyhearosis virus (hereinafter abbreviated as AcNPV) belonging to the nuclear polyhedrosis virus (hereinafter abbreviated as NPV) was used.
  • plasmid pAcYMl was used as a transfer vector for baculovirus.
  • This vector is the highest in the plasmid vector containing ATG of ATG, which is the polyhydrin gene start codon (base sequence near the cloning position is AAAAAAACCTA TAAAT A CGGATCCG (SEQ ID NO: 1), underlined is the restriction enzyme BamHI recognition site) It is known that expression efficiency is obtained.
  • the point of the baculovirus expression system lies in the promoter of the nuclear inclusion body protein called polyhydrin. Different forces depending on the gene to be expressed Many of them are at the end of virus infection, and about 50% of the total cellular protein is said to be the target protein. In this system, protein synthesis is performed in insect cells, which are eukaryotes. Therefore, unlike prokaryotes such as E.
  • the present inventors further attempted production of MAb against NP. Compared with polyclonal antibodies, the advantages of MAb are summarized in the following three points (1) to (3).
  • Antibody-producing hyperpridoma can be stored in liquid nitrogen like other cell lines, and antibodies can be adjusted as needed by the experimenter.
  • the current diagnostic methods have many points to be improved in terms of sensitivity, rapidity, specificity, etc.
  • the AGP test which is regarded as a diagnostic criterion for AIV, is unsuitable for multi-sample processing and has many antibodies and antigens. Need the amount of. In other words, it is important to develop a highly sensitive and specific diagnostic system that can process multiple specimens. In addition, AIV is sensitive to various mammals and birds. Therefore, it is desirable to have a method capable of monitoring these various animal species.
  • AIV NP which is considered to be highly conserved among AIV strains, for example, in Noculus virus, and intensively studied the possibility of supplying antigens for screening.
  • MAb was prepared, and the usefulness of competitive ELISA as a highly sensitive detection system for AIV was studied intensively. Attempts were made to establish a highly sensitive serological diagnostic system using NP, and the present invention was completed.
  • the present invention is an invention of an influenza infection test drug according to the following 1 to 7 and an influenza infection test method according to the following 8 to 14:
  • An influenza infection test drug characterized by using an influenza virus nucleoprotein (NP) as an AIV (avian influenza virus) antibody detection antigen.
  • NP influenza virus nucleoprotein
  • AIV avian influenza virus
  • the nucleoprotein (NP) gene of influenza virus is isolated from AIV (avian influenza virus), and the nucleoprotein (NP) expressed using a baculovirus expression system is used as an AIV antibody detection antigen as described in 1 above Influenza screening medicine.
  • the nucleoprotein (NP) expressed as NP_His by adding the His-tag sequence to the nucleoprotein (NP) gene of AIV A / Duck / Aomori / 478/02 (HlNl) is used as the AIV antibody detection antigen.
  • the influenza infection screening agent according to 1 or 2.
  • influenza infection test agent according to any one of 1 to 3 above, wherein an influenza virus nucleoprotein (NP) is used as an immobilized antigen in an enzyme-linked immunosorbent assay (ELISA).
  • NP influenza virus nucleoprotein
  • ELISA enzyme-linked immunosorbent assay
  • Influenza virus nucleoprotein is used as an immobilized antigen in enzyme-linked immunosorbent assay (E LISA), and at least the immobilized antigen of (1) above
  • influenza test agent according to any one of (1) to (4), which comprises an antinuclear protein monoclonal antibody (NP MAb) that specifically binds to a nucleoprotein (NP) of influenza virus.
  • NP MAb antinuclear protein monoclonal antibody
  • influenza test agent for influenza infection according to 4 or 5 above, wherein the immobilized antigen is immobilized on an ELISA plate.
  • a method for detecting influenza infection characterized by using an influenza virus nucleoprotein as an immobilized antigen in an enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • nucleoprotein ( ⁇ ) isolated from the AIV of the influenza virus nucleoprotein ( ⁇ ) gene and expressed using the baculovirus expression system is used as an immobilized antigen in the enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • Influenza virus nucleoprotein ( ⁇ ) is used as an immobilized antigen in enzyme-linked immunosorbent assay (ELISA), and at least the immobilized antigen (1) and
  • NP MAb antinuclear protein monoclonal antibody
  • Influenza virus nucleoprotein as an immobilized antigen for enzyme-linked immunosorbent assay (ELISA),
  • NP MAb antinuclear protein monoclonal antibody that specifically binds to the nucleoprotein (NP) of (1) above is added, and the antinuclear protein monoclonal antibody (NP M Ab) is added to the above (2).
  • NP M Ab antinuclear protein monoclonal antibody
  • Influenza virus nucleoprotein was used as an immobilized antigen for enzyme-linked immunosorbent assay (ELISA),
  • NP MAb antinuclear protein monoclonal antibody that specifically binds to the nucleoprotein (NP) of (1) above is added, and the antinuclear protein monoclonal antibody (NP M Ab) is added to the above (2). Compete with the test sera added in the next step.
  • the diagnostic system using AIV NP His according to the present invention showed high sensitivity and specificity.
  • competitive ELISA using MAb showed high utility.
  • Competitive ELISAs can save time because they can handle multiple samples. Because it uses a recombinant protein, it is necessary to use a virus. Furthermore, by immobilizing the antigen on the ELISA plate and storing it, the test can be facilitated and the time can be further shortened. There is no need to purify highly purified antigens like indirect ELISA. And monitoring in various mammals and birds could be tested in the same way.
  • preservation of the ELISA plate on which the antigen is immobilized is particularly preferable because sensitivity and specificity do not decrease, because it facilitates inspection and facilitates supply to other inspection institutions.
  • AIV NP His was purified using a His-tag.
  • purified antigen is used, non-specific reaction of ELISA can be effectively suppressed.
  • purification takes a lot of time and the amount of antigen is also significantly reduced.
  • Non-purified competitive ELISA can handle multiple samples more efficiently.
  • the diagnostic system according to the present invention can be widely used by conducting competitive ELISA using sera from various animal species.
  • the strain AZDuckZAomori Z478Z02 (H1N1), which will be described later, can be obtained by amplifying the full length of each segment by RTPCR. It ’s not limited.
  • the NP gene-specific primer was designed to add a histidine tag to the C-terminus of NP. Insert the PCR product into pCR2.1 It was transformed into the patent cells.
  • the expression of the AIV NP gene is not particularly limited, it is preferably carried out by a baculovirus expression system.
  • Recombinant baculovirus is prepared by inserting AIV NP His cDNA into the baculovirus transfer vector pAcYMl, then transforming it, and preparing recombinant virus by co-transformation of pAIV NP His-pAcYMl and AcRP23-LacZ. did.
  • This virus was plaque-cloned and named AcAI NP His.
  • the obtained AcAI NP His was infected to Sf9 cells to express AIV NP His.
  • AIV NP His was purified from AcAI NP His-infected cells using a Ni column.
  • the present inventors used the expressed AIV NP His, and examined the usefulness of ELISA and AGP tests.
  • AIV immunized chicken sera showed significantly higher values than the field normal chicken sera used in the serum dilutions performed and were performed in all field normal chicken sera used. No non-specific positive reaction was observed in the serum dilution.
  • sedimentation lines were formed only with AIV-immune chicken sera, and nonspecific positive reactions were not observed with field normal chicken sera. All of the field ostrich sera were negative. These results were the same as those using RNP antigen. From these results, it is considered that the expressed NP can be widely used as an anti-AIV antibody detection antigen in various detection methods.
  • MAb 1 was obtained from mice immunized with purified AIV A / budgerigar / Aichi / l / 77 (H3 N8) (available from the Society for Animal Biological Products). 1E5 (light chain ⁇ , heavy chain IgGl) was obtained. The MAb 11E5 was concentrated and the reactivity was examined. In the ELISA using purified AIV NP His as an antigen, the reaction was performed up to a 1.0 ⁇ 1 O 5- fold dilution. In Western blotting using AIV AZbudgerigar / AichiZlZ77 (H3N8) and purified AI V NP His as antigens, no band was observed at any position. The following are considered to be negative for Western blotting.
  • influenza NP is stable as an oligomer.
  • the NP oligomers are non-covalently bound and are resistant to SDS resistance, a wide temperature range and the presence of salts (1M Na CI, 1 M KC1) and denaturants (8M urea). Heating above 80 ° C And dissociates into NP monomer at pH 5 or lower. In other words, the three-dimensional structure may have changed due to the effect of 100 ° C heating or ME + during the antigen preparation stage.
  • the present inventors examined the application of competitive ELISA using AIV NP His and MAb 11E5.
  • competitive ELISA all AIV immunized chicken sera showed almost 100% inhibition, and no inhibition reaction was observed in SPF chicken sera, field normal chicken sera, and ostrich sera.
  • 5 actual H9N2-infected chicken sera with AGP values of 2 to 8 showed inhibition rates of 100%, 34%, 100%, 91%, and 80%, respectively. Therefore, it was decided that more than 30% of inhibition was positive based on the average value and standard deviation of the inhibition rate of negative specimens and the inhibition rate of experimentally infected chicken serum.
  • the competitive ELISA positive limit / AGP test positive limit is about 1 to 10 times, compared to AGP test A competitive ELISA was found to be more sensitive.
  • a highly sensitive and highly specific avian influenza virus (AIV) diagnostic system has been developed that can handle multiple samples in place of the gel precipitation (AGP) test.
  • AI is highly conserved among AIV strains It expresses nucleoprotein (NP), which is considered to be useful for the diagnosis of cancer, enables the supply of antigens for screening, and further reveals the production of monoclonal antibody (MAb) against NP and the usefulness of competitive ELISA using it I made it.
  • His-tag sequence was added to the NP gene of AIV A / Duck / Aomori / 478/02 (HI Nl) and expressed as NP-His using baculovirus and insect cells.
  • Anti-NP MAb (IgG1, ⁇ ) was prepared by immunizing AIV A / budgeriger / Aichi / 1Z77 (H3N8). As mentioned above, the NP of NP_His combined with MAb is arbitrary.
  • AIV (H9N2 subtype) experimentally infected chicken serum was distributed by the Institute for Animal Health (Tsukuba, Ibaraki). Eighty chicken sera subject to AI killing were distributed from the Central Animal Health Center in Yamaguchi Prefecture. Ostrich serum is from Yamagata Asahi Ostrich Industrial Center (Yamagata Pref. From Himachi). Field normal chicken serum was distributed from Aomori Portley (Hachinohe City, Aomori Prefecture). SPF chicken serum was distributed from the Institute for Chemotherapy and Serum Therapy (Akira Kumamoto, Kumamoto Prefecture). The prepared anti-AIV immunized chicken serum was used.
  • a virus containing 1 to 12 subtypes of HA and 1 to 9 subtypes of NA was used as a reference virus strain. These strains were provided by Professor Hiroshi Kita, Department of Microbiology, graduate School of Veterinary Medicine, Hokkaido University.
  • AZbudgerigar / Aichi / l / 77 (hereinafter abbreviated as A strain) was purchased from the Association for Animal Biological Products (Tokyo).
  • Tokyo For the NP gene clone, we used the strain A / Duck / Aomori / 478/02 isolated from soil in Aomori Prefecture.
  • Strain A was phosphate buffered saline (0.14M NaCl, 2mM KC1, 3mM Na HPO, 1.5mM KH
  • the supernatant was discarded, and the pellet was resuspended in 0.08% sodium azide sodium (hereinafter abbreviated as NaN) in PBS and stored at 4 ° C for 3 days.
  • the pellet was agitated well and centrifuged at 30,000xg for 5 minutes.
  • the supernatant was collected, sucrose was added to 10%, and 20 to 80% sucrose solution was layered on the density gradient, and centrifuged at 80,000 ⁇ g for 90 minutes at 4 ° C. Collect each fraction and save 30ml of PBS, 80,000xg, 90 minutes, 4. Centrifuged at C and discarded the supernatant.
  • the pellet was resuspended in lml of 0.08% NaN-added PBS to obtain purified AZbudgerigar / AichiZlZ77.
  • the agar gel was prepared according to “Diagnosis method of chicken virus disease” (Sadaharu Horiuchi. Diagnosis method of chicken virus disease. ⁇ • 595-606.1982.). Phosphate buffer (0.02M Na HPO, ImM KH PO, p
  • H7.4 was mixed with 8.0% NaCl and 1.0% Bacto-Agar. After that, 10% NaN was burned 1.0%. Put 25ml of agar in the petri dish, and the agar will harden and force gel puncher (hole 5m m x 7 well), agar was cut out and aspirated to make a reaction agar plate.
  • a cultured egg culture purified strain A (hereinafter referred to as RNP antigen) was used.
  • AGP antigen was placed in the center well of the agar gel, anti-AIV chicken serum was added every other well as positive sera, and test sera were placed at 30 ⁇ l / well, respectively, and observed at room temperature for 2 days. The reciprocal of the maximum serum dilution at which sedimentation lines were confirmed was taken as the AGP value.
  • the HI test was performed according to the “OIE MANUAL OF DIAGNOSTIC TESTS AND VA CCINES FOR TERRESTRIAL ANIMALS. HIGHLY PATHOGENIC AVIAN INFLUE NZA. OIE Terrestrial Manual. CHAPTER 2.1.14.2004”.
  • a 96-well U-type microplate (96U W / OUT LID SH MICROWELL PLATE: Nalge Nunc International, NY, USA), dilute the virus solution 25 ⁇ 1 with 0.15M physiological saline two-fold, and then add physiological saline 25 After mixing the same amount with ⁇ 1, the same amount was mixed with 50 ⁇ 1 of 0.5% chicken erythrocyte solution. After standing at room temperature for 1 hour, the reciprocal of the highest dilution factor in which phlegm was observed was determined as the evaluation.
  • the 4 ⁇ unit antigen was determined using 1HA unit as the highest dilution virus solution at the time of positive ⁇ determination (National Institute of Preventive Health) Society. Serum reaction. Pp. 192-201.1982. Revised 2nd edition, Virus Experimental Studies. Tokyo, Maruzen Co., Ltd., “OIE Manual”).
  • test serum 25 ⁇ 1
  • physiological saline physiological saline
  • test serum 25 ⁇ 1
  • physiological saline physiological saline
  • 50 ⁇ ⁇ of 0.5% chicken erythrocyte solution was mixed with 50 ⁇ ⁇ of 0.5% chicken erythrocyte solution and sensitized for 1 hour at room temperature.
  • the reciprocal of the highest dilution factor for inhibiting aggregation was taken as the HI value.
  • the restriction enzyme BamHI site that does not exist in the original gene at both ends of the AIV NP open reading frame (hereinafter abbreviated as ORF), and the purification of the resulting protein 3 ' It was designed to add a histidine tag (hereinafter referred to as “His-tag”) sequence to the terminal side.
  • His-tag a histidine tag
  • RNA is abbreviated and RT-PCR using AI-specific primer Unil 2 (Hoffmann et al., Arch Virol. 146: 2275- 2289.2001.) Is abbreviated as complementary DNA (hereinafter referred to as cDNA).
  • cDNA complementary DNA
  • cDNA complementary DNA
  • NP-specific primers set denaturation at 94 ° C, primer annealing temperature at 58 ° C, cDNA synthesis at 72 ° C, and stepl (94 ° C x 4 min) for 1 cycle. , St cycle 2 (94 ° CX 20 seconds, 58 ° CX 30 seconds, 72 ° CX 7 minutes), 30 cycles, St cycle 3 (72 ° CX 7 minutes) (Journal of Virological Methods. 97: 13-22.2001).
  • the AIV NP His gene amplified by PCR was ligated to pCR2.1, a plasmid vector, using TA Cloning Kit (Invitrogen, CA, USA). This was transduced into the host E. coli JM109 (Takara Shuzo Co., Ltd., Kyoto) using the Heat Shoe k method, and 50 g / m 1 ampicillin, 0.12% X-gal (5-Bromo-4- Chloro-3-indolyl- ⁇ —d — Galactoside (Takara Shuzo Co., Ltd.)-Added TYM agar medium (2% bacto_tryptone, 0.5% yeast ext ract, 0.1% glucose, 10 mM MgSO ⁇ 7 ⁇ 0, 1.5% bacto agar) was cultured at 37 ° C.
  • ⁇ -galatatosidase metabolizes X-gal and produces a blue pigment, but when the AIV NP His gene is inserted into the LacZ region of the vector, / 3-galatatosidase is not produced and white colonies are observed .
  • white colonies are selected and cultured in TYM liquid medium (2% bacto-tryptone, 0.5% yeast extract, 0.1% glucose, 10 mM MgSO-7H O).
  • PAIV NP His_pCR2.1 was obtained. The plasmid was extracted from this, digested with BamHI and confirmed by Agarose gel electrophoresis for AIV NP His gene insertion.
  • the BamHI digested plasmid gene and primers (AIV NPF and AIV NPR His) were used to perform a sequencing reaction by the Dideoxy Terminator method (Sanger et al., J. Mol. Biol. 94: 41.1975.).
  • Dye Terminator Cycle Sequencing Kit FS Perkin-Elmer
  • ddNTP labeled fluorescent dideoxynucleotide
  • the pAIV NP His_pCR2.1 obtained by cloning was digested with BamHI (Takara Shuzo Co., Ltd., Kyoto) and developed by agarose gel electrophoresis, and the AIV NP His gene fragment was excised together with the gel.
  • the AIV NP His gene was purified by ethanol precipitation after phenol treatment.
  • the obtained fragment was ligated to BAcHI digested baculovirus transfer vector pAcYMl (Matsuura et al., J. Gen. Virol. 68: 1233-1250.1987.) Using Takara DNA Ligation Kit (Takara Shuzo Co., Ltd.) Subsequently, the host E.
  • coli JM109 (Takara Shuzo Co., Ltd.) was transduced by the Heat Shock method. This was cultured on a TYM agar medium supplemented with 50 ⁇ g / ml ampicillin. Large-scale culture was performed from the appearing colonies in a TYM liquid medium supplemented with 50 ⁇ g / ml ampicillin to obtain pAIV NP His—pAcYMl. The plasmid was extracted, and the AIV NP His gene was confirmed by agarose gel electrophoresis after digestion with BamHI.
  • a PCR method using a plasmid containing the AIV NP His gene as a saddle type was performed.
  • a BaclN primer (5'-tgataacc atctcgcaaa-3 '(SEQ ID NO: 4)) with a recognition site between the polyhydrin promoter of pAcYMl and the cloning site and an AIV complementary to the 3' end of the AIV NP His gene NPR His primer was used.
  • amplification is observed at a band of about 1,500 bp only when inserted in the forward direction with respect to the polyhydrin promoter.
  • IPLB_Sf21AE (hereinafter abbreviated as Sf21 cells) established with ovarian force of Spodoptera frugiperda, or Sf9 cells with cloned Sf21 cell force.
  • Sf21 cells consist of TC-100 insect medium (Gibco BRL, Gaithersburg, MD, USA), hereinafter abbreviated as TC100 (0%), and fetal calf serum (hereinafter abbreviated as FCS). % Maintained at TC100 (10%) and used for cloning. It was. Sf9 cells are cultured using ESF921 medium (Expression System LLC, Woodland, CA, USA: hereinafter abbreviated as ESF921) at 28 ° C by spin culture method to produce high-titer virus. Used for expression. Baculovirus and Sf21 cells were dispensed from the NER C Institute of Virology (Oxford, UK) and Sf9 cells were purchased from Expression Systems L LC (Woodland, CA, USA).
  • pAIV NP His-pAcYMl (10 ⁇ g / ml) 1 ⁇ 1, Ac8RP23-LacZ DNA digested with Eco8 II and linearized (1 ⁇ g / ml) 1 ⁇ 1, TC I 00 (0%)
  • Add 6 ⁇ l of the mixed solution (DNA mixed solution), TC 100 (0% FCS) medium 8 ⁇ 1, and ribofuctin (GIBCO BRL) 8 / il mixed solution (lipofectin mixed solution) for 30 minutes at room temperature. Reacted.
  • the DNA mixture and the lipofectin mixture were mixed and allowed to react at room temperature for 15 minutes, and then transferred to Sf21 cells to produce recombinant viruses by homologous recombination.
  • Multiplicity of infection (hereinafter abbreviated as M0I) is about 5 Plaque Forming Unites (hereinafter abbreviated as PFU) in Sf9 cells (1.5 X 10 6 cells) cultured in a single layer with AcAI NP His. ) Inoculated with / cell, cultured at 28 ° C using ESF921 medium, and the cells and supernatant were collected 1, 2, 3, 4 and 5 days after inoculation. In addition, a culture solution (hereinafter abbreviated as Mock) was similarly inoculated as AcNPV or negative control, and the cells and supernatant were collected after 5 days.
  • PFU Plaque Forming Unites
  • CBB staining Kumashi brilliant blue staining solution (45% methanol, 10% acetic acid, 0.025% Coomassie brilliant blue) (hereinafter abbreviated as CBB staining) or Western blotting.
  • anti-polyhistidine mouse serum (SIGMA, Stuois, MO, USA: hereinafter abbreviated as a poly His) was used as a primary antibody and allowed to react at room temperature for 3 hours. After the reaction, it was washed 3 times with a blocking solution, and the secondary antibody was reacted at 37 ° C for 2 hours.
  • the secondary antibody was prepared by diluting peroxidase-labeled anti-mouse IgG rabbit serum (Transduction Laboratories, Lexington, KY, USA). After the reaction, the plate was washed 3 times with PBS and developed with a BM blue POD substrate (Boehringer Mannheim, Mannheim, Germany).
  • the column was washed with Wash Buffer, fractionated into 1 ml fractions using Imidazole adjusted to 50, 500, and 1,000 mM concentrations, and the His-tag added protein was eluted. This was developed with 12.5% SDS_PAGE, and after confirmation by CBB staining, the fraction in which the protein was confirmed was desalted through a PD10 column (Amersham Pharmacia Biotech, Tokyo) equilibrated with PBS. This was similarly developed on 12.5% SDS-PAGE, confirmed by CBB staining and Western blotting, and then sterile filtered to obtain purified AIV NP His. The protein content of purified AIV NP His was measured using the Micro BCA Protein Assay Reagent Kit (PIERCE, Rockford, IN, USA).
  • Kida et al. Kera et al., Virology. 122: 38-47.1982 ⁇ .
  • Seed purified AIV NP His (90 ⁇ g / ml) as an antigen on an ELISA 96-well plate (SUMILON, Sumitomo Bakelite Co., Ltd., Tokyo) at 50 / l / well, 2 hours at room temperature or 24 hours at 4 ° C Left to stand.
  • BSA10 Bovine serum albmin fraction V (Nacalai Testa Co., Ltd., Kyoto Prefecture)
  • lOmgZml is added to PBS in order to recover the antigen and prevent non-specific reaction to the well portion where the antigen is not coated. .2) was added in 100 ⁇ l aliquots, allowed to stand at room temperature for 1 hour or 4 ° C for 24 hours, and washed 3 times with PBST (PBS with 0.05% Tween20, pH 7.4).
  • Test serum diluted 40, 160, 640, or 2,560 times with BSA5T (caroten to PBST so that BSA fraction V is 5 mg / ml) at a time, and let stand at room temperature for 1 hour. Placed.
  • Purified AIV A / budgerigar / Aichi ZlZ77 (H3N8) was used as an immunogen for the production of monoclonal antibodies.
  • Purified virus at a final concentration of 0.05. /. It was inactivated with formalin and stored at 4 ° C.
  • Freund's complete adjuvant (SIGMA) was used as an adjuvant, and an equal volume of purified virus diluted 4 times with PBS was mixed to prepare an emulsion.
  • the prepared immunogen was inoculated into the abdominal cavity of the mouse at 200 ⁇ 1 / head. Emulsions treated in the same way were boosted several times every 2 weeks from the initial immunization in the same manner, and the mice were dissected 3 days after the final boost.
  • FO cells Mouse myeloma cells (J Immunol Methods. 35 (1_2): 1_21.1980: hereinafter abbreviated as FO cells) in RPMI 1640 medium (Nissi, Tokyo) plus 10% FCS (hereinafter 10% And abbreviated as RPMI 1640) at 37 ° C. and 5% CO.
  • FO cells were also removed from the cell culture routine, and the cells were collected by centrifugation at lOOxg for 8 minutes, resuspended in 10% RPMI 1640, adjusted to 2.0 ⁇ 10 7 cells / ml, and subjected to fusion.
  • mice were euthanized by blood sampling under ether anesthesia, and the blood was allowed to stand at 37 ° C, 5% C 1 hour, and further at 4 ° C for 24 hours. Serum was separated after 24 hours and stored at -20 ° C.
  • the mouse spleen after the euthanasia treatment was removed, and the spleen cells were separated by pressing the inner cylinder of a disposable syringe and pressing the spleen on a metal mesh.
  • Spleen cells were supplemented with 10% RPMI 1640, and spleen cells were washed twice at 100 ⁇ g for 8 minutes. Washed spleen cells were adjusted to 1.0 ⁇ 10 8 cells / ml with 10% RPMI 1640.
  • mouse lymphokine was prepared (KANE, M. M. and BANKb, JN Immunoassay Making and manipulating hybndoma cells. 5.1 Facilities and media Protocol. 11 37-40.). Euthanize uninoculated BALB / c mice Thereafter, the spleen was separated by the same method as described above, and the separated spleen cells were cultured in 10% RPMI 1640 medium containing 2.5 x lZml lipopolysac charide endotoxin (SIGMA: LPS) 3 7 ° C 5% CO 4 days Cultured. After centrifuging at 390xg for 5 minutes, the supernatant is recovered and 26mm Sy
  • rmge Filter 0.22 ⁇ 1 (Corning Incorporated Corning, NY 4831 ermany for ermany) After thoughtful extinction, Cehumchu 1 ⁇ bu (Greiner bio—one Frickenhausen 1, Germany) ⁇ ⁇ ; 3 ⁇ 4T / mainly at 20 ° C When used, it was diluted 20-fold with HT medium (SIGMA) and used at 100 ⁇ l / well.
  • SIGMA HT medium
  • PEG polyethylene glycol 4000
  • the cell concentration was adjusted to 10 6 cells / ml with HAT medium (SIGMA), seeded in a 96-well plate (96 Wells w / Lid Flat Bottom: Greiner) at 100 ⁇ l / well, and a high-pridoma was selected.
  • the obtained hyperidoma was treated with AIV antigen (A / Aichi / 2/68) and crude purified AIV NP provided by Professor Hiroshi Kida, graduate School of Veterinary Medicine, Hokkaido University. Screening by ELISA, NP antibody-producing hybridoma was cloned twice by limiting dilution using HT medium supplemented with 5% lymphokine. After cloning, the isotype was examined and cultured in large quantities.
  • ELISA was performed using crudely purified AIV NP as an antigen, hyperpridoma culture supernatant as a primary antibody, and peroxidase-labeled anti-mouse IgG rabbit serum (CHEMICON International Inc. CA USA) as a secondary antibody.
  • AIV A / budgerigar / Aichi / 1/77 H3N8
  • PBS pH 7.2
  • SDS_PAGE electrophoresis and Western blotting were performed.
  • MAb-producing hyperpridoma culture supernatant was used, Peroxidase-labeled anti-mouse IgG rabbit serum (Transduction Laooratones, Lexington, KY, USA) diluted 1000-fold with block solution was used as the body.
  • Isotypes were determined using Immuno pure Monoclonal Antibody Isotype kit I (PIERCE) based on the product manual. Apply 50 coating antibody solutions supplied with the kit to a 96-well plate (SUMILON) for ELISA for 2 hours or 4 at room temperature. Allowed to stand at C for 24 hours. Remove the solution and add 125 ⁇ 1 blocking solution37. C. left for 1 hour. After washing 4 times with a 125 / i 1 wash buffer, 50 ⁇ l each of the high-pridoma culture supernatant containing MAb and the positive control were added, and reacted at 37 ° C. for 1 hour.
  • PIERCE Immuno pure Monoclonal Antibody Isotype kit I
  • anti-mouse specific isotypes IgG1, IgG2a, IgG3, IgM, IgA, K, E
  • Usagi serum and normal Usagi serum as a negative control were added dropwise.
  • anti-mouse IgG was dropped and reacted at 37 ° C for 1 hour.
  • 50 ⁇ l of peroxidase-labeled anti-rabbit IgG goat serum was added and reacted at 37 ° C for 1 hour.
  • the cloned MAb-producing hybridoma was cultured using a large culture flat (INTEGRA Cel ILineCL 1000) (IBS INTEGRA BIOSCIENCES, INTEGRA Biosciences AG'Switzerland). The cells were subcultured at a time when the number of cells was about 2 ⁇ 10 7 to 4 ⁇ 10 7 cells / ml, and the culture supernatant was collected. Mass culture flat (INTEGRA CellLineCLlOO) force MAb was purified from the collected MAb-producing hybridoma culture supernatant using Montage TM life science kits (MILLIPORE.com, MA, USA).
  • INTEGRA Cel ILineCL 1000 IBS INTEGRA BIOSCIENCES, INTEGRA Biosciences AG'Switzerland
  • the formula is 100- (100 X [test serum value—blank value (BSA5T) Z control value (MAb only) —blank value (BSA5T)])). A suppression rate of 30% or more was considered positive. In addition, the reciprocal of the maximum serum dilution that showed more than 30% inhibition was taken as the competitive ELISA value.
  • the obtained PCR product was ligated to pCR2.1 using a TA Cloning Kit and transformed into E. coli JM109, which is a recombinant cell.
  • White colonies containing the target gene were selected using White-Blue Selection, and enrichment culture was performed.
  • Plasmids were extracted from the resulting Escherichia coli, digested with BamHI, and developed by 1% agarose gel electrophoresis. AI compared to lOObp DNA Ladder (Takara Shuzo Co., Ltd.)
  • AIVNP His—pCR2.1 was digested with BamHI, and the fragment containing AIV NP His was ligated to the baculovirus transfer vector pAcYMl using the Takara DNA Ligation Kit. Furthermore, the cells were transformed into a competent cell and the plasmid was extracted from E. coli. The extracted plasmid was digested with BamHI and electrophoresed with 1% agarose gel. As a result, about 1,500 bp of inserted gene and about 10,000 bp of pAcYMl vector DNA were confirmed.
  • the plasmid pAIV NP His_pAcYMl and AcRP23-LacZ DNA were cotransfected, and the culture supernatant was subjected to black cloning in the presence of X-gal. Plaques that showed white color by white-blue selection were plaque-cloned three times. After several passages in Sf21 cells, high-titer virus was obtained and used as seed virus. As a result of Black Atsey, The titer of virus was 2.0 ⁇ 10 8 PFU / ml. This was designated as recombinant baculovirus AcAI NP His.
  • the obtained AcAI NP His was inoculated to 1.5 ⁇ 10 6 cell / ml 300 ml of Sf 9 cells at 5 PFU / cell and collected at a time when it fell below 5.0 ⁇ 10 5 cell / ml.
  • Western blotting using polyHis a band of approximately 56 kDa was observed in AcAI NP His-infected cells, but no specific band was observed in AcNPV-infected cells and Mock.
  • Mass culture was performed and purification was performed using a Ni column.
  • AIV NP His adsorbed on the Ni column was eluted with Imidazole, developed by SDS-PAGE, and confirmed by CBB staining. Fractions in which a band of about 56 kDa was observed when dissolved at 500 mM were pooled, desalted through a PD10 column, and similarly subjected to CBB staining and Western blotting. Fractions in which a band of about 56 kDa was observed were pooled again and filtered through a 0.45 / im filter to obtain purified AIV NP His. As a result of protein quantification, purified AIV NP His with 90 ⁇ g / ml and total protein amount of 270 ⁇ g was obtained from 100 ml of Sf9 cell culture pellet.
  • the 12 AIV immunized chicken sera showed significantly higher values than the 10 normal normal chicken sera used at the serum dilutions of 40, 160, 640, and 2560. In addition, all the normal normal chicken sera used did not show any nonspecific positive reaction in the serum dilution performed.
  • the positive range was calculated from the mean value and standard deviation of field normal chicken sera, and a positive reaction was defined as a value equal to or greater than three times the standard deviation of the value in field normal chicken serum.
  • Sedimentation lines were confirmed only in 12 samples of AIV immunized chicken sera, and no nonspecific positive reaction was observed in 164 samples of field chicken sera. All 448 field ostrich sera were negative. These results were the same as the test results using RNP antigen.
  • mice immunized with purified AIV A / budgerigar / Aichi / 1/77 (H3N8)
  • Ten MAb-producing hybridomas were obtained from spleen cells and myeloma cells.
  • 8 types of culture supernatant that react with AIV NP antigen are considered as hybridomas that produce MAbs that react with NP, and 2 types that react with AIV antigen (A / AichiZ2Z68) but do not react with AIV NP antigen.
  • These MAb isotypes were IgGl, IgG2, IgA, and IgM.
  • IgG that reacts with the target NP was confirmed in 1E5 and 6E10.
  • 6E10 two types of MAb-producing hybridomas, light chain ⁇ , heavy chain IgM and IgG2a, were cloned five times using the limiting dilution method. It was mixed. Since further separation was considered difficult in terms of time, the cells were stored frozen in liquid nitrogen. 1 1E5 was light chain ⁇ and heavy chain IgGl.
  • the cloned MAb-producing hybridoma 11E5 was cultured in large quantities with INTEGRA CellLineCL 1000 to obtain 30 ml of culture supernatant. Furthermore, as a result of purifying the culture supernatant using Montage (trademark) life science kits (MILLIPORE.com, MA, USA), 400 ⁇ l of concentrated MAb 11E5 was obtained.
  • AIV immunized chicken sera were diluted 100-fold, and other sera were diluted 5-fold and used for ELISA.
  • the blank mean value was 0.218
  • the control mean value was 1.311
  • H4N5 immune serum value was 0.201
  • H5N3 immune serum value was 0.23
  • H9N 2 immune serum value was 0.194
  • H1 1N6 immune serum The value was .204.
  • the SPF chicken serum had a blank average value of 0.137, a control average value of 1.046, sample 1 of 1.035, sample 2 of 1.045, and sample 3 of 0.993.
  • SPF serum sample 3 inhibition rate (%) 100- (100 X [0.993-0.137
  • competitive dilutions can detect serum dilutions of approximately 100-fold to 500-fold (competitive ELISA values 100-500), and AGP studies approximately 10-fold to 250-fold (AGP Values of 10 to 250) were detectable (Table 1).
  • competitive ELIS A positive limit ZAGP test positive limit was about 1 to 10 times.
  • FIG. 1 shows setting of Cut Off value of competitive ELISA according to the present invention.

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Abstract

L'invention concerne un réactif de détection et un procédé de détection de l'infection par l'influenza, au moyen d'une protéine nucléaire (NP) d'un virus de l'influenza en tant qu'antigène de détection d'anticorps AIV. De préférence, la protéine nucléaire, (NP) dans laquelle une séquence marqueur d'histidine est ajoutée à un gène de protéine nucléaire (NP) d'AIV A/Duck/Aomori/478/02(H1N1) et ledit gène est exprimé en tant que NP-His au moyen d'un baculovirus et d'une cellule d'insecte, est utilisée en tant que l'antigène de détection de l'anticorps AIV. Selon l'invention, il est possible d'obtenir un réactif de détection et un procédé de détection d'infection de l'influenza qui permettent un traitement multi-échantillon, constituant un substitut au test de précipitation sur gel d'agar (PGA), et sont extrêmement sensibles et extrêmement spécifiques.
PCT/JP2006/321891 2006-04-13 2006-11-01 Réactif de détection et procédé de détection de l'infection par l'influenza WO2007122760A1 (fr)

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