JPH11180998A - Polypeptide for detecting borna disease virus antibody - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a polypeptide that contains a specific amino acid sequence, shows high specificity to the anti-BDV antibody and is useful as a diagnostic reagent for infections. SOLUTION: This compound is a polypeptide constituting the uncleoprotein (p40) or the polymerase cofactor (p24) and has the amino acid sequence of formulas I-VI or a substantially equivalent sequence thereto. A biological sample and one or more of these polypeptide are mixed to form an immune reaction product. In a preferred embodiment, this product is labeled and the labeled product is measured to detect the BDV antibody in the biological specimen.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a reagent for diagnosing infectious diseases for humans and animals. More specifically, the present invention relates to a peptide for detecting a Borna disease virus antibody, a detection method thereof, and a reagent used for the detection.

[0002]

BACKGROUND OF THE INVENTION Borna's disease is a viral encephalomyelitis that occurs in horses throughout the four seasons in Germany and surrounding countries. Symptoms such as cerebral palsy and emotional disorders are present, and are fatal when following an acute course. Lesions are common in the brainstem nucleus, especially in the midbrain, and form inclusions called Joest-Degen bodies in the nuclei of the nerve cells. Borna Disease V
irus, following BDV) is a virus belonging to Bornaviridae family, the genomic single-stranded about 9 kb, be RNA virus negative strand has been clarified. BDV naturally infects not only horses but also sheep, cattle, cats, ostriches, and the like, and can experimentally infect mice, birds, and monkeys more widely. Serological tests have also confirmed the presence of antibodies to BDV in humans (Rott, R. et al., Science, 228 , 755-756, 1
985) In particular, it has been reported that patients with neuropsychiatric disorders such as schizophrenia and depression have a higher rate than healthy subjects, and have received attention. The entire nucleotide sequence of the virus is found in Cubitt et al.
B., et al., J. Virol., 68 , 1382-1396, 1994), Brie
se et al. (Briese, T., et al., Proc. Natl. Acad. Sci. US
A, 91 , 4362-4366, 1994), and a nuclear protein (p4
0), polymerase cofactor (p24), matrix (gp
18), envelope (gp56) and polymerase (p180) proteins are known. As a method for diagnosing BDV infection, serologic methods for detecting anti-BDV antibodies in blood, RT-PC
Genetic techniques for directly detecting BDV genomic RNA by the R method have been used. As a method for detecting an anti-BDV antibody,
Indirect fluorescent antibody method (Amsterdam, JD, et al., Arch.Gen.
Psychiatry, 42 , 1093-1096, 1985) and Western blotting (Fu, ZF, et al., J. Affect. Disord., 27 ,
61-68, 1993), immunoprecipitation (Bode, L. et al., J. Med.
irol., 36 , 309-315, 1992), ELISA method (Weisman,
Y., et al., Lancet, 344 , 1232-1233, 1994), flow cytometry using monocytes in the peripheral blood of patients (Bode,
L. et al., Nature Medicine, 1 , 232-236, 1995). Although there are various methods for detecting antibodies as described above, anti-BDV antibody titers in humans are generally very low, making it difficult to discriminate between specific and non-specific reactions. However, a reliable standard antibody measurement method has not been established yet. As a method for directly detecting genomic RNA of BDV, RTN can be detected from mononuclear cells in human peripheral blood.
-A method for detecting a fragment of a viral gene by PCR has been reported (Kishi, M., et al., FEBS Lett.,
364 , 293-297, 1995). Although the method of directly detecting the genome is a highly accurate method, BDV is originally a neurotrophic virus, so that BDV genomic RNA is not necessarily detected in peripheral blood mononuclear cells of all infected individuals. Absent. In addition, the extremely high sensitivity of the RT-PCR method is always accompanied by the risk of a false positive reaction due to cross-contamination, and reliable and universal data has not been accumulated.

[0003]

An object of the present invention is to provide an anti-B
An object of the present invention is to provide an antigen peptide having high specificity to an antibody upon detection of a DV antibody, and to provide a measurement method and a measurement reagent thereof.

[0004]

DISCLOSURE OF THE INVENTION The present inventors have focused on peptides constituting BDV nuclear protein (p40) and polymerase cofactor (p24), and have focused on p40 3-20, 48-68, 310-323.
It has been found that a polypeptide having the amino acid sequence at positions 338-358 and a polypeptide having the amino acid sequence at positions 41-55 and 59-79 of p24 cause a specific immune reaction with an antiserum against BDV. completed. That is, the present invention relates to (1) a nucleic acid sequence encoding a polypeptide constituting the nucleoprotein (p40) or polymerase cofactor (p24) of the Borna disease virus, which comprises the sequence described in SEQ ID NOS: 1 to 6. A polypeptide consisting of or a polypeptide substantially equivalent thereto, (2) a method for detecting a Borna disease virus antibody using the polypeptide as an antigen, and (3) a method for detecting a Borna disease virus antibody comprising the polypeptide as a component. It is a detection reagent. In addition, a polypeptide substantially equivalent to the polypeptide of the present invention described in SEQ ID NOs: 1 to 6 is also included in the present invention. Substantially equivalent means having a binding activity to the Borna disease virus antibody, and includes deletion, substitution and addition of amino acids.

[0005]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail.
Using the peptide according to the present invention, anti-BDV in a biological sample
The presence or absence of the p40 antibody and / or the anti-BDV p24 antibody can be assayed by an immunological technique, and infection diagnosis of the virus is possible. Biological samples include, for example, human and animal serum, plasma, spinal fluid, lymph, saliva, serum, cells, and the like. Known methods may be applied as the immunological method, such as electrochemiluminescence immunoassay, enzyme immunoassay, radioimmunoassay, Western blot,
Passive particle agglutination, passive hemagglutination, latex agglutination, chemiluminescence immunoassay, and the like. The measurement method by the electrochemiluminescence immunoassay will be described below as an example.
Components of the measurement system are a carrier for immobilizing the antigen peptide,
A test sample, a labeling antibody, an electrochemiluminescent substance as a labeling substance, and the like. The method for producing the antigenic peptide is not particularly limited, and includes methods such as ordinary chemical synthesis, extraction from BDV-infected cells, and expression in genetically modified Escherichia coli and the like, but a synthetic method capable of obtaining a highly specific peptide. Is preferred. Examples of the synthesis method include a solid phase synthesis method. Further, in order to facilitate the adsorption of the peptide to the carrier, an amino acid residue such as cysteine or lysine may be added to the terminal of the peptide. As a carrier for immobilizing a peptide, any carrier such as a magnetically responsive particle, a microtiter plate, a plastic bead, an erythrocyte, a gelatin particle, a polyamino acid particle, a latex particle, a nylon membrane, and a nitrocellulose membrane can be used. , May be appropriately selected according to the measurement method. In the case of the electrochemiluminescence method, magnetic particles, latex particles and the like are preferable. As a labeling antibody, a human IgG antibody, a human IgM antibody, a human IgA antibody and the like are used. As an electrochemiluminescent substance as a labeling substance, ruthenium-tris-bi-
Pyridyl, osmium-tris-bi-pyridyl, cadmium-tris-bi-pyridyl, rhenium-tris-bi-
Pyridyl and the like. The procedure of the measurement is as follows. The antigen peptide is immobilized on magnetically responsive particles and reacted with the sample. After washing, it is reacted with an anti-human IgG antibody labeled with an electrochemiluminescent substance. After further washing,
Electric energy is applied to the magnetically sensitive particles, and the amount of light emitted from the electrochemiluminescent substance is measured. The measurement reagent according to the present invention comprises at least one of the polypeptides shown in SEQ ID NOs: 1 to 6 as an essential component, and comprises a labeling antibody, a standard antigen, an enzyme, a substrate, and the like. When the peptide and the electrochemiluminescent substance are contained at the same time, they may be in a bonded state. Further, depending on the convenience of measurement, an appropriate antigen diluent, reaction diluent, washing solution, luminescence auxiliary solution, etc. may be included.

[0006] The recombinant BDVp4 used in the present invention
0 antigen protein and recombinant BDVp24 antigen protein
It can be prepared by a conventional genetic recombination technique. For example, p40 cDNA (Cubitt, B., et al., J. V
irol., 68 , 1382-1396, 1994). pB is a plasmid vector that incorporates cDNA.
Examples include luescript II SK, pBR322 and pUC18 derived from Escherichia coli, and psH15 derived from Bacillus subtilis. Any of these can be used as long as they are retained and replicated and amplified in host cells. The vector thus obtained is introduced into an appropriate host, for example, Escherichia coli, to prepare a transformant. As a method for introducing a vector into a host, for example, the competent cell method (J. Mol. Biol., 53 , 154, 1
970), protoplast method (Proc. Natl. Acad. Sci. USA,
75 , 1929, 1978), calcium phosphate method (Science, 221 ,
551, 1983), in vitro packaging method (Proc. Nat.
l. Acad. Sci, USA, 72 , 581, 1975), the virus vector method (Cell, 37 , 1053, 1984) and the like. Examples of Escherichia coli that can be a host include Escherichia coli
HB101, JM109 and the like. When the cDNA vector is a plasmid, it can be retained in host cells using the calcium chloride method or calcium chloride / rubidium chloride method (Molecular Cloning, A Laboratory Manua
l, Cold Spring Harbor Laboratory, p249, 1982). Then, after these transformants are grown by a usual culture method, p40 antigen protein can be obtained from the culture (Mole
cular Cloning, A Laboratory Manual, Cold Spring Har
bor Laboratory, 1982).

[0007]

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples. Example 1 Preparation of BDV Synthetic Peptide Cubitt et al. (Cubitt, B., et al., J. Virol., 68 , 1382-1)
396, 1994) from the amino acid sequence of the BDV nucleoprotein (p40) and polymerase cofactor (p24) sites.
Woods method (Hopp, TP, et al., Proc. Natl. Acad. Sc.
USA, 78 , 3824-3828, 1981), 13 sites with high hydrophilicity were selected (FIGS. 1 and 2). For each amino acid sequence, a peptide was synthesized by the solid phase synthesis method, and the purity was 95% using reverse phase liquid chromatography.
Purification was performed as described above.

Example 2 Screening of BDV Synthetic Peptide Recombinant BDV p40 antigen or Recombinant B
Rabbits were inoculated with the DVp24 antigen to prepare anti-BDVp40 serum and anti-BDVP24 serum, respectively. Normal rabbit serum was used as a control. Thirteen types of peptides synthesized in Example 1 were dissolved in a 0.05 M borate buffer (pH 9.5) to a concentration of 100 μg / ml. To 1 ml of each peptide solution, 30 mg of magnetically responsive beads (Dynabeads M-450 ^™ , manufactured by Dynal) were added and mixed at 37 ° C. overnight to prepare 13 types of peptide-bound beads. 200 μl of chicken serum in a reaction tube,
20 μl of rabbit antiserum and each peptide-bound bead (1 mg / m
l) 25 μl was added and reacted at 30 ° C. for 9 minutes. After washing the beads, an anti-rabbit IgG antibody solution (1
(μg / ml), and reacted at 30 ° C. for 9 minutes. After further washing the beads, the electric energy to the light emitting electrolyte after the addition 250 [mu] l, the amount of light emitted from the trapped ruthenium complex beads dedicated measuring instrument (Picolumi 8220 ^TM,
(Manufactured by Sanko Junyaku Co., Ltd.). As a result, the peptides No. 1, 2, 7 and No. 8 react specifically with the anti-BDVp40 rabbit serum, and the peptides No. 10 and No. 11 react specifically with the anti-BDV p24 rabbit serum. (Tables 1 and 2). The amino acid sequences of these peptides are shown in SEQ ID NO: 1 (No. 1), 2 (No. 2), 3 (No. 7), 4 (No. 8), 5 (N
o.10) and 6 (No. 11).

[0009]

[Table 1]

[0010]

[Table 2]

[0011]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 18 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Pro Lys Arg Arg Leu Val Asp Asp Ala Asp Ala Met Glu Asp Gln Asp 15 10 15 Leu Tyr

SEQ ID NO: 2 Sequence length: 21 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Gly His Glu Lys Asp Ile Arg Gln Asn Ala Val Ala Leu Leu Asp Gln 15 10 15 Ser Arg Arg Asp Met 20

SEQ ID NO: 3 Sequence length: 14 Sequence type: amino acid Topology: linear Sequence type: peptide sequence Ser Lys Lys Glu Asn Pro Thr Met Ala Gly Tyr Arg Ala Ser 15 10

SEQ ID NO: 4 Sequence length: 20 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Arg Tyr Arg Arg Arg Glu Ile Ser Arg Gly Glu Asp Gly Ala Glu Leu 15 10 15 Ser Gly Glu Ile 20

SEQ ID NO: 5 Sequence length: 15 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Gln Pro Val Asp Gln Leu Leu Lys Asp Leu Arg Lys Asn Pro Ser 15 10 15

SEQ ID NO: 6 Sequence length: 21 Sequence type: amino acid Topology: Linear Sequence type: Peptide sequence Asp Pro Asp Gln Arg Thr Gly Arg Glu Gln Leu Ser Asn Asp Glu Leu 15 10 15 Ile Lys Lys Leu Val 20

[0017]

[Brief description of the drawings]

FIG. 1 shows the hydrophilic pattern of the amino acid sequence of the nuclear protein (p40) of BDV and the site of the synthesized peptide.

FIG. 2 is a diagram showing a hydrophilic pattern of a polymerase cofactor (p24) amino acid sequence of BDV and a site of a synthesized peptide.

Claims (3)

[Claims] 1. A polypeptide comprising the nucleoprotein (p40) or polymerase cofactor (p24) of the Borna disease virus, comprising a polypeptide comprising the amino acid sequence shown in SEQ ID NOs: 1 to 6, or a polypeptide comprising the same. A substantially equivalent polypeptide. 2. A method for detecting an antibody against a polypeptide constituting a nucleoprotein (p40) and / or a polymerase cofactor (p24) of a Borna disease virus in a biological sample, comprising: (B) mixing at least one of a polypeptide comprising the amino acid sequence shown in any one of 1 to 6 or a polypeptide substantially equivalent thereto to generate an immunoreactant;
A method for detecting a Borna disease virus antibody, comprising the steps of: labeling the immunoreactant; and (c) measuring the label. 3. A polypeptide comprising the nucleoprotein (p40) and / or polymerase cofactor (p24) of the Borna disease virus, comprising a polypeptide comprising the amino acid sequence shown in SEQ ID NOs: 1 to 6, or A reagent for detecting a Borna disease virus antibody, comprising at least one of polypeptides substantially equivalent thereto as a component.