JPH07147986A - Human parvovirus gene, polypeptide coded with the same and use - Google Patents

Abstract

PURPOSE:To provide the gene coding a human parvovirus (HPV) antigen protein, enabling to massively produce the antigen protein, capable of being utilized for the high sensitive measurement of the HPV and the HPV-resistant antibody in a specimen, and useful for the diagnoses of HPV-originated diseases, etc. CONSTITUTION:The serum of a patient infected with infectious erythema is diluted with a phosphate-buffered physiological saline (pH: 7.2), mixed with proteinase K and sodium dodecyl sulfate, subjected to their reaction at 37 deg.C for 1hr, mixed with phenol, chloroform and isoamyl alcohol, and subsequently centrifuged to recover the supernatant liquid. The supernatant liquid is added to ethanol and subsequently allowed to stand at -20 deg.C to obtain the precipitates of human parvovirus DNA. The DNA is multiplied by a PCR method, and then treated with a restriction enzyme to obtain the objective human parvovirus gene enabling the mass production of the human parvovirus antigen protein and enabling the measurement the human parvovirus or the human parvovirus- resistant antibody in a specimen in high sensitivity.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel human parvovirus gene involved in the pathogenesis of various diseases such as acute erythroblast wax, chronic bone marrow failure, abortion, fetal edema, liver injury, hemorrhagic fever, arthritis and rheumatism. , A polypeptide it encodes and its use.

[0002]

2. Description of the Related Art Parvovirus is a single-stranded linear DNA.
It has a genome, and each end of the genome has a palindromic (Pa
llindromic) DNA sequence, the smallest virus group (Parvoviridae) with a characteristic hairpin loop structure.
Family).

Parvoviridae virus particles have a cubic symmetry, exhibit Cubic symmetry, have 32 capsomeres, are small viruses with a diameter of about 20-25 nm, do not have an envelope, and are ether-resistant. is there. It is also a virus that is relatively resistant to heat and drying [Intervirology, 23, 61-73 (1985)]. Viral replication and capsid formation occur in the nucleus of infected cells.

Vertebrate parvoviruses are divided into autonomous and defective groups.

The autonomous parvovirus <Parvovirus> can continue to replicate in the host cell, but the defective parvovirus <Depevirus (Depevirus)
ndovirus)> requires coinfection by a helper virus. Autonomous animal parvoviruses include:
Bovine (BPV), Pig (PPV), Dog (CPV), Cat (FPL)
V), rat (H-1), mink (ADV), and human (HPV
) Are known, and generally have similar pathogenicity, but no immunological cross-reactivity has been found between these viruses.
Therefore, it is important that the virus is derived from human parvovirus when diagnosing the human parvovirus, conducting an epidemiological survey and developing a vaccine.

Human parvovirus was accidentally found in blood for transfusion in 1975 [Lancet, i: 72-73 (1975)], but its pathogenicity was unknown. Subsequently, it was reported that the acute erythroblast wax that occurred in sickle cell anemia was due to human parvovirus [Lancet, i: 664-665 (1981)]. Also,
It has been confirmed that it is a causative virus of erythema infectious disease that prevails in children [Lancet, i: 1378 (1983)], and it becomes a source of infection, which also affects adults and causes diseases with arthritis as the main symptom. I understood. Furthermore, a case of abortion due to fetal edema was reported after the epidemic of contagious erythema [Lancet, ii: 1033-1034].
(1984)], it was proved that human parvovirus infection in pregnant women causes stillbirth and edema of the fetus, and fetal death cases due to human parvovirus infection were also reported in Japan [Journal of Obstetrics and Gynecology, 40:99]. -100 (1988)]. And even in healthy adults, it is known to cause indefinite eruption, hemorrhagic fever-like disease, and arthritis [History of Medicine, 142: 530-5
32 (1987)], and recently, a relationship with rheumatism has been speculated [Arch.Intern.Med.148: 2587-2589 (1988)].

[0007] As described above, human parvovirus is a clinically extremely interesting virus and may affect human life. Therefore, the necessity of treatment and preventive measures has become clear. .

[0008] However, human parvovirus started from in vitro culture using human primary bone marrow cells, and culture using red blood cells obtained from patients with chronic myelogenous leukemia or erythroblasts in fetal liver cells. Is possible, but
A culture system sufficient for industrial use has not yet been found. Therefore, it is difficult to obtain the human parvovirus antigen, the generalization of the diagnostic system has been delayed, and clinical tests cannot be easily performed at present.

Human parvovirus is generally caused by nasal infection, but it is unusually transmitted by blood transfusion and infusion of blood products. It was proved in the in vitro hematopoietic stem cell culture system that the infected target cells were erythroid progenitor cells [Nature, 302: 426-429 (1983)].

When infected with human parvovirus, viremia occurs after one week, but fever, myalgia, and malaise only appear, and symptoms such as erythema and arthralgia appear from about 2 to 3 weeks after infection. To be observed. These symptoms are believed to be due to immune complexes with viral antibodies. Therefore, it is difficult to detect the viral antigen because the virus is almost exterminated when the symptoms appear.

Therefore, the proof of human parvovirus infection is
Since it is difficult to detect viral antigens, it is performed by detecting anti-human parvovirus antibody in serum.

Diagnosis of human parvovirus is carried out at a limited number of facilities by the following method using an antigen-antibody reaction system in which virus particles are purified from the sera of viremia during the epidemic of infectious erythema. It's just happening. Immuno-electric countercurrent method (medical history, 135: 317-318 (1985)), enzyme immunoassay method (medical history, 134: 909-910 (198)
5)], Radioimmunoassay [Journal of Infectious Diseases, 58: 1213
-1220 (1985)]. However, the immunoelectric countercurrent method has a low detection sensitivity, and when an enzyme immunoassay method or a radioimmunoassay method is used, the lack of the amount of viral antigen becomes a problem.

Under such circumstances, the acquisition of a useful and large amount of viral antigen is useful and indispensable for the development of a diagnostic system for human parvovirus.

In general, a method for obtaining a viral antigen is a method in which a virus is propagated in a cell culture, or a gene recombination method, for example, cloning a viral gene,
It is roughly classified into a method of expressing a large amount of an antigen protein and non-infectious virus particles.

The cell culture system is erythroid cells [J. Infect. Dis. 160: 548 (198) obtained from a patient with chronic myelogenous leukemia.
9)], erythroblast cells in fetal liver cells [J. Virol. 63: 2422-24
26 (1989)] and can be cultured only in a limited cell line. However, the virus recovery rate is extremely low, and the number of virus target cells in normal bone marrow is extremely low, making it difficult to culture other than fresh primary culture, and the cell culture system has a limited range of use. Recently, sensitivity to one of the established cell lines was confirmed, but the growth rate of the virus is extremely low in this system [Clinical and Microbial, 16: 177-186 (1989)]. Thus, it is impossible to obtain a useful and large amount of human parvovirus antigen by the method using the human parvovirus cell culture system.

On the other hand, the human parvovirus gene is required for obtaining useful and large-scale expression of viral antigens by the gene recombination method. At present, the entire sequence of the human parvovirus B19 strain gene derived from sickle cell anemia patient sera infected with human parvovirus has been reported [J. Virol., 58: 921-936 (1986)]. From the analysis of the nucleotide sequence of the human parvovirus gene cloned in the United States, it is found that VP-1 (molecular weight about 84 KDa) and VP-2 constitute viral particles as viral structural proteins.
There are only two types of proteins (molecular weight of about 58 KDa), and the host infected with human parvovirus can detect these VP-1 and V
It has been confirmed that the antigen-antibody reaction is induced by targeting P-2 [J. Virol., 61: 2627 (1987)].

Therefore, a report example in which a part of the human parvovirus structural protein gene was amplified from the serum of an infectious erythema patient by the PCR method and cloned [JP-A-4-88985]
And a partial gene region of the human parvovirus structural protein gene, a reported example using an enzyme immunoassay with an antigen expressed in Escherichia coli [J. Clin. Microbiol.,
30: 305-311 (1992)].

However, these are reported examples of a part of the human parvovirus structural gene, and utilize only a part of the human parvovirus structural protein. And
The human parvovirus antibody detection using this enzyme immunoassay had a lower detection rate than RIA using human parvovirus particles purified from the serum of an infectious erythema patient as an antigen.

[0019]

Therefore, an object of the present invention is to enable large-scale culture of human parvovirus antigen protein and to measure human parvovirus or anti-human parvovirus antibody in a sample with high sensitivity. It is to provide a means to make possible.

[0020]

Therefore, the present inventors have
As a result of an examination to obtain a gene region encoding a non-structural gene and a structural gene of human parvovirus, it was found that the known human parvovirus gene, amino acid level and D
Human parvo containing a non-structural gene and a structural gene, which is useful for the development of a novel and diagnostic system having several structures different at the NA level and is capable of producing, for example, an empty particle of human parvovirus (Empty particle) As a result of the examination using the enzyme immunoassay with the full-length structural proteins VP-1 and VP-2 capable of obtaining the viral gene region and expressed from this gene region, the human parvovirus structural proteins VP-1 and VP were found. -2
Of immunological reaction of
It was found that the enzyme immunoassay using e had the same detection sensitivity and specificity as the enzyme immunoassay using human parvovirus particles, and that Tn5 expressed more human parvovirus protein than insect cell Sf9. The present invention has been completed and the present invention has been completed.

That is, the present invention provides the human parvovirus gene represented by the above formula (1). The present invention also provides a human parvovirus VP-1 structural gene encoding the amino acid sequence of the above formula (2). further,
The present invention provides a human parvovirus VP-2 structural gene encoding the amino acid sequence of the above formula (3). The present invention also provides a human parvovirus nonstructural (NS) gene encoding the amino acid sequence of the above formula (4). The present invention further provides a recombinant vector containing any of the above genes of the present invention and capable of expressing the gene in a host cell. Furthermore, the present invention provides a transformant transformed with the above recombinant vector of the present invention. The present invention also provides a polypeptide having the amino acid sequence represented by any of the above formulas (2) to (4). Furthermore, the present invention provides virion-like structures of human parvoviruses composed of these polypeptides. The present invention also provides an antibody using any of the polypeptides of the present invention or the virus particle-like structure of the present invention as an immunogen, and detects human parvovirus in a sample by immunoassay using the antibody. Provide a way to detect. Further, the present invention provides a method for detecting an anti-human parvovirus antibody in a sample by an immunoassay using the above-mentioned polypeptide of the present invention or the above-mentioned virus particle-like structure of the present invention as an antigen. Furthermore, the present invention uses a pair of oligonucleotides each hybridizing with a part of the gene represented by the above formula (1) to amplify the gene represented by the formula (1) or a part thereof by the PCR method, A method for detecting human parvovirus in a sample, which comprises detecting the amplified gene or a part thereof.

As will be described in more detail later, the above equation (1)
The nucleotide sequences of the genes represented by (4) to (4) are novel because they are different from the nucleotide sequences of the conventionally known human parvovirus genes. By using this full-length gene to express a polypeptide and immunoassay using the gene, human parvovirus or anti-human parvovirus antibody in a sample can be detected with high sensitivity. In addition, although it is preferable to use the full-length genes described in each formula as described above, it is possible that even if a part of these genes is lacking, detection can be performed with the same high sensitivity. It is undisputedly recognized by the vendor. Therefore,
Even if some of these genes are deleted, substituted or inserted, those that can be used in the same manner as the genes of formulas (1) to (4) are described in formulas (1) to (4). It is substantially the same as what was done and is intended to fall within the scope of the present invention.

As described above, the present invention provides a recombinant vector containing the gene of the present invention and capable of expressing the gene in a host cell. Such a recombinant vector can be easily prepared by inserting the above gene into the cloning site of a commercially available vector by a conventional method. As the vector, a vector for E. coli such as pUC19 can be used, and as will be apparent from the examples described below, a baculovirus vector (J. General Virology, Vol.68, pp.1233-1250, (1987)
It is also preferable to use.

The transformant of the present invention can be obtained by transforming a host cell with the above-mentioned recombinant vector of the present invention by a conventional method. As the host cell, a host of the constructed recombinant vector is used. That is, when a gene is inserted into an E. coli vector to construct a recombinant vector, E. coli serves as a host. Insect cells can be preferably used when a recombinant vector is constructed using a baculovirus vector. In particular, Tn5 (In
High Five (trade name) manufactured by Invitrogen is preferably used.

As described above, the present invention provides a polypeptide having the amino acid sequence of the above formulas (2) to (4). As in the case of the above, even if a part of the polypeptide having the amino acid sequence described in formulas (2) to (4) is deleted, substituted or inserted, the formulas (2) to (4) Which can be used in the same manner as the polypeptide having the amino acid described in (1) to (4).
It is substantially the same as the polypeptide having the amino acid described in 1. and is intended to fall within the scope of the present invention. As described in detail in Examples below, human parvovirus virus particle-like structures can be constructed from these polypeptides, and these virus particle-like structures are also included in the scope of the present invention.

Using these polypeptides or virus particle-like structures as antigens, anti-human parvovirus antibodies in a sample can be detected by immunoassay. The immunoassay method itself is well known in the art, and in the present invention, various conventionally well-known immunoassay methods, that is, enzyme immunoassay methods if classified based on the label used,
A radioimmunoassay method, a fluorescent antibody analysis method, and the like can be adopted, and a competitive method, a sandwich method, and the like can be adopted by classifying in a measurement format. The inventors of the present invention have found that in the body of a patient infected with human parvovirus, anti-VP-2 antibody is abundant in the early stage of infection and rapidly decreases in the latter stage of infection, whereas anti-VP-1 antibody is infected. It was found that there were few in the early stage but many in the late stage of infection, and the antibody titer slowed down. Therefore, VP-1 and VP-2
When an immunoassay is performed using a mixture of polypeptides as an antigen, anti-human parvovirus antibody can be detected in the early and late stages of infection, and the detection rate can be increased. In this case, the mixing ratio of VP-1 and VP-2 is 10 :.
1 to 1:10 is preferable, and 1: 2 to 1: 4 is more preferable. Since the above virus particle-like structure contains both VP-1 and VP-2 polypeptides, it is possible to bring about the above detection rate improving effect by using this as an antigen. Further, since there is a difference in reactivity and antibody amount between the anti-VP-1 IgG antibody and the anti-VP-2 IgG antibody, the IgG antibody is detected by the IgG antibody detection using the VP-1 antigen solid phase plate and the VP-2 antigen solid phase plate. It was possible to estimate the time of infection only by detection.

Further, using the above-mentioned polypeptide or virus particle-like structure as an immunogen, an antibody against the polypeptide or virus particle-like structure is prepared by a conventional method, and this antibody is used in an immunoassay method. It is also possible to detect human parvovirus in a sample. As the immunoassay, any well-known method as described above can be adopted.

Further, as mentioned above, the present invention hybridizes with a part of the gene represented by the above formula (1).
A method for detecting human parvovirus in a specimen, which comprises amplifying the gene represented by the formula (1) or a part thereof by PCR using a pair of oligonucleotides and detecting the amplified gene or a part thereof. I will provide a. The DNA primer (oligonucleotide) used in this method is not particularly limited, but it is preferable to use at least one selected from the following DNA primer group 1 and DNA primer group 2. DNA primer group 1 D-1: GCTGCCATGTGGGAGCTTCTAATC D-4: TTCCCGCCTTATGCAAATGGGCAGC DNA primer group 2 U-2: GTGTTAGGCTGTCTTATAGGTACA U-4: CTTGTATTCATGGTCTACTAAC

The present invention will be described in more detail below, but the present invention is not limited to the following.

1. Human parvovirus gene region including nonstructural and structural genes Human parvovirus is a virus having a linear single-stranded DNA genome of about 5.4 kb in length, and each end of the genome is a palindromic DNA. Depending on the array
It ends with a characteristic hairpin loop structure.

In order to obtain a gene region containing a non-structural gene and a structural gene of human parvovirus, for example, humans are routinely prepared from virus particles purified by sucrose density gradient centrifugation from serum of a patient with erythema infectiousum (EI). Parvovirus DNA is extracted and purified for cloning.

The following method is also possible. It is the human parvovirus B19 Au strain gene sequence [J. Virol. 58: 921-936 (1986)] and Wi strain [Virolo reported in Europe and America.
gy, 157: 534-538 (1987)], using a DNA primer synthesized according to the polymerase chain reaction [PCR method;
ture, 324: 163 (1986)], the target gene region of the human parvovirus including the nonstructural gene and the structural gene can be amplified.

Specifically, for example, DN comprising a partial sequence of the gene is added to purified human parvovirus DNA.
A consisting of a primer and a partial sequence complementary to the gene
Two kinds of DNA primers, NA primer, are annealed, and the DNA between the DNA primers is prepared by a conventional method.
To be amplified.

The most preferred primers are as described above. When PCR is carried out using this primer, the gene region containing the non-structural gene and the structural gene of human parvovirus can be amplified. The obtained DNA fragment is inserted into a general cloning vector such as pUC19 (manufactured by Takara Shuzo Co., Ltd.) and the like, and plasmid DNA is extracted from the obtained clone, and the DNA sequence incorporated in the vector is treated with dideoxy. By determining by the method or the like, it is possible to determine the entire base sequence of the gene region including the target non-structural gene and structural gene of human parvovirus.

According to the present invention, the gene region containing the non-structural gene and the structural gene of human parvovirus has the nucleotide sequence represented by the above formula (1), and the structural gene VP-1 is represented by the above formula. The nucleotide sequence and amino acid sequence represented by (2), the structural gene VP-2 is represented by the formula (3), and the nonstructural gene NS is represented by the formula (4). It had a base sequence and an amino acid sequence. These formulas (1) to (4)
In the nucleotide sequence represented by, 41 positions at the base level and 10 to 13 positions at the amino acid level were different from the sequence of the known human parvovirus B19 strain (Au strain, Wi strain).

2. Human parvovirus structural protein V
Construction of P-1 and VP-2 and non-structural protein NS and construction of antigen and antibody detection system using them VP-1 (84 KDa) is present in virus particles as a viral structural protein And VP-2 (58), which is the main component of virus particles in quantity.
KDa), and the host produces antibodies against VP-1 and VP-2. Therefore, an antibody against human parvovirus can be detected by using the VP-1 and VP-2 antigens.

Human parvovirus VP-1, VP-2
In order to obtain the antigen, the human parvovirus antigen must be secured. However, until now, there have been only reports of human parvovirus cell culture systems using bone marrow cells of patients with specific genetic diseases [Blood. 70: 384-391 (1987)], and the virus recovery rate Since it is extremely bad, its range of use was actually limited.

Therefore, full-length human parvovirus structural proteins VP-1 and VP-2 were prepared as follows by using the human parvovirus nonstructural gene of the present invention and the gene region containing the structural gene.

A gene region containing a human parvovirus non-structural gene and a structural gene, the above formula (1) and the human parvovirus structural gene VP-1, the above formula (2) and the human parvovirus structural gene VP-2, the above formula ( 3) is cleaved with a restriction enzyme or the like that is most convenient for protein expression, and a prokaryotic (eg Escherichia coli etc.) expression vector, eukaryotic (eg CHO cell etc.) expression vector, insect cell (eg Sf9, Tn5 etc.) Re-cloning into an expression vector to express.

The expressed full-length human parvovirus structural proteins VP-1 and VP-2 were insoluble and required purification. Therefore, the expressed protein was solubilized with a urea buffer, purified by a combination of appropriate adsorption, ion exchange, molecular sieving, hydrophobicity, isoelectric point, and chromatography, and the full-length human parvovirus structural proteins VP-1, VP- were purified. Got 2.

The expressed and purified full-length human parvovirus structural proteins VP-1 and VP-2 were diluted with a carbonate buffer (pH 9.5) to an optimum concentration, and then subjected to ELISA.
A human parvovirus antibody detection enzyme immunoassay reagent was prototyped by immobilizing it on a polystyrene microplate for commercial use.

Using this prototype enzyme immunoassay reagent, anti-human parvovirus I in patients with erythema infectiousum was used.
The change over time of the gG antibody was examined and the following was found. Full-length human parvovirus structural proteins VP-1, VP-
IgG antibodies against 2 also appear, but IgG against full-length human parvovirus structural protein VP-1
It was found that the antibody has a longer duration of antibody amount than the IgG antibody against the full-length human parvovirus structural protein VP-2.

Here, the coding region for the protein of the full-length human parvovirus structural gene VP-2 is completely contained in the C-terminal portion of the full-length human parvovirus structural gene VP-1. Therefore, it was previously believed that the human parvovirus structural protein VP-1 alone was sufficient for the detection of human parvovirus antibodies.

However, since it was revealed that the immunological reactivity of the full-length human parvovirus structural proteins VP-1 and VP-2 was different, the full-length human parvovirus can be more reliably diagnosed. It was found that the full-length human parvovirus structural protein VP-2 as well as the human parvovirus structural protein VP-1 is required.

3. Empty par using baculovirus
Expression of particles, purification, and construction of antibody detection system using empty particles Human parvovirus particles are structural proteins VP-
1 and VP-2 are known.
[J. Virol., 58: 921-936 (1986)] When the structural proteins VP-1 and VP-2 are individually expressed in the same cell, the structural proteins VP-1 and VP-2 are expressed in the same cell. It was found that a virus particle-like structure (Empty particle) was created. (Proc.Natl.Acad.Sci.USA, 86: 7601-7
605 (1989))

Therefore, expression of virus particle-like structures was carried out using an expression system using baculovirus. Empty
Expression of particles was performed by the following method using the human parvovirus nonstructural gene of the present invention and the gene region containing the structural gene.

A gene region containing a human parvovirus non-structural gene and a structural gene, the above formula (1) and the human parvovirus structural gene VP-1, the above formula (2) and the human parvovirus structural gene VP-2, the above formula ( 3) is cleaved with a restriction enzyme or the like that is most convenient for expression of full-length VP-1 and full-length VP-2 proteins, and then eukaryotic cell (eg, CHO cell) expression vector, insect cell (eg, S cell).
f9, Tn5, etc.) and cloned into an expression vector for expression.

Next, from the examination of human parvovirus protein expression using insect cells (Sf9, Tn5), it was confirmed that the expression level was higher in Tn5 cells than in Sf9 cells.

Since the expressed Empty particles have a virus particle-like structure, a method for purifying virus particles can be applied. For example, it can be purified by a sucrose density gradient centrifugation method, a cesium chloride density gradient centrifugation method, or the like.

The expressed and purified Empty particles are diluted with carbonate buffer (pH 9.5), PBS and the like to an optimum concentration and then solid-phased on a polystyrene microplate for ELISA to detect human parvovirus antibody enzyme immunoassay reagent. Was produced.

The expressed and purified Empty particl
e is diluted to the optimum concentration with PBS etc.
An enzyme immunoassay reagent was prepared by the icle capture method.

Using this prototype enzyme immunoassay reagent, human parvovirus antibody detection was performed using serum at the time of epidemic erythema infestation. As a result, detection sensitivity and specificity almost equal to those of the enzyme immunoassay using human parvovirus particles purified from patient serum were obtained. Therefore, the baculovirus-expressed protein Em of the present invention is
Since the enzyme immunoassay reagent using pty particles has the highest detection sensitivity and specificity, it was found to be a more reliable and clinically meaningful human parvovirus antibody detection reagent than before.

[0053]

INDUSTRIAL APPLICABILITY The gene region containing the human parvovirus non-structural gene and structural gene of the present invention is transformed with the whole sequence or a DNA fragment obtained by the PCR method into a known expression vector, and then transformed with the expression vector. By expressing in a host cell, a recombinant antigen that can be used for diagnosis such as radioimmunoassay and enzyme immunoassay can be obtained. In addition, the IgG antibody against human parvovirus structural gene VP-1 is compared with the IgG antibody against human parvovirus structural gene VP-2.
Based on the long-lasting antibody amount, it is possible to develop a human parvovirus antibody detection system capable of inferring the time of infection using only IgG antibody. In addition, at the early stage of infection, the antibody against human parvovirus structural protein VP-2 is more human parvovirus structural protein VP-.
The human parvovirus structural protein VP-
By mixing 1 and VP-2, a human parvovirus antibody detection reagent having a higher detection rate than conventional anti-human parvovirus antibody detection systems can be developed. In addition, since the enzyme immunoassay using the baculovirus-expressed protein Empty particle shows the same detection sensitivity and specificity as the enzyme immunoassay using human parvovirus particles, it is more reliable and clinically meaningful than before. A new human parvovirus antibody detection reagent can be developed. Insect cells (Sf9, Tn5)
From the examination of the expression level using S.
It was found that the expression level was higher than that using f9 cells, and a high-yield large-scale expression system of human parvovirus antigen can be established.

The present invention will be described in more detail based on the following examples, but the invention is not intended to be limited to these examples.

[0055]

EXAMPLES Example 1 Cloning of human parvovirus gene (1) Preparation of human parvovirus genomic DNA Serum (serum No. 8 and No. 10) of two patients infected with human parvovirus at the time of epidemic of infectious erythema, respectively. About 200 μl was diluted 10-fold with phosphate buffered saline [PBS] (pH 7.2). To the diluted serum, protein nause K having a final concentration of 100 μg / ml and sodium dodecyl sulfate [SDS] having a final concentration of 1% were added and reacted at 37 ° C. for about 1 hour. Then, the reaction solution was divided into equal amounts, and TE saturated phenol,
Chloroform and isoamyl alcohol (25:24:
An equal amount of 1) was added and mixed. After mixing, 15,000 rp
The supernatant was recovered by centrifugation for about 10 minutes. Glycogen at a final concentration of 1 µg / ml was added to the recovered supernatant, 1/10 amount of 3M sodium acetate was added to the supernatant, and then 2.5 times the amount of ethanol of the whole solution was added to -20 ° C. Then, the mixture was left standing overnight in ethanol precipitation.

Ethanol precipitation solution was added to 15,000 rp
Centrifuge for about 10 minutes. The precipitate was washed with 70% ethanol and then dried. After drying to dryness, TE buffer (10 mM
It was dissolved in 20 μl of Tris-HCl (pH 7.5) -1 mM EDTA).

(2) Amplification of human parvovirus gene and determination of nucleotide sequence Human parvovirus genome DN obtained in Example 1- (1)
Using 1 μl of solution A, the method of Saiki et al. [Nature, 324: 1
63 (1986)], the gene region containing the human parvovirus nonstructural gene and the structural gene was amplified by the PCR method using the Gene Amp PCR Reagent Kit (manufactured by Takara Shuzo). That is, 50 mM KCl, 10 mM Tris-HCl
(PH 8.3), 5 mM MgCl2, 0.1% gelatin in a reaction solution containing human parvovirus DNA solution 1μ
l and the following two types of DNA primers 100p each
moles and Taq polymerase 5 units,
The final volume was 100 μl. D-4: TTCCCGCCTTATGCAAATGGGCAGC U-2: GTGTTAGGCTGTCTTATAGGTACA FIG. 1 shows two types of DNA primer positions. The amplified fragment contains a human parvovirus non-structural gene and a gene region containing a structural gene (F), a gene region containing a human parvovirus structural gene (VP), and a gene region containing a human parvovirus non-structural gene (NS).

This reaction solution was added at (95 ° C., 1 minute)-(55
After carrying out 30 cycles of amplification reaction in a cycle of (° C, 2 minutes)-(72 ° C, 3 minutes), a further polymerase reaction was carried out at 72 ° C, 5 minutes to complete the DNA extension reaction. The reaction solution was fractionated by 1% agarose gel electrophoresis.
A PCR amplification product of about 4.7 Kb was confirmed. About this 4.
7Kb PCR amplification product is Geneclean II [BIO101]
After recovering the DNA with a DNA Blunting Kit (manufactured by Takara Shuzo), the DNA was further smoothed.

Next, 5 μg of pUC19 was digested with 5 units of the restriction enzyme SmaI for 2 hours at 37 ° C., and the 5 ′ end was dephosphorylated with alkaline phosphatase (BRL).

The PCR-amplified fragment and the pUC19 fragment were combined with Ligati.
on Kit (Takara Shuzo Co., Ltd.), and the CaCl ₂ method [Molecular Cloning, p250 Cold Spring Harbar Labora
was introduced into E. coli XL1-Blue. The 10 transformants obtained were appropriately selected, and the alkali of Birnboim et al.
DN by SDS method [Nucleic Acids Res., 7: 1513 (1979)]
A clone was purified and cloned with a PCR amplified fragment by restriction enzyme mapping (from serum No. 10: pUC19 / F8, serum N).
Origin of o.8: pUC19 / C2) was obtained. In addition, these transformants (Escherichia coli XL1-Blue (pUC19 / F8) and
Escherichia coli XL1-Blue (pUC19 / C2) has been deposited at the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology, and the deposit numbers are 131
No. 25 and 13124, Microtechnology Research Institute.

Plasmid DNA was purified from the obtained clones, and for each PCR amplified fragment, Sequenase Versio
n 2.0 DNA Sequencing Kit (United State Biochemical
(Manufactured by Corporation) or a DNA sequencer 373A manufactured by ABI company was used to determine the nucleotide sequence (FIGS. 2 to 10). As a result, the gene region containing non-structural genes and structural genes was 4,677 bp, and the gene region containing structural genes was 2,756 bp.
The gene region including bp and nonstructural gene was 3,383 bp. Therefore, a comparison was made with the known human parvovirus gene sequences (described above) analyzed in the United States and Europe (Tables 1 to 3).

[0062]

[Table 1]

[0063]

[Table 2]

[0064]

[Table 3]

From Tables 1 to 3, in the base sequences and amino acid sequences represented by the formulas (1) to (4), 41 positions at the base level and 10 to 13 positions at the amino acid level are known human parvovirus B19 strains. (Au strain, Wi strain). Further, these results suggest that human parvoviruses have regional differences between Europe and the United States and Japan, and it is found that the formulas (1) to (4) of the present invention are novel human parvovirus genes. It was

Example 2 VP-1 and V using E. coli
Expression of P-2 Antigen (1) Preparation of Expression Vector Having Multiple Cloning Sites PL promoter expression vector pPLN-CSF214CΔ95 5 μg described in JP-A-4-82900 was digested with restriction enzymes NdeI and H
indIII 1% after digesting with 5units each at 37 ℃ for 2 hours
A 3.5 Kb DNA fragment was fractionated by agarose gel electrophoresis, and the DNA was recovered by Geneclean II. Next, pUC19 was similarly digested with restriction enzymes NdeI and HindIII, and then subjected to 1% agarose gel electrophoresis to recover a 0.27 Kb fragment. 3.
The 5 Kb fragment and the 0.27 Kb fragment were ligated using Ligation Kit (manufactured by Takara Shuzo) and introduced into Escherichia coli JM109.

The 10 transformants obtained were appropriately selected,
Purify the DNA by the alkali-SDS method and map it with the restriction enzymes NdeI and HindIII to obtain the desired plasmid pP.
LN-MCS was obtained (Fig. 11).

(2) Preparation of VP-2 antigen expression vector 5 μg of the plasmid pUC19 / F8 obtained in Example 1 was digested with the restriction enzyme HindIII 5 units at 37 ° C. for 2 hours, and then 5.1 Kb of 1% agarose gel electrophoresis was performed. Fractionate the DNA fragments and
The DNA was recovered with clean II. This fragment was self-ligated and introduced into E. coli JM109. The 10 transformants thus obtained were appropriately selected, the DNA was purified by the alkali-SDS method, and the target plasmid pVP was obtained by mapping with the restriction enzymes PstI and HindIII.

10 μg of the plasmid pVP was digested with the restriction enzyme EcoRI 10
It was digested with units at 37 ° C. for 2 hours, and phenol extraction-ethanol precipitation was performed. After lysing the DNA, the restriction enzyme PstI
After partial digestion with 1 unit at 37 ° C. for 10 minutes, 1% agarose gel electrophoresis was performed. Fractionation of 1.8 Kb fragment
The DNA was recovered with clean II. Then plasmid pPLN-MCS
3 μg of 5 μg with 10 units each of NdeI and EcoRI restriction enzymes
After digestion at 7 ℃ for 2 hours, 1% agarose gel electrophoresis 3.
A 5 Kb DNA fragment was fractionated and the DNA was recovered by Geneclean II. Furthermore, the following oligonucleotides were synthesized using a DNA synthesizer. 5'-TATGACTTCAGTGAATTCTGCA-3 '5'-GAATTCACTGAAGTCA-3' Each of the above oligonucleotides was phosphorylated at the 5'end with MEGALABEL (Takara Shuzo), and then the phosphorylated oligonucleotide was annealed.

The 1.8 Kb fragment, the 3.5 Kb fragment and the annealed oligonucleotide were ligated and introduced into E. coli N4830-1. Twenty-four transformants obtained are appropriately selected, DNA is purified by the alkali-SDS method, and the restriction enzymes NdeI, Pst
Target plasmid by mapping with I and EcoRI
We obtained pVP200 (Fig. 12).

(3) Preparation of VP-1 antigen expression vector 10 μg of plasmid pVP was digested with restriction enzymes HindIII and BamHI at 10 units each at 37 ° C. for 2 hours, and a 1.5 Kb DNA fragment was fractionated by 1% agarose gel electrophoresis. And Genecl
DNA was recovered with ean II. Then plasmid M13mp19 RF
Similarly, 5 μg was subjected to 1% agarose gel electrophoresis with restriction enzymes HindIII and BamHI to recover a 7.2 Kb fragment.
After ligating the 1.5 Kb and 7.2 Kb fragments, E. coli JM109
Infected. Selection of recombinant phage was carried out in the presence of Xgal in La
cZ was used as an index. Twelve white plaques were appropriately selected and re-infected with Escherichia coli JM109. The RF-type DNA was purified by the alkali-SDS method, and the restriction enzymes HindIII and BamHI were used.
The desired plasmid p19-VP was obtained by mapping with.

Next, Escherichia coli JM109 was infected with the plasmid p19-VP to obtain phage. From this, single-stranded phage DNA was purified. In addition, an oligonucleotide having the following sequence was synthesized as a mutation-introducing primer. After annealing the 5'-TTACTCATATGCTACAAAGCTTGGCG -3 'single-stranded phage DNA and the mutagenic primer, T7-GEN (United States Biochemical Corporati
on (USB) manufactured by using site-directed mutation
d mutagenesis). site-directed mutagenesi
The method of S followed the protocol of USB company. The mutagenesis site is as shown in FIG. Twenty-four of the obtained phages were appropriately selected and E. coli JM109 was reinfected. RF type DNA was purified by the alkali-SDS method, and the target plasmid mp19-VP1 was obtained by mapping with the restriction enzyme NdeI and DNA sequencing.

20 μg of the plasmid mp19-VP1 was digested with the restriction enzyme Nd
After digesting with eI and BglI in 20units at 37 ℃ for 2 hours,
A 0.6 Kb DNA fragment was fractionated by 1% agarose gel electrophoresis, and the DNA was recovered by Geneclean II. Plasmid pV
After digesting P 10 μg with the restriction enzyme BstXI 10 units for 2 hours at 55 ° C, and further digesting with the restriction enzyme BglI 10 units for 2 hours at 37 ° C, fractionate the 1.7 Kb DNA fragment by 1% agarose gel electrophoresis and use the Geneclean II to separate the DNA. Recovered. Furthermore, plasmid pVP200 (10 μg) was digested with restriction enzyme BstXI 20 units to obtain 5
After digesting at 5 ℃ for 2 hours, further 3 with restriction enzyme NdeI 20 units
After digestion at 7 ℃ for 2 hours, 1% agarose gel electrophoresis 3.
The 8 Kb DNA fragment was fractionated, and the DNA was recovered by Geneclean II. The above 0.6 Kb, 1.7 Kb and 3.8 Kb DNA fragments were ligated and introduced into Escherichia coli N4830-1. 20 transformants obtained are appropriately selected, and DNA is selected by the alkali-SDS method.
Was purified and the desired plasmid pVP100 was obtained by mapping with the restriction enzymes NdeI, BstXI and PvuII (Fig. 1).
4).

(4) Expression of VP-1 and VP-2 antigens Escherichia coli N4830-1 [pVP100] and Escherichia coli N4830-1 [pVP200] were expressed.
1 ml of LB medium containing 50 μg / ml Ampicilin at 30 ℃, 1
Cultured for 6 hours. 1 ml of the preculture liquid was added to 10 ml of fresh LB
0 ml was inoculated. After culturing at 30 ° C for about 3 hours, 65
100 ml of LB medium warmed to ° C was added, and the mixture was further cultured at 42 ° C for 3 hours. After culturing, the cells were collected by centrifugation and collected.

A part of the bacterial cells was used as Phosphate Buffered-Saline.
After suspending in (PBS) and crushing the cells with an ultrasonic crusher, Laemm
SDS according to the method of Li [Nature, 227: 680 (1970)]
Polyacrylamide gel electrophoresis [SDS-PAGE]
I went. After the migration, Western blotting was performed using serum of infectious erythema patient [Proc. Natl. Acad. Sci. USA., 76: 311].
6-3120 (1979)]. As a result, the molecular weight is 84,000 and
Reacted to 58,000 positions. Therefore, VP-1 and VP-2
The expression of the antigen was confirmed.

In addition, the sonicated cells were treated with 5,500 g of 1
Centrifugation was performed for 0 minutes to separate a supernatant fraction and a precipitate fraction. Each fraction was subjected to SDS-polyacrylamide gel electrophoresis and stained with Coomassie Brilliant Blue (CBB). As a result, the VP-1 and VP-2 antigens were found in the precipitated fraction and formed an inclusion body. .

(5) Culturing of Escherichia coli expressing VP-1 and VP-2 antigen E. coli N4830-1 [pVP100] was added to L containing 50 μg / ml Ampicilin.
The cells were cultured in 2 ml of B medium at 30 ° C. for 7 hours. Next, using a 2 L fermentor, 1 L of JAR medium (0.7% Na ₂ HPO ₄ , 0.3% KH ₂ PO
₄ , 0.5% (NH ₄ ) ₂ SO ₄ , 0.1% sodium citrate, 0.02%
_{MgSO 4 · 7H 2 O, 2.5} % glucose, 0.4% yeast extract, 0.
4% casamino acid, 0.4% histidine, 0.4% isoleucine,
0.4% valine, 50 μg / ml ampicillin) at 30 ℃, about 16
Incubated for hours. Further, it was cultivated at 35 ° C. in JAR medium (40 L) using a 70 L fermentor. When OD ₅₅₀ = 5 was reached, casamino acid was added to a final concentration of 2% and histidine, isoleucine, and valine were added to a final concentration of 0.4%, respectively, and the mixture was cultured at 42 ° C for 3 hours.

The culture broth was concentrated with an ultrafiltration device and the bacterial cells were added to 5
L crush buffer (50mM Tris-HCl (pH8.0), 10mM EDTA, 30
Suspended in mM NaCl). The cells were disrupted using a Manton Gaulin homogenizer (7,500 PSI x 3 times). next
The precipitate was collected by centrifugation at 5,500 g for 30 minutes. This precipitate was added to 2.5 L of Detergent buffer (50 mM Tris-HCl (pH8.0),
Suspended in 50 mM NaCl, 10 mM EDTA, 0.5% Triton X-100),
After stirring at 4 ° C for 30 minutes, the precipitate was collected by centrifugation at 5,500g for 15 minutes. The suspension-centrifugation operation with this Detergent buffer was performed 3 times. The obtained precipitate (inclusion body; IBs) was stored at -20 ° C.

Escherichia coli N4830-1 [pVP200] was similarly obtained to obtain an inclusion body.

(6) Purification of VP-1 and VP-2 Antigens Inclusion of VP-1 obtained in Example 2- (5)
The body and wet weight of 50 mg were dissolved in 1 ml of IBs lysis buffer (8 M urea, 10 mM DTT, 1 mM EDTA, 20 mM Tris-HCl (pH 7.0)). Add 1 ml of this solution to the SDS sample buffer (130 mM Tr
is-HCl (pH6.8), 4% SDS, 15% Ficoll Type400 (Pharmac
ia), 10% β-mercaptoethanol, 0.002% bromophenol) and heat treated at 80 ° C for 20 minutes. From this sample ProSieve Gel System (FMC
The VP-1 antigen was purified using the product manufactured by K.K. Electrophoresis is performed according to the instruction manual to fractionate the protein in the sample for each molecular weight, and only the portion containing the VP-1 antigen is excised and extracted with an extraction buffer (8M urea, 10 mM DTT, 1 mM EDTA, 20
It was extracted from the gel with mM Tris-HCl (pH 7.0)). The gel plate used had a size of 14.5 cm x 16.5 cm x 2 mm. The extracted protein was electrophoresed using the Pharst System (Pharmacia) and silver-stained. As a result, a band was observed only at the molecular weight position (84,000) of the VP-1 antigen. The VP-2 antigen was similarly purified, and the molecular weight position of the VP-2 antigen (58,000)
Only recognized band.

Example 3 Preparation of VP-1 Antigen Derivative (1) Preparation of VP-1 Antigen Derivative Vector N-terminal side 46 amino acids and C-terminal side of VP-1 antigen (781 amino acids) of the formula (2) of the present invention A VP-1 antigen derivative (637 amino acids) with 98 amino acids deleted was prepared.

20 μg of pUC19 / F8 obtained in Example 1- (2)
Digested with 20 units of Ssp I restriction enzyme at 37 ℃ for 2 hours, then 1%
A 1.9 Kb VP-1 DNA fragment was fractionated by agarose gel electrophoresis, and the DNA was recovered by Geneclean II.

Next, 5 μg of the plasmid pKK233-2 (Pharmacia) was digested with 5 units of the restriction enzyme Nco I for 2 hours at 37 ° C., and the ends were made blunt with a Blunting Kit (Takara Shuzo).

The ligation kit was prepared by using the fragment of about 1.9 Kb and the vector.
Was ligated with E. coli and introduced into E. coli XL1-Blue. The 10 transformants thus obtained were appropriately selected, the DNA was purified by the alkali-SDS method, and the target plasmid pVP300 was obtained by mapping with the restriction enzymes PstI and HindIII (FIG. 15).

(2) Expression of VP-1 antigen derivative E. coli XL1-Blue [pVP300] was cultured in 1 ml of LB medium containing 50 μg / ml ampicillin at 37 ° C. for 16 hours. 1 ml of the preculture liquid was inoculated into 100 ml of fresh LB medium. 37
After culturing at ℃ for about 2 hours, the final concentration of 1 mM IPTG was added, and then culturing at 37 ℃ for another 3 hours. After culturing, Example 2
-As a result of examining the expression status by the method shown in (4), VP-
It was confirmed that the 1-antigen derivative (named VP-1 / SspI antigen) also formed an inclusion body. Furthermore, by Western blotting and a fluorescent antibody indirect method, the reaction was performed at the position of the molecular weight of 70,000. Therefore VP
It was found that the −1 / SspI antigen also has the same antigenicity as the VP-1 and VP-2 antigens.

Example 4 VP using baculovirus
-1 and VP-2 antigen expression (1) Preparation of recombinant transfer vector 20 μg of pUC19 / F4 obtained in Example 1- (2) was treated with the restriction enzyme Hi.
After digesting with ndIII 20 units for 2 hours at 37 ° C., a 2.5 Kb VP-1 DNA fragment was fractionated by 1% agarose gel electrophoresis, and the DNA was recovered by Geneclean II. Recovered VP-
One fragment was made blunt-ended with the Blunting Kit.

Similarly, 20 μg of pUC19 / F8 was added to the restriction enzyme HpaII.
20 units each with SalI and SalI at 37 ℃ for 2 hours, then 1
A 1.8 Kb VP-2 DNA fragment was fractionated by% agarose gel electrophoresis, and the DNA was recovered by Geneclean II. VP-
The two fragments were made blunt-ended with the Blunting Kit.

On the other hand, the transfer vector pAcYM1 [J. Gen. Virol., 6
8: 1233 (1987)] 5 μg was digested with a restriction enzyme BamHI at 37 ° C. for 2 hours and then blunted with a Blunting Kit. Further, the 5'end was dephosphorylated with alkaline phosphatase (BRL).

The VP-1 and VP-2 fragments were respectively transformed into pAcY
After binding M1, E. coli XL1-Blue was infected. 20 transformants obtained are appropriately selected, and alkali-SDS is selected.
DNA was purified by the method and the target plasmids pAcVP1 and pAcVP2 were obtained by mapping with restriction enzymes (Fig. 1
6).

(2) Preparation of recombinant virus Using a φ35 mm dish (manufactured by Sumitomo Bakelite Co., Ltd.), a cell line derived from night moth larvae: Sf9 (Spodopterd frugiperd)
a) Cells are cultured in medium containing 10% fetal calf serum (FCS) (TC-10).
0, or Grace's medium), and cell line: Tn
5: (Trichoplusia ni) cells were cultured in serum-free medium (TC-100 or Grace's medium) for 3 to 4 days. Then φ3
Using a 5 mm dish (Sumitomo Bakelite Co., Ltd.), 1
The cultured cells described above were adjusted so that the amount of the medium was × 10 ⁶ / dish and the amount of the medium was about 1.5 ml. After leaving still for 30 to 40 minutes in this state, the above medium was aseptically removed and replaced with 1.5 ml serum-free medium (EX-CELL 400 or Grace's medium).

AcNPV (Kinura subfamily, Autograp
ha californica nuclear polyhedorosis virus) DNA
About 20 ng / μl 1 μl, transfer vector 2-3 μg / μl Concentration 1 μl, sterile distilled water 6 μl total 8 μl and Lipofectin (BRL) 5 μl, sterile distilled water 3 μl total 8 μl
After gently mixing 1 l in an Eppendorf tube, 1 drop was gently added to the whole serum-free medium (co-transfection). Then, it was stored in a moisture chamber and cultured at 26 to 26.5 ° C for 3 days.

After co-transfection, about 1 ml of the culture supernatant on the 3rd day was transferred to a tube, and 20 μl of each of them was diluted to 10 ⁻¹ to 10 ⁻³ , and 100 μl of them was diluted to 1 × 10 ^6.
Approximately 1 by inoculating cells adjusted to a dish (φ35 mm)
Let stand for a time to allow adsorption. Then, discard the virus solution 1
% Low melting point agar (Sea Plaque: Takara Shuzo) 2m
After layering and coagulation, 1 ml of medium (in the case of Sf9 cells, use TC-100 or Grace's medium containing 10% FCS,
In the case of Tn5 cells, TC-100 or Grace's serum-free medium is used. ) And cultured at 26-26.5 ° C for 3-4 days, 1 ml of 0.01% (W / V) neutral red in 10 mM phosphate buffered saline (10 mM PBS) was added, and plaques were stained to give a transparent recombinant. Was selected.

Plaques considered to be recombinants obtained by the plaque assay were collected together with the gel using a Pasteur pipette, suspended in 400 μl of the cell culture medium and sufficiently stirred, and then at about 3000 rpm for about 5 minutes. The agarose gel was precipitated under the conditions described above, and the supernatant was added to each medium up to 10 ^-1 to 10 ^-3 as in the plaque assay (TC-100 or Grace's medium containing 10% FCS in the case of Sf9 cells was used).
In the case of n5 cells, TC-100 or Grace's serum-free medium is used. ) With a low melting point agar.
Recombinant virus was purified by performing the assay.

Plaque purif
The titer of the recombinant obtained by y) was increased by infecting cells. Also at this time, about 1 × 10 ⁶ Sf9 cells and Tn5 were similarly added to the φ35 mm dish.
Prepare the cells, leave them for about 30 minutes, discard most of the culture medium, add about 100 μl of the obtained recombinants, and gently mix with shaking at about 15 minutes intervals for a total of 4 times for about 1 hour. This operation was performed over.

Next, 1.5 ml of medium containing 10% FCS (TC-100 or Grace's) for Sf9 cells and 1.5 ml of serum-free medium (TC-100 or Grace's) for Tn5 cells were added.
Culture for ~ 4 days. 75 using the supernatant obtained at this time
Cells were similarly prepared using 2 cm ² culture bottles, and 1 ml of the recombinant virus solution whose titer had been increased once was inoculated. After culturing for 3 to 4 days, the resulting supernatant was It was a high titer recombinant for structural gene expression.

The number of cells was adjusted to about 1 × 10 ⁶ / dish in a φ35 mm dish, most of the medium was aspirated, and a high titer recombinant (about 1 × 10 ⁷ pfu / ml) was added to the M.0.I. This operation was continued for 4 hours with a total of 4 inoculations of 5 to 10 and gentle shaking at intervals of about 15 minutes.

After that, the medium (10% FCS in the case of Sf9 cells was used)
TC-100 or Grace's medium containing Tc5 is used, and in the case of Tn5 cells, TC-100 or Grace's serum-free medium is used. )
0.5-1 ml was added and cultured at 26 to 26.5 ° C. for 3 to 4 days to express human parvovirus structural genes (VP-1, VP-2 and VP-1 + VP-2).

The expressed protein was expressed in 10% S with human parvovirus particles purified from serum of infectious erythema patient as a control.
After electrophoresis for about 2 hours at 20 mA using DS-PAGE, CB
B staining was performed to confirm the molecular weight and expression level. As a result, an expressed protein having the same molecular weight as VP-1 and VP-2 of human parvovirus was confirmed. It was also found that the expression level was higher in Tn5 cells than in SF-9 cells.

The antigenicity was confirmed by Western blotting using a serum of infectious erythema patient and a fluorescent antibody indirect method. As a result, the presence of a band that specifically reacts with the expressed protein and the presence of fluorescent color were confirmed, and it was confirmed that the expressed proteins were human parvovirus structural proteins VP-1 and VP-2.

(3) Human Parvovirus Expression Protein Emp
Empty parti using ty particle purification and electron microscopy
Confirmation of cle Using insect cells Sf9, Tn5, etc., preferably Tn5
Using the cells, a human parvovirus structural protein was expressed in a large amount in a plastic cell culture bottle (Sumitomo Bakelite Co., Ltd.) having a surface area of 75 cm 2 and used for purification. A large amount of expressed protein (75 cm ² plastic cell culture bottles for 20 bottles) was harvested together with the cultured cells by pipetting using a cell culture solution, and two 500 ml centrifugal precipitation bottles (Hitachi) were used. (About 300 ml) and transfer them to 1000-3000 rpm
Cell components were precipitated by centrifugation at 20 ° C for 15 minutes, and the supernatant was discarded. The precipitated cells were suspended in 10 ml of PBS (-) (phosphate buffered saline, Nissui Pharmaceutical Co., Ltd.) or Hanks' BSS (Hank's buffered saline, homemade) and mixed well, and then 2 using an ultralow temperature refrigerator at -80 ° C. The freeze-thaw operation was repeated 3 times. Next, freeze-thaw cell suspension was added to 5 plastic Spitz tubes (Eiken Kikai Co., Ltd.) with 2 ml each.
Ultrasonic treatment (manufactured by Olympus Corporation) was performed 2 to 4 times in total under the conditions of 100 to 500 W, 0 ° C., and 5 to 20 minutes.
At this time, about 50 μl each was collected from each cell suspension,
After dripping on a slide glass, a cover glass was placed and observed with an optical microscope (manufactured by Nikon) at a magnification of 200 times to confirm the crushed state of cells. Here, in order to separate the cell components from the cell suspension, about 10000 rpm, 20 ° C, 1
Centrifugal precipitation for 5 minutes was performed. About 10 ml of the supernatant obtained by this operation was used as a sample for the following purification operation. First, 13PA tube for ultracentrifugation (manufactured by Hitachi Koki Co., Ltd.)
40% sucrose (W / V) (PBS (-) or Hanks'BS
2 to 3 ml of (suspended in S) (manufactured by Nacalai Tesque, Inc.) is dispensed and the above sample is layered on these sucrose cushions, and ultracentrifugation is performed at about 35,000 rpm, 20 ° C. for 16 to 20 hours to exclude trace minor proteins. A human parvovirus-expressed protein was also precipitated with. Since the precipitated human parvovirus-expressed protein was adhered to the tube bottom of the 13PA tube, 2 ml each of PBS (-) was added dropwise and mixed well by pipetting and shaking. Further, at this time, since large molecules were visually confirmed, ultrasonic treatment was performed at 100 to 500 W, 0 ° C. for 2 to 10 minutes to prepare a suspension solution. Next, in advance ultrapure water (mi
li-Q Millipore) with a density of 1.2, 1.3, 1.4 (g
/ Cm ³⁾ it becomes as prepared CsCl solution (Ju Nacalai Tesque) in descending order of density - 2,5,2 from the tube bottom of the probe
After overlaying each with 1 ml and preforming the gradient, 2 ml of the sample was overlaid on 2 ultracentrifugation tubes to obtain about 35
Purification by the first ultracentrifugation was performed under the conditions of 000 rpm, 20 ° C., and 40 to 45 hours. After ultracentrifugation, each sample was separated into about 20 fractions using a perista pump or the like, and the anti-VP1 guinea pig antibody (1.5 μg / m
l) An antigen search was performed using a solid-phased plate and an anti-VP1 guinea pig POD-labeled antibody (homemade). as a result,
The horizontal axis is fraction NO. And the vertical axis is O.F. D. 492/63
When the measurement results at 0 nm were graphed, a single-peaked curve was obtained, and thus the second ultracentrifugation operation and antigen retrieval were similarly performed using the peak-containing fraction. At this time, the weight percentage of CsCl in each fraction was measured by a saccharimeter (made by ATAGO), and each CsCl solution with a density of 1.2, 1.3, 1.4 (g / cm ³ ) serving as a reference. The density of each fraction solution was converted from the value of the weight percent of the above and converted into a graph. At this time, the horizontal axis was the fraction No. and the vertical axis was the density value of each fraction. as a result,
It was confirmed that the density decreased from the tube bottom of the tube toward the tube mouth. Further, from these relationships, it was confirmed that the density of the fraction solution containing the antigen was about 1.3 g / cm ³ . Next, this sample was PBS in a 13PA tube.
Diluted 15 to 20 times with (-) and about 35,000 rp
Ultracentrifugation was performed at 20 ° C. for 10 to 20 hours to precipitate the purified expressed protein of human parvovirus. These expressed proteins were finally suspended in 1 to 2 ml of PBS (-) to prepare an expressed protein stock solution. For confirmation by an electron microscope, using the expressed protein solution as a sample, negative staining was performed with 4% Uranic acetate, and a microscope was performed with a transmission electron microscope (manufactured by Hitachi) at a magnification of 50,000 times. As a result, about 2 as shown in FIG.
3nm hollow circular virus-like structure called "Empty"
We confirmed the existence of "particles".

Example 5 Escherichia coli expressed antigens VP-1, VP
-Human parvovirus IgG antibody detection using -2 antigen (1) Preparation of VP-1, VP-2 antigen solid phase plate VP-expressed in Escherichia coli described in Example 2- (6)
1. VP-2 antigen with 50 mM carbonate buffer (pH 9.5)
Dilute to a concentration of 1-10 μg / ml, dispense into polystyrene flat microplates (Nunc) at 100 μl / well, 4 ° C.
I left it overnight. The microplate, which had been left standing for 18 hours or more, was washed 3 to 4 times with 200 μl / well of PBS containing 0.05% Tween20 at the final concentration.
SA) and 200 μl / well of 10 mM PBS containing 0.05% Tween 20 were added and left to stand overnight at 4 ° C. to prepare VP-1 and VP-2 solid phase microplates.

(2) Detection of anti-human parvovirus IgG antibody using serum of infectious erythema patients After removing the plate preservation solution in the antigen solid phase microplate well described above, the serum of infectious erythema patients was supplemented with 0.05% BSA.
It was diluted 200 times with 10 mM PBS containing 0.05% Tween20, and 100 μl of the diluted solution was added to the wells of the antigen solid phase microplate, and the mixture was reacted at room temperature (15 ° C to 25 ° C) for about 1 hour. After the reaction, add 3% with 200 μl / well of 5 mM PBS containing 0.05% Tween20.
Washed ~ 4 times. Then, 5 mM PBS containing 0.05% Tween20
100 μl / well of anti-human IgG goat peroxidase-labeled antibody (MBL) diluted approximately 40,000 times with
The reaction was carried out at room temperature for about 1 hour. After the reaction, the cells were washed 3 to 4 times with 200 μl / well of 5 mM PBS containing 0.05% Tween20. After washing, 0.1M containing 3.3mg / ml O-phenylenediamine
Add 100 μl / well of substrate solution containing 0.02% hydrogen peroxide solution to citric acid-phosphate buffer (pH 5.0), and add about 30 at room temperature.
Let react for minutes. After the reaction, 100 μl / well of 1.5 N sulfuric acid was added to stop the reaction, and the OD value of each well was measured at a main wavelength of 492 nm and a sub wavelength of 630 nm using a colorimeter for a microplate (Table 5). ).

[0103]

[Table 5] Viral particles are purified from patient serum.

From these results, the enzyme immunoassay using the full-length human parvovirus structural protein VP-1 was found to be better than the full-length human parvovirus structural protein VP.
The IgG antibody detection rate was higher than that of the enzyme immunoassay using -2.

However, since it was found that there was serum having a higher reactivity to the full-length human parvovirus structural protein VP-2 than to the full-length human parvovirus structural protein VP-1, it was found that the human parvovirus structural Not only the protein VP-1, but also the human parvovirus structural protein VP-2 is added to the optimum concentration, preferably 1-10 μg / ml of VP-1 and 1-20 μg / ml of VP-2.
ml was added and solid-phased on an ELISA microplate by the method of Example 5- (1). Therefore, using serum that becomes negative in the enzyme immunoassay using the VP-1 antigen and positive in the enzyme immunoassay using the VP-2 antigen, IgG using a microplate in which VP-1 and the VP-2 antigen are mixed and solid-phased is used. Antibody detection was performed (Table 6).

[0106]

[Table 6] As a result, it was possible to efficiently detect serum that strongly reacts with either the VP-1 or VP-2 antigen. That is, the serum immunoassay using the VP-1 (or VP-2) antigen was positive, and the serum immunoassay using the full-length human parvovirus structural protein VP-2 (or VP-1) antigen was negative, In the enzyme immunoassay in which VP-1 and VP-2 antigens were mixed, all were found to be positive.

(3) Detection of anti-parvovirus IgG antibody using VP-1 antigen derivative VP-1 expressed in Escherichia coli as described in Example 3- (2)
Antigen derivative with 50 mM carbonate buffer (pH 9.5) 1-10 μ each
It was diluted to a concentration of g / ml, dispensed at 100 µl / well on a polystyrene flat microplate (manufactured by Nunc), and left at 4 ° C overnight. The microplate that had been left standing for 18 hours or more was washed 3 to 4 times with 200 μl / well of PBS containing Tween20 at a final concentration of 0.05%, and then the final concentration of 0.05% bovine serum albumin (BSA) and 0.05 were added.
200 μl / well of 10 mM PBS containing 10% Tween 20 was added and left at 4 ° C. overnight to prepare a VP-1 antigen derivative solid phase microplate.

[0108] Next, anti-parvovirus IgG antibody was detected by the above-mentioned microplate using the serum at the time of infectious erythema epidemic. First, the plate preservation solution in the VP-1 antigen derivative solid phase microplate well was removed, and then the serum of infectious erythema patient was added with 0.05% BSA and 0.05% Tween20.
Dilute 200-fold with 10 mM PBS, add 100 μl to the wells of the solid phase microplate of the antigen, and mix at room temperature (15 ° C-25 ° C).
The reaction was carried out at (° C.) for about 1 hour. After the reaction, the wells were washed 3 to 4 times with 200 μl / well of 5 mM PBS containing 0.05% Tween20. Then,
Anti-human IgG goat peroxidase-labeled antibody (MBL) diluted approximately 40,000 times with 5 mM PBS containing 0.05% Tween20.
100 μl / well) and reacted at room temperature for about 1 hour.
After the reaction, add 3% with 5 mM PBS containing 0.05% Tween20 (200 μl / well).
Washed ~ 4 times. After washing, 0.1M citric acid-phosphate buffer (pH 5.0) containing 3.3 mg / ml O-phenylenediamine
Substrate solution containing 0.02% hydrogen peroxide in 100 μl / well
In addition, the reaction was carried out at room temperature for about 30 minutes. After reaction, 1.5N
The reaction was stopped by adding 100 μl / well of sulfuric acid, and the main wavelength was 492 nm and the sub wavelength was 6 using a microplate colorimeter.
The OD value of each well was measured at 30 nm (Table 4).

[0109]

[Table 4] Viral particles are purified from patient serum.

From this result, anti-human parvovirus IgG was detected rather than enzyme immunoassay using virus particles.
The antibody detection rate was found to be quite low. In addition, from the results of Example 5- (2), the enzyme immunoassay using the full-length human parvovirus structural protein VP-1 is more partially expressed than the enzyme immunoassay using the VP-1 antigen derivative. IgG
It was found that the antibody detection rate was high and the agreement rate with the enzyme immunoassay using virus particles was also high. This means that the same expressed antigen, eg VP-1 antigen and VP-1
In the case of the antigen derivative, the number and the three-dimensional structure of the antigen site seemed to be the cause of the difference in the detection rate of the anti-human parvovirus IgG antibody. Therefore, it is important for the expressed antigen used for the enzyme immunoassay that the number and the three-dimensional structure of the antigen site are close to the number and the three-dimensional structure of the antigen site of the virus particle. Therefore, even with the same expressed antigen, the full-length expressed antigen was considered to satisfy the above condition more than the partially expressed antigen.

(4) Examination of time course change of anti-human parvovirus IgG antibody using serum of infectious erythema patients Using the antigen solid phase microplate described in Example 5- (1), Example 5- (2). Anti-human parvovirus Ig in serum of an infectious erythema patient according to the described enzyme immunoassay method
The change in G antibody over time was examined (FIG. 18). In addition, VP-
1 was 5 μg / ml, VP-2 was 10 μg / ml, M
ix is 5 μg / ml for VP-1 and 10 μg / ml for VP-2.
It was solid-phased in ml.

As a result, IgG antibodies against the VP-1 and VP-2 antigens both appear, but the IgG antibody against the VP-1 antigen has a longer lasting antibody amount than the IgG antibody against the VP-2 antigen. I understood.

By the way, the coding region of the protein of VP-2 antigen is completely contained in the C-terminal portion of VP-1 antigen. Therefore, it has long been considered that the VP-1 antigen alone is sufficient for detecting human parvovirus antibodies. However, it was found that the VP-1 and VP-2 antigens had a difference in immunological reactivity and a difference in the persistence of IgG antibody amount, and thus a more reliable diagnosis of human parvovirus was made. Found that an enzyme immunoassay in which VP-2 antigen was added was required, not an enzyme immunoassay in which only VP-1 antigen was used. That is, the VP-1 and VP-2 antigen mixed system is more VP-
It was found that the detection rate of IgG antibody against human parvovirus was higher than that of the 1 or VP-2 antigen alone system.

Example 6 Escherichia coli expressed antigens VP-1, VP
-2 anti-human parvovirus IgM antibody detection using antigen-2 (1) anti-human parvovirus structural protein VP-1,
Peroxidase labeling against VP-2 antibody VP-expressed in E. coli as described in Example 2- (6)
1. 200 μg / ml of VP-2 antigen (1 ml each) and 1 ml of a complete adjuvant (manufactured by Difco) were mixed in equal amounts to prepare an emulsion and immunize a guinea pig. Then, 1 ml of incomplete adjuvant (manufactured by Difco) was mixed with the above-mentioned antigen solution in an equal amount to prepare an emulsion, and booster immunization was performed twice. After boosting, the guinea pigs were exsanguinated. The blood was serum-separated, and after ammonium sulfate fractionation, it was treated with 50 mM Tris-HCl (pH 7.6) at 4 ° C.
It was dialyzed overnight. Next, it was subjected to DEAE Sepharose chromatography equilibrated with 50 mM Tris-HCl (pH 7.6), and U
The V wavelength was monitored at 280 nm, and the OD peak was collected to be a DEAE-purified IgG antibody fraction.

This IgG antibody fraction was labeled with peroxidase by the periodoic acid modification method [enzyme immunoassay method, 2:91 (1982)]. That is, the peroxidase was dissolved in distilled water so as to have a concentration of 4 mg / ml. Then, 0.2 ml of 0.1 M sodium periodate was added, the mixture was reacted at room temperature for about 20 minutes, and dialyzed against 1 mM sodium acetate buffer (pH 4.0) overnight. After dialysis, 0.
Add 0.02 ml of 2M sodium carbonate buffer (pH 9.5) to adjust the pH to 9-
At the same time as 9.5, add 8 mg of the IgG antibody purified as described above.
added.

The reaction was carried out at room temperature for about 2 hours, 0.1 ml of 4 mg / ml sodium borohydride was added, and the reaction was carried out at 4 ° C. for about 2 hours. After the reaction, gel filtration with Sephacryl S-200 was performed using 10 mM phosphate buffer, and UV wavelength was 28
The peroxidase-labeled antibody fraction was collected by monitoring at 0 nm.

(2) Detection of anti-human parvovirus IgM antibody using 88 samples of serum at the time of epidemic of erythema infectio Anti-human IgM monoclonal antibody (manufactured by MILS) was used.
Dilute 500-1000 times with mM carbonate buffer (pH 9.5) and dispense 100 μl / well to Nunc polystyrene flat plate split microplate (high binding type) at 4 ° C.
Let stand for 18 hours or more. After standing, place the microplate at 0.
After washing 3 to 4 times with 200 μl of 5 mM PBS containing 05% Tween20, 200 μl of 5 mM PBS containing 0.5% BSA and 0.05% Tween20
The wells were added and left at 4 ° C. overnight to prepare an anti-human IgM monoclonal solid phase microplate. Next, an anti-human parvovirus IgM antibody detection reagent was experimentally produced using this anti-human IgM monoclonal antibody solid phase microplate.

Next, the anti-human parvovirus IgM antibody detection was carried out using 88 samples of serum at the time of epidemic of infectious erythema using the trial-produced anti-human parvovirus IgM antibody detection reagent. The plate stock solution in the antigen solid phase microplate wells described above was removed, and then the serum of infectious erythema erythema was diluted 200-fold with 10 mM PBS containing 0.2% BSA and 0.05% Tween20, and 100 μl of the diluted solution was used. 100 μl / well was added to a human IgM monoclonal antibody solid phase microplate and reacted at room temperature (15 ° C. to 25 ° C.) for about 1 hour. After the reaction, add 3% with 200 μl / well of 5 mM PBS containing 0.05% Tween20.
Washed 4 times. Then, the full-length human parvovirus structural proteins VP-1 and VP-2 purified as described above were added to 0.2%.
The mixture was diluted with 5 mM PBS containing 5% BSA and 0.05% Tween 20 to a concentration of 5 μg / ml, added 100 μl / well, and reacted at room temperature for about 1 hour. After the reaction, the wells were washed 3 to 4 times with 200 μl / well of 5 mM PBS containing 0.05% Tween20. 100 μl / well of peroxidase-labeled guinea pig antibodies against the VP-1 and VP-2 antigens of the above description, Example 5- (1), which had been diluted 2000 to 5000 times with 5 mM PBS containing 0.5% BSA and 0.05% Tween 20, were added. The reaction was carried out at room temperature for about 1 hour. After reaction, 0.05% Tw
Washed 3-4 times with 200 μl / well of 5 mM PBS containing een20. After washing, contains 3.3mg / ml O-phenylenediamine
0.1M citric acid-phosphate buffer (pH 5.0), final concentration 0.02
Substrate solution containing 100% hydrogen peroxide was added at 100 μl / well and reacted at room temperature for about 30 minutes. After the reaction, add 1.5N sulfuric acid to 10
The reaction was stopped by adding 0 μl / well, and the OD value of each well was measured with a colorimeter for a microplate using a main wavelength of 492 nm and a sub wavelength of 630 nm (Table 7).

[0119]

[Table 7]

As a result, when compared with the enzyme immunoassay for detecting an anti-human parvovirus IgM antibody using human parvovirus particles purified from the serum of an infectious erythema patient, the detection sensitivity was slightly low but the specificity was high.

Therefore, it was found that the structural protein expressed in Escherichia coli of the present invention is useful for detecting anti-human parvovirus IgM antibody.

Example 7 Baculovirus Expressed Protein Emp
Detection of anti-human parvovirus IgG antibody using ty particles (1) Preparation of baculovirus-expressed protein Empty particle solid phase plate Baculovirus-expressed protein E described in Example 4- (3)
The mpty particles were each diluted with 10 mM PBS (pH 7.2) to a concentration of 0.1 to 10 μg / ml, dispensed at 100 μg / well on polystyrene flat microplates (Nunc), and allowed to stand at 4 ° C. for 1 to 2 days. I put it. The microplate left to stand for 1 to 2 days had a final concentration of 0.05% Tween.
After washing 3 to 4 times with 200 μl / well of PBS containing n20, bovine serum albumin (BS
A) and 10 mM PBS containing 0.05% Tween 20
200 μl / well was added and left at 4 ° C. overnight to prepare a baculovirus-expressing antigen Empty particles solid phase microplate.

(2) Detection of anti-human parvovirus IgG antibody using 88 specimens of erythema infectious erythema patient After removing the plate stock solution in the antigen solid phase microplate well described above, the serum of infectious erythema patient was adjusted to 0. 5% BS
10 mM PBS containing A and 0.05% Tween 20
Dilute 200-fold with (pH 7.2), add 100 μl thereof to the well of the antigen solid phase microplate, and add at room temperature (15
The reaction was performed at about 1 to 25 ° C for about 1 hour. After reaction, 0.05
Washed 3-4 times with 200 μl / well of 5 mM PBS containing% Tween20. Then, 0.05% Twee
Approximately 20,000 to 40,000 with 5 mM PBS containing n20
100 μl / well of anti-human IgG goat peroxidase-labeled antibody (MBL) diluted 1-fold was added, and the mixture was diluted to about 1 at room temperature.
Reacted for hours. After the reaction, the wells were washed 3 to 4 times with 200 μl / well of 5 mM PBS containing 0.05% Tween20. After washing, 0.1 M citrate-phosphate buffer (pH 5.3) containing 3.3 mg / ml O-phenylenediamine.
0) substrate solution containing 0.02% hydrogen peroxide solution added to 10)
0 μl / well was added and reacted at room temperature for about 30 minutes. After the reaction, 1.5 N sulfuric acid was added in an amount of 100 μl / well to stop the reaction, and the OD value of each well was measured at a main wavelength of 492 nm and a sub wavelength of 630 nm using a colorimeter for a microplate (Table 8). .

[0124]

[Table 8]

From these results, when the results of the IgG antibody detection enzyme immunoassay using human parvovirus particles purified from the serum of infectious erythema patients and the IgG antibody detection enzyme immunoassay using baculovirus expressed protein Empty particles were compared, The positive match rate was 100% and the negative match rate was 100%. From the above,
Baculovirus expressed protein Empty particle I
The enzyme immunoassay for detecting gG antibody is used for the detection of specificity and specificity of other human parvovirus Ig.
Since it is higher than the G antibody detection enzyme immunoassay,
More reliable IgG antibody detection is now possible.

Example 8 Baculovirus Expressed Protein Emp
Detection of anti-human parvovirus IgM antibody using ty particles (1) Anti-baculovirus expressed protein Empty particles
Peroxidase labeling of antibody Baculovirus-expressed protein E described in Example 4- (3)
mpty particles 1 to 10-500 μg / ml each
And 1 ml of complete adjuvant (manufactured by Difco) were mixed to prepare an emulsion, and the guinea pig was immunized. Then incomplete adjuvant (Difco) 1 ml
Was mixed with the above-mentioned antigen solution in an equal amount to prepare an emulsion, and booster immunization was performed twice. After boosting, the guinea pigs were exsanguinated. The blood is serum-separated, and after ammonium sulfate fractionation, 50 mM
It was dialyzed against Tris-HCl (pH 7.6) at 4 ° C overnight. It was then subjected to DEAE Sepharose chromatography equilibrated with 50 mM Tris-HCl (pH 7.6), monitored at UV wavelength of 280 nm and OD.
Was collected and used as a DEAE-purified IgG antibody fraction.

This IgG antibody fraction was labeled with peroxidase by the periodoic acid modification method [enzyme immunoassay, 2:91 (1982)]. That is, the peroxidase was dissolved in distilled water so as to have a concentration of 4 mg / ml. Next, 0.2 ml of 0.1 M sodium periodate was added, and the mixture was reacted at room temperature for about 20 minutes, and 1 mM sodium acetate buffer (pH 4.0) was added.
Dialyzed overnight. After dialysis, 0.02 ml of 0.2 M sodium carbonate buffer solution (pH 9.5) was added, and the pH was adjusted to 9 to 9.
5 mg and 8 mg of IgG antibody purified as described above
added.

After reacting at room temperature for about 2 hours, 0.1 ml of 4 mg / ml sodium borohydride was added and reacted at 4 ° C. for about 2 hours. After the reaction, gel filtration with Sephacryl S-200 was performed using 10 mM phosphate buffer,
The peroxidase-labeled antibody fraction was collected by monitoring at UV wavelength of 280 nm.

(2) Detection of anti-human parvovirus IgM antibody using 88 serum samples at the time of epidemic erythema infestation Anti-human IgM monoclonal antibody (manufactured by MILS) was used as 5
500-100 with 0 mM carbonate buffer (pH 9.5)
It was diluted 0 times, and 100 μl / well was dispensed to a polystyrene flat type split microplate (high binding type) manufactured by Nunc Co., Ltd., and left still at 4 ° C. for 18 hours or more. After standing, the microplate contains 5 mM PBS containing 0.05% Tween 20.
After washing 3 to 4 times with 200 μl / well, 0.5% BSA
And 5 mM PBS 200 containing 0.05% Tween 20
μl / well was added and left overnight at 4 ° C. to prepare an anti-human IgM monoclonal solid phase microplate. Next, an anti-human parvovirus IgM antibody detection reagent was experimentally produced using this anti-human IgM monoclonal antibody solid phase microplate.

Next, using this prototype anti-human parvovirus IgM antibody detection reagent, anti-human parvovirus IgM antibody detection was performed using 88 serum samples from the epidemic of infectious erythema. First, the plate stock solution in the anti-human IgM monoclonal antibody solid phase microplate well described above was removed. Then, 0.2% B of infectious erythema patient serum was added.
10 mM PBS containing SA and 0.05% Tween 20
200-fold, and 100 μl / well was added to the anti-human IgM monoclonal antibody solid phase microplate described above, and the mixture was reacted at room temperature (15 ° C to 25 ° C) for about 1 hour. After the reaction, 5 mM PBS 2 containing 0.05% Tween 20
Wash 3-4 times with 00 μl / well. Then, the baculovirus-expressed antigen Empty particles purified as described above
5m containing 0.5% BSA and 0.05% Tween 20
Dilute to 0.05-5.0 μg / ml concentration with MPBS,
It was added at 100 μl / well and reacted at room temperature for about 1 hour. After the reaction, 5 mM containing 0.05% Tween 20
Washed with PBS 200 μl / well 3-4 times. 0.
5mMP containing 5% BSA and 0.05% Tween 20
Baculovirus-expressing antigen Empty parti of the above-mentioned Example 8- (1), which was diluted 2000 to 20000 times with BS.
1 peroxidase-labeled guinea pig antibody against cle
00 μl / well was added and reacted at room temperature for about 1 hour. After the reaction, 5 mM PBS containing 0.05% Tween 20
Washed 3-4 times with 200 μl / well. After washing, 3.
0.1 M containing 3 mg / ml O-phenylenediamine
100 μl of a substrate solution obtained by adding hydrogen peroxide solution having a final concentration of 0.02% to a citric acid-phosphate buffer solution (pH 5.0)
/ Well was added and reacted at room temperature for about 30 minutes. After the reaction
The reaction was stopped by adding 100 μl / well of 1.5 N sulfuric acid, and the OD value of each well was measured using a microplate colorimeter with a main wavelength of 492 nm and a sub wavelength of 630 nm (Table 9).

[0131]

[Table 9]

From these results, the results of the IgM antibody detection enzyme immunoassay using human parvovirus particles purified from the serum of infectious erythema patients and the IgM antibody detection enzyme immunoassay using baculovirus-expressed protein empty particles were compared. The positive match rate was 100% and the negative match rate was 100%.

Based on the above, the baculovirus-expressed protein Emp
Since the IgM antibody detection enzyme immunoassay using ty particles has higher detection sensitivity and specificity than the human parvovirus antibody detection enzyme immunoassay such as literature, more reliable antibody detection became possible.

Example 9 Human Parvovirus Antigen Detection (1) Preparation of Antibody Solid Phase Microplate Against VP-1 and VP-2 Antigens The anti-human parvovirus guinea pig antibody described in Example 6- (1) was added to a 50 mM carbonate buffer ( at pH 9.5),
Diluted to 0.1-5.0 μl / ml. After dilution, add 100μ to a polystyrene flat microplate (Nunc)
1 / well was dispensed, and the mixture was allowed to stand at 4 ° C for 18 hours or more. After standing, the microplate was washed 3 to 4 times with 5 mM PBS containing 0.05% Tween 20 at 200 μl / well, and 200 μl / well of 5 mM PBS containing 0.5% BSA and 0.05% Tween 20 was added at 4 ° C. overnight. The plate was left to stand to prepare an antibody solid phase microplate.

(2) Human Parvovirus Antigen Detection Using Serum During Infectious Erythema Epidemic Next, human parvovirus antigen was detected using serum during the infectious erythema epidemic. The plate preservation solution in the antibody solid phase microplate well described above was removed, and then 0.2% of serum at the time of epidemic erythema infestation was removed.
10 mM PBS containing BSA and 0.05% Tween 20
Was diluted 200-fold with 100 μl / well to the anti-human parvovirus antibody solid phase microplate described above, and reacted at room temperature (15 ° C to 25 ° C) for about 1 hour. After the reaction
5 mM PBS 200 containing 0.05% Tween 20
Wash 3-4 times with μl / well.

0.05% BSA and 0.05% Tween
100 μl / well of peroxidase-labeled guinea pig antibody against VP-1 and VP-2 antigens diluted 2000-5000 times with 5 mM PBS containing 20 was added and reacted at room temperature for about 1 hour. After the reaction, 0.05% Tween2
The cells were washed 3 to 4 times with 200 μl of 5 mM PBS containing 0 / well.

After washing, 0.1 M citric acid-phosphate buffer solution (pH: 3.3 mg / ml O-phenylenediamine)
To 5.0), 100 μl / well of a substrate solution containing a final concentration of 0.02% hydrogen peroxide solution was added and reacted at room temperature for about 30 minutes. After the reaction, 100 μl / well of 1.5N sulfuric acid was added to stop the reaction, and the OD value of each well was measured using a microplate colorimeter with a main wavelength of 492 nm and a sub wavelength of 630 nm (Table 10). .

[0138]

[Table 10]

An enzyme immunoassay using the anti-human parvovirus monoclonal antibody solid phase microplate [37th Annual Meeting of the Virology Society of Japan (1989)] was used as a positive control.

As a result, it was found that human parvovirus antigen detection using an anti-human parvovirus antibody immunized with the VP-1 and VP-2 antigens was useful.

Example 10 Detection of Human Parvovirus Gene by PCR Method Human parvovirus DNA was extracted by the method described in Example 1- (1) using serum at the time of epidemic of infectious erythema. Next, from among the following DNA primer group 1 and DNA primer group 2, the human parvovirus PCR amplification primer DNA pairs (D-4 and U-2 and D-1 and U-4) were used in Example 1- ( 2) The human parvovirus gene region was amplified according to the method described. The positions of four types of primers are shown in FIG.

DNA primer group 1 D-1: GCTGCCATGTGGGAGCTTCTAATC D-4: TTCCCGCCTTATGCAAATGGGCAGC DNA primer group 2 U-2: GTGTTAGGCTGTCTTATAGGTACA U-4: CTTGTATTCATGGTCTACTAAC Amplification of 4.6 Kb when amplified with D-4 and U-2 primers A fragment was obtained, while a 2.6 Kb amplified fragment was obtained when amplified with the D-1 and U-4 primers (Table 11).

[0143]

[Table 11]

From the results of Table 11, human parvovirus D
Since the amplification of the NA fragment and the result of the enzyme immunoassay antigen detection of Example 9- (2) were in agreement, a DNA primer was prepared based on the base sequence of the human parvovirus gene and amplified by the PCR method. It was found that the human parvovirus gene can be detected by detecting the DNA fragment.

The symbols and symbols of the polypeptides and base sequences in the specification and the figures below are as follows, and are commonly used in the art.

[0146]

[Table 12]

[0147]

[Table 13]

HPV: Human parvovirus DNA: Deoxyribonucleic acid RNA: Ribonucleic acid EDTA: Ethylenediaminetetraacetic acid SDS: Sodium dodecyl sulfate IPTG: Isopropyl β-D-galactoside Xgal: 5-Bromo-4-chloro-3-indolyl-
β-D-galactoside PCR: Polymerase chain reaction Ap ^r : Ampicillin resistance gene LacZ: β-galactosidase gene VP-1: Human parvovirus structural protein gene V
P-1 VP-2: Human parvovirus structural protein gene V
P-2 NS: human parvovirus nonstructural gene N ': lambda phage N protein gene corresponding to 33 amino acids at the N-terminus of N protein Ptac: Tac promoter PL: PL promoter

[Brief description of drawings]

FIG. 1 is a schematic diagram showing a cloning region of a gene of the present invention.

FIG. 2 shows the base sequence of the gene of the present invention and the first part of the amino acid sequence of the polypeptide encoded by the gene.

FIG. 3 is a diagram showing a continuation of FIG. 2;

FIG. 4 is a diagram showing a sequence subsequent to that of FIG. 3;

FIG. 5 is a diagram showing a continuation of FIG. 4;

FIG. 6 is a diagram showing a sequence subsequent to that of FIG. 5;

FIG. 7 is a diagram showing a sequence subsequent to that of FIG. 6;

FIG. 8 is a diagram showing a sequence subsequent to that of FIG. 7;

FIG. 9 is a diagram showing a sequence subsequent to that of FIG. 8;

FIG. 10 is a view showing a sequence subsequent to that of FIG. 9;

FIG. 11 is a schematic diagram of the construction of plasmid pPLN-MCS.

FIG. 12 is a schematic diagram of the construction of plasmid pVP200.

FIG. 13 is a schematic diagram showing site-specific mutations at the time of producing a VP-1 expression vector.

FIG. 14 is a schematic diagram of the construction of plasmid pVP100.

FIG. 15 is a schematic diagram of the construction of plasmid pVP300.

FIG. 16 is a schematic diagram of the construction of plasmids pAcVP1 and pAcVP2.

FIG. 17 is an electron micrograph of virus particle-like structures produced by baculovirus.

FIG. 18 is a diagram showing a time course of IgG antibodies against VP-1 and VP-2 in patients with erythema infectiousum.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location C12N 5/10 C12P 21/02 9282-4B C12Q 1/68 A 9453-4B 1/70 9453-4B G01N 33/53 D 33/569 L // (C12P 21/02 C12R 1:19) (C12P 21/02 C12R 1:91) (C12Q 1/68 C12R 1:91) (72) Inventor Zenji Matsuura Saitama Prefecture Kamifukuoka City 1-3-5-406 (72) Inventor Hiroyuki Ogawa 5-25-18 Morino, Machida-shi, Tokyo Denrakucho Town Dormitory (72) Hideharu Shimizu 4-16 Tamagawa Gakuen, Machida-shi, Tokyo 44 (72) Kimio Kamata, Inventor 1-2-2 Minamihonmachi, Gosen, Niigata Denka Seiken Co., Ltd. Niigata Plant (72) Daisuke Kurosawa 1-2-2 Minamihonmachi, Gosen, Niigata Denka Seiken Co., Ltd. Niigata Factory

Claims (21)

[Claims] 1. A human parvovirus gene represented by the following formula (1). ## EQU1 ## Formula (1) 10 20 30 40 50 60 TTCCCGCCTT ATGCAAATGG GCAGCCATTT TAAGTGTTTT ACTATAATTT TATTGGTCAG 70 80 90 100 110 120 TTTTGTAACG GTTAAAATGG GCGGAGCGTA GGCGGGGACT ACAGTATGC TTGACTTCTT TTGATCTTCTGCTTTCAGTTCTTTTTACTAG GCTAACTAAC AGGTATTTAT ACTACTTGTT AACATACTAA CATGGAGCTA TTTAGAGGGG 250 260 270 280 290 300 TTCTTCAAGT TTCTTCTAAT GTTCTGGACT GTGCTAACGA TAATTGGTGG TGCTCTTTAC 310 320 330 340 350 360 TGGATTTAGA CACTTCTGAC TGGGAACCAC TAACTCATAC TAACAGACTA ATGGCAATAT 370 380 390 400 410 420 ACTTAAGCAG TGTGGCTTCT AAGCTTGACT TTACCGGGGG GCCACTAGCA GGGTGCTTGT 430 440 450 460 470 480 ACTTTTTTCA AGTAGAATGT AACAAATTTG AAGAAGGCTA TCATATTCAT GTGGTTATTG 490 500 510 520 530 540 GGGGGCCAGG GTTAAATCCC AGAAACCTCA CTGTGTGTGT AGAGGGGTTA TT ATA TAG 610 CAT 609 CAT ACT CAT ACT CAT ACT CAT ACT AAATAC 670 680 690 700 710 720 CTTTAAATGT TGTATGGTGT GTTACTAATA TTGATGGATA TATAGATACC TGTATTTCTG 730 740 750 760 770 780 CTACTTTTAG AAGGGGAGCT TGCCATGCCA AGAAACCCCG CATTACCACA GCCATAAATG 790 800 810 820 830 840 ATACTAGTAG TGATGCTGGG GAGTCTAGCG GCACAGGGGC AGAGGTTGTG CCATTTAATG 850 860 870 880 890 900 GGAAAGGAAC TAAGGCTAGC ATAAAGTTTC AAACTATGGT AAACTGGCTG TGTGAAAACA 910 920 930 940 950 960 GAGTGTTTAC AGAGGATAAG TGGAAACTAG TTGACTTTAA CCAGTACACT TTACTAAGCA 970 980 990 1000 1010 1020 GTAGTCACAG TGGAAGTTTT CAAATTCAAA GTGCACTAAA ACTAGCAATT TATAAAGCAA 1030 1040 1050 1060 1070 1080 CTAATTTAGT GCCTACTAGC ACATTTTTAT TGCATACAGA CTTTGAGCAG GTTATGTGTA 1090 1100 1110 1120 1130 1140 TTAAAGACAA TAAAATTGTT AAATTGTTAC TTTGTCAAAA CTATGACCCC CTATTGGTGG 1150 1160 1170 1180 1190 1200 GGCAGCATGT GTTAAAGTGG ATTGATAAAA AATGTGGCAA AAAAAATACA CTGTGGTTTT 1210 1220 1230 1240 1250 1260 ATGGGCCGCC AAGTACAGGA AAAACAAACT TGGCAATGGC CATTGCTAAA AGTGTTCCAG 1270 1280 1290 1300 1310 1320 TATAGGACATGAGTTA TTCCATT TAATGATGTA GCAGGGAAAA 1330 1340 1350 1360 1370 1380 GCTTGGTGGT CTGGGATGAA GGTATTATTA AGTCTACAAT TGTAGAAGCT GCAAAAGCCA 1390 1400 1410 1420 1430 1440 TTTTAGGCGG GCAACCTACC AGGGTAGATC AAAAAATGCG TGGAAGTGTA GCTGTGCCTG 1450 1460 1470 1480 1490 1500 GAGTACCTGT GGTTATAACC AGCAATGGTG ATATTACTTT TGTTGTAAGC GGGAACACTA 1510 1520 1530 1540 1550 1560 CAACAACTGT ACATGCTAAA GCCTTAAAAG AGCGCATGGT AAAGTTAAAC TTTACTGTAA 1570 1580 1590 1600 1610 1620 GATGCAGCCC TGACATGGGG TTACTAACAG AGGCTGATGT ACAACAGTGG CTTACATGGT 1630 1640 1650 1660 1670 1680 GTAATGCACA AAGCTGGGAC CACTATGAAA ACTGGGCAAT AAACTACACT TTTGATTTCC 1690 1700 1710 1720 1730 1740 CTGGAATTAA TGCAGATGCC CTCCACCCAG ACCTCCAAAC CACCCCAATT GTCACAGACA 1750 1760 1770 1780 1790 1800 CCAGTATCAG CAGCAGTGGT GGTGAAAGCT CTGAAGAACT CAGTGAAAGC AGCTTTTTTA 1810 1820 1830 1840 1850 1860 ACCTCATCAC CCCAGGCGCC TGGAACACTG AAACCCCGCG CTCTAGTACG CCCATCCCCG 1870 1880 1890 1900 1910 1920 GGACCAGTTC AGGAGAATCA TTTGTCGGAA GCCCAGTTTC CTCCGAAGTT GTAGCTGCAT 1930 1940 1950 1960 1970 1980 CGTGGGAAGA AGCCTTCTAC ACACCTTTGG CAGACCAGTT TCGTGAACTG TTAGTTGGGG 1990 2000 2010 2020 2030 2040 TTGATTATGT GTGGGACGGT GTAAGGGGTT TACCTGTGTG TTGTGTGCAA CATATTAACA 2050 2060 2070 2080 2090 2100 ATAGTGGGGG AGGCTTGGGA CTTTGTCCCC ATTGCATTAA TGTAGGGGCT TGGTATAATG 2110 2120 2130 2140 2150 2160 GATGGAAATT TCGAGAATTT ACCCCAGATT TGGTGCGGTG TAGCTGCCAT GTGGGAGCTT 2170 2180 2190 2200 2210 2220 CTAATCCCTT TTCTGTGCTA ACCTGCAAAA AATGTGCTTA CCTGTCTGGA TTGCAAAGCT 2230 2240 2250 2260 2270 2280 TTGTAGATTA TGAGTAAAGA AAGTGGCAAA TGGTGGGAAA GTGATGATAA ATTTGCTAAA 2290 2300 2310 2320 2330 2340 GCTGKGTATC AGCAATTTGT GGAATTTTAT GAAAAGGTTA CTGGWACAGA CTTAGAGCTT 2350 2360 2370 2380 2390 2400 ATTCAAATAT TAAAAGATCA TTATAATATT TCTTTAGATA ATCCCCTAGA AAACCCATCC 2410 2420 2430 2440 2450 2460 TCTCTGTTTR ACTTAGTTGC TCGTATTAAA AATAACCTTA AAAACTCTCC AGACTTATAT 2470 2480 2490 2500 2510 2520 AGTCATCATT TTCAAAGTCA TGGACAGTTA TCTGACCACC CCCATGCCTT ATCATCCAGT 2530 2540 2550 2560 2570 2580 AGCAGTAATG CAGAACCTA G AGGAGAAAAT GCAGTATTAT CTAGTGAAGA CTTACACAAG 2590 2600 2610 2620 2630 2640 CCTGGGCAAG TTAGCGTACA ACTACCCGGT ACTAACTATG TTGGGCCTGG TAATGAGTTA 2650 2660 2670 2680 2690 2700 CAAGCTGGGC CCCCGCAAAG TGCTGTTGAC AGTGCTGCAA GGATTCATGA CTTTAGGTAT 2710 2720 2730 2740 2750 2760 AGCCAACTGG CTAAGTTGGG AATAAATCCA TATACTCATT GGACTGTAGC AGATGAAGAG 2770 2780 2790 2800 2810 2820 CTTTTAAAAA ATATAAAAAA TGAAACTGGG TTTCAAGCAC AAGTAGTAAA AGACTACTTT 2830 2840 2850 2860 2870 2880 ACTTTAAAAG GTGCAGCTGC CCCTGTGGCC CATTTTCAAG GAAGTTTGCC GGAAGTTCCC 2890 2900 2910 2920 2930 2940 GCTTACMACG CCTCAGAAAA ATACCCAAGC ATGACTTCAG TTAATTCTGC AGAAGCCAGC 2950 2960 2970 2980 2990 3000 ACTGGTGCAG GAGGGGGGGG CAGTAATCCT GTCAAAAGCA TGTGGAGTGA GGGGGCCACT 3010 3020 3030 3040 3050 3060 TTTAGTGCCA ACTCTGTAAC TTGTACATTT TCCAGACAGT TTTTAATTCC ATATGACCCA 3070 3080 3090 3100 3110 3120 GAGCACCATT ATAARGTGTT TTCTCCCGCA GCAAGTAGCT GCCACAATGC CAGTGGAAAA 3130 3140 3150 3160 3170 3180 GAGGCAAAGG TTTGCACCAT TAGTCCCATA ATGGGATACT CAACCCCATG GAGAT ATTTA 3190 3200 3210 3220 3230 3240 GATTTTAATG CTTTAAATTT ATTTTTTTCA CCTTTAGAGT TTCAGCACTT AATTGAAAAT 3250 3260 3270 3280 3290 3300 TATGGAAGCA TAGCTCCTGA TGCTTTAACT GTAACCATAT CAGAAATTGC TGTTAAGGAT 3310 3320 3330 3340 3350 3360 GTTACAGACA AAACTGGAGG GGGGGTACAG GTTACTGACA GCACTACAGG GCGCCTATGC 3370 3380 3390 3400 3410 3420 ATGTTAGTAG ACCATGAATA CAAGTACCCA TATGTGTTAG GGCAAGGTCA GGATACTTTA 3430 3440 3450 3460 3470 3480 GCCCCAGAAC TTCCTATTTG GGTATACTTT CCCCCTCAAT ATGCTTACTT AACAGTAGGA 3490 3500 3510 3520 3530 3540 GATGTTAACA CACAAGGAAT TTCTGGAGAC AGCAAAAAAT TAGCAAGTGA AGAATCAGCA 3550 3560 3570 3580 3590 3600 TTTTATGTTT TGGAACACAG TTCYTTTCAG CTTTTAGGTA CAGGAGGTAC AGCAACTATG 3610 3620 3630 3640 3650 3660 TCTTATAAGT TTCCTCCAGT GCCCCCAGAA AATTTAGAGG GCTGCAGTCA ACACTTTTAT 3670 3680 3690 3700 3710 3720 GAAATGTACA ATCCCTTATA CGGATCCCGC TTAGGGGTCC CTGACACATT AGGAGGTGAC 3730 3740 3750 3760 3770 3780 CCAAAATTTA GATCTTTAAC ACATGAAGAC CATGCAATTC AGCCCCAAAA CTTCATGCCA 3790 3800 3810 3820 3830 3840 GGGC CACTAG TAAACTCAGT GTCTACAAAG GAGGGAGACA GCTCTAATAC TGGAGCTGGA 3850 3860 3870 3880 3890 3900 AAAGCCTTAA CAGGCCTTAG CACAGGTACC TCGCAAAACA CTAGAATATC CTTACGSCCT 3910 3920 3930 3940 3950 3960 GGGCCAGTGT CTCAGCCATA CCACCACTGG GACACAGATA AATATGTCAC AGGAATAAAT 3970 3980 3990 4000 4010 4020 GCCATTTCTC ATGGTCAGAC CACTTATGGT AACGCTGAAG ACAAAGAGTA TCAGCAAGGA 4030 4040 4050 4060 4070 4080 GTGGGTAGAT TTCCAAATGA AAAAGAACAG CTAAAACAGT TACAGGGTTT AAACATGCAC 4090 4100 4110 4120 4130 4140 ACCTATTTYC CCAATAAAGG AACCCAGCAA TATACAGATC AAATTGAGCG CCCCCTAATG 4150 4160 4170 4180 4190 4200 GTGGGTTCTG TATGGAACAG AAGAGCCCTT CACTATGAAA GCCAGCTGTG GAGTAAAATT 4210 4220 4230 4240 4250 4260 CCAAATTTAG ATGACAGTTT TAAAACTCAG TTTGCAGCCT TAGGAGGATG GGGTTTGCAT 4270 4280 4290 4300 4310 4320 CAGCCACCTC CTCAAATATT TTTAAAAATA TTACCACAAA GTGGGCCAAT TGGAGGTATT 4330 4340 4350 4360 4370 4380 AAATCAATGG GAATTACTAC CTTAGTTCAG TATGCTGTGG GAATTATGAC AGTAACTATG 4390 4400 4410 4420 4430 4440 ACATTTAAAT TGGGGCCCCG TAAAGCTACG GGACGGTGGA ATCCTCAACC TGGAGTATAT 4450 4460 4470 4480 4490 4500 CCCCCGCACG CAGCAGGTCA TTTACCATAT GTACTATATG ACCCCACAGC TATAGATGCA 4510 4520 4530 4540 4550 4560 AAACAACACC ACAGACATGG ATATGAAAAG CCTGAAGAGT TGTGGACAGC CAAAAGCCGT 4570 4580 4590 4600 4610 4620 GTGCGCCCAT TGTAAACACT CCCCACCGTG CCCTCAGCCA GGATGCGTAA CTAAACGCCC 4630 4640 4650 4660 4670 4680 ACCAGTACCA CCCAGACTGT ACCTGCCCCC TCCTGTACCT ATAAGACAGC CTAACAC (Where M is A or C and R is A or
G and W are A or T, S is C or G, Y is C or T, and K is G or T base. ) 2. A human parvovirus VP-1 structural gene encoding the amino acid sequence of the following formula (2). Embedded image Formula (2) 1 10 Met Ser Lys Glu Ser Gly Lys Trp Trp Glu Ser Asp Asp Lys Phe 20 30 Ala Lys Ala X ₁ Tyr Gln Gln Phe Val Glu Phe Tyr Glu Lys Val 40 Thr Gly Thr Asp Leu Glu Leu Ile Gln Ile Leu Lys Asp His Tyr 50 60 Asn Ile Ser Leu Asp Asn Pro Leu Glu Asn Pro Ser Ser Leu Phe 70 X ₂ Leu Val Ala Arg Ile Lys Asn Asn Leu Lys Asn Ser Pro Asp 80 90 Leu Tyr Ser His His Phe Gln Ser His Gly Gln Leu Ser Asp His 100 Pro His Ala Leu Ser Ser Ser Ser Ser Asn Ala Glu Pro Arg Gly 110 120 Glu Asn Ala Val Leu Ser Ser Glu Asp Leu His Lys Pro Gly Gln 130 Val Ser Val Gln Leu Pro Gly Thr Asn Tyr Val Gly Pro Gly Asn 140 150 Glu Leu Gln Ala Gly Pro Pro Gln Ser Ala Val Asp Ser Ala Ala 160 Arg Ile His Asp Phe Arg Tyr Ser Gln Leu Ala Lys Leu Gly Ile 170 180 Asn Pro Tyr Thr His Trp Thr Val Ala Asp Glu Glu Leu Leu Lys 190 Asn Ile Lys Asn Glu Thr Gly Phe Gln Ala Gln Val Val Lys Asp 200 210 Tyr Phe Thr Leu Lys Gly Ala Ala Ala Pro Val Ala His Phe Gln 220 Gly Ser Leu Pro Glu Val Pro Ala Tyr X ₃ Ala Ser Glu L ys Tyr 230 240 Pro Ser Met Thr Ser Val Asn Ser Ala Glu Ala Ser Thr Gly Ala 250 Gly Gly Gly Gly Ser Asn Pro Val Lys Ser Met Trp Ser Glu Gly 260 270 Ala Thr Phe Ser Ala Asn Ser Val Thr Cys Thr Phe Ser Arg Gln 280 Phe Leu Ile Pro Tyr Asp Pro Glu His His Tyr Lys Val Phe Ser 290 300 Pro Ala Ala Ser Ser Cys His Asn Ala Ser Gly Lys Glu Ala Lys 310 Val Cys Thr Ile Ser Pro Ile Met Gly Tyr Ser Thr Pro Trp Arg 320 330 Tyr Leu Asp Phe Asn Ala Leu Asn Leu Phe Phe Ser Pro Leu Glu 340 Phe Gln His Leu Ile Glu Asn Tyr Gly Ser Ile Ala Pro Asp Ala 350 360 Leu Thr Val Thr Ile Ser Glu Ile Ala Val Lys Asp Val Thr Asp 370 Lys Thr Gly Gly Gly Val Gln Val Thr Asp Ser Thr Thr Gly Arg 380 390 Leu Cys Met Leu Val Asp His Glu Tyr Lys Tyr Pro Tyr Val Leu 400 Gly Gln Gly Gln Asp Thr Leu Ala Pro Glu Leu Pro Ile Trp Val 410 420 Tyr Phe Pro Pro Gln Tyr Ala Tyr Leu Thr Val Gly Asp Val Asn 430 Thr Gln Gly Ile Ser Gly Asp Ser Lys Lys Leu Ala Ser Glu Glu 440 450 Ser Ala Phe Tyr Val Leu Glu His Ser Ser Phe Gln Leu Leu Gly Four 60 Thr Gly Gly Thr Ala Thr Met Ser Tyr Lys Phe Pro Pro Val Pro 470 480 Pro Glu Asn Leu Glu Gly Cys Ser Gln His Phe Tyr Glu Met Tyr 490 Asn Pro Leu Tyr Gly Ser Arg Leu Gly Val Pro Asp Thr Leu Gly 500 510 Gly Asp Pro Lys Phe Arg Ser Leu Thr His Glu Asp His Ala Ile 520 Gln Pro Gln Asn Phe Met Pro Gly Pro Leu Val Asn Ser Val Ser 530 540 Thr Lys Glu Gly Asp Ser Ser Asn Thr Gly Ala Gly Lys Ala Leu 550 Thr Gly Leu Ser Thr Gly Thr Ser Gln Asn Thr Arg Ile Ser Leu 560 570 Arg Pro Gly Pro Val Ser Gln Pro Tyr His His Trp Asp Thr Asp 580 Lys Tyr Val Thr Gly Ile Asn Ala Ile Ser His Gly Gln Thr Thr 590 600 Tyr Gly Asn Ala Glu Asp Lys Glu Tyr Gln Gln Gly Val Gly Arg 610 Phe Pro Asn Glu Lys Glu Gln Leu Lys Gln Leu Gln Gly Leu Asn 620 630 Met His Thr Tyr Phe Pro Asn Lys Gly Thr Gln Gln Tyr Thr Asp 640 Gln Ile Glu Arg Pro Leu Met Val Gly Ser Val Trp Asn Arg Arg 650 660 Ala Leu His Tyr Glu Ser Gln Leu Trp Ser Lys Ile Pro Asn Leu 670 Asp Asp Ser Phe Lys Thr Gln Phe Ala Ala Leu Gly Gly Trp Gly 680 690 Leu H is Gln Pro Pro Pro Gln Ile Phe Leu Lys Ile Leu Pro Gln 700 Ser Gly Pro Ile Gly Gly Ile Lys Ser Met Gly Ile Thr Thr Leu 710 720 Val Gln Tyr Ala Val Gly Ile Met Thr Val Thr Met Thr Phe Lys 730 Leu Gly Pro Arg Lys Ala Thr Gly Arg Trp Asn Pro Gln Pro Gly 740 750 Val Tyr Pro Pro His Ala Ala Gly His Leu Pro Tyr Val Leu Tyr 760 Asp Pro Thr Ala Ile Asp Ala Lys Gln His His Arg His Gly Tyr 770 780 Glu Lys Pro Glu Glu Leu Trp Thr Ala Lys Ser Arg Val Arg Pro 781 Leu (wherein X ₁ is Gly or Val, X ₂ is Asn or Asp, and X ₃ is His or Asn. 3. A human parvovirus VP-2 structural gene encoding the amino acid sequence of the following formula (3). [Formula 3] 1 10 Met Thr Ser Val Asn Ser Ala Glu Ala Ser Thr Gly Ala Gly Gly 20 30 Gly Gly Ser Asn Pro Val Lys Ser Met Trp Ser Glu Gly Ala Thr 40 Phe Ser Ala Asn Ser Val Thr Cys Thr Phe Ser Arg Gln Phe Leu 50 60 Ile Pro Tyr Asp Pro Glu His His Tyr Lys Val Phe Ser Pro Ala 70 Ala Ser Ser Cys His Asn Ala Ser Gly Lys Glu Ala Lys Val Cys 80 90 Thr Ile Ser Pro Ile Met Gly Tyr Ser Thr Pro Trp Arg Tyr Leu 100 Asp Phe Asn Ala Leu Asn Leu Phe Phe Ser Pro Leu Glu Phe Gln 110 120 His Leu Ile Glu Asn Tyr Gly Ser Ile Ala Pro Asp Ala Leu Thr 130 Val Thr Ile Ser Glu Ile Ala Val Lys Asp Val Thr Asp Lys Thr 140 150 Gly Gly Gly Val Gln Val Thr Asp Ser Thr Thr Gly Arg Leu Cys 160 Met Leu Val Asp His Glu Tyr Lys Tyr Pro Tyr Val Leu Gly Gln 170 180 Gly Gln Asp Thr Leu Ala Pro Glu Leu Pro Ile Trp Val Tyr Phe 190 Pro Pro Gln Tyr Ala Tyr Leu Thr Val Gly Asp Val Asn Thr Gln 200 210 Gly Ile Ser Gly Asp Ser Lys Lys Leu Ala Ser Glu Glu Ser Ala 220 Phe Tyr Val Leu Glu His Ser Ser Phe Gln Leu Leu Gly T hr Gly 230 240 Gly Thr Ala Thr Met Ser Tyr Lys Phe Pro Pro Val Pro Pro Glu 250 Asn Leu Glu Gly Cys Ser Gln His Phe Tyr Glu Met Tyr Asn Pro 260 270 Leu Tyr Gly Ser Arg Leu Gly Val Pro Asp Thr Leu Gly Gly Asp 280 Pro Lys Phe Arg Ser Leu Thr His Glu Asp His Ala Ile Gln Pro 290 300 Gln Asn Phe Met Pro Gly Pro Leu Val Asn Ser Val Ser Thr Lys 310 Glu Gly Asp Ser Ser Asn Thr Gly Ala Gly Lys Ala Leu Thr Gly 320 330 Leu Ser Thr Gly Thr Ser Gln Asn Thr Arg Ile Ser Leu Arg Pro 340 Gly Pro Val Ser Gln Pro Tyr His His Trp Asp Thr Asp Lys Tyr 350 360 Val Thr Gly Ile Asn Ala Ile Ser His Gly Gln Thr Thr Tyr Gly 370 Asn Ala Glu Asp Lys Glu Tyr Gln Gln Gly Val Gly Arg Phe Pro 380 390 Asn Glu Lys Glu Gln Leu Lys Gln Leu Gln Gly Leu Asn Met His 400 Thr Tyr Phe Pro Asn Lys Gly Thr Gln Gln Tyr Thr Asp Gln Ile 410 420 Glu Arg Pro Leu Met Val Gly Ser Val Trp Asn Arg Arg Ala Leu 430 His Tyr Glu Ser Gln Leu Trp Ser Lys Ile Pro Asn Leu Asp Asp 440 450 Ser Phe Lys Thr Gln Phe Ala Ala Leu Gly Gly Trp Gly Leu His Four 60 Gln Pro Pro Pro Gln Ile Phe Leu Lys Ile Leu Pro Gln Ser Gly 470 480 Pro Ile Gly Gly Ile Lys Ser Met Gly Ile Thr Thr Leu Val Gln 490 Tyr Ala Val Gly Ile Met Thr Val Thr Met Thr Phe Lys Leu Gly 500 510 Pro Arg Lys Ala Thr Gly Arg Trp Asn Pro Gln Pro Gly Val Tyr 520 Pro Pro His Ala Ala Gly His Leu Pro Tyr Val Leu Tyr Asp Pro 530 540 Thr Ala Ile Asp Ala Lys Gln His His Arg His Gly Tyr Glu Lys 550 554 Pro Glu Glu Leu Trp Thr Ala Lys Ser Arg Val Arg Pro Leu 4. A human parvovirus nonstructural (NS) gene encoding the amino acid sequence of the following formula (4). Formula (4) 1 10 Met Glu Leu Phe Arg Gly Val Leu Gln Val Ser Ser Asn Val Leu 20 30 Asp Cys Ala Asn Asp Asn Trp Trp Cys Ser Leu Leu Asp Leu Asp 40 Thr Ser Asp Trp Glu Pro Leu Thr His Thr Asn Arg Leu Met Ala 50 60 Ile Tyr Leu Ser Ser Val Ala Ser Lys Leu Asp Phe Thr Gly Gly 70 Pro Leu Ala Gly Cys Leu Tyr Phe Phe Gln Val Glu Cys Asn Lys 80 90 Phe Glu Glu Gly Tyr His Ile His Val Val Ile Gly Gly Pro Gly 100 Leu Asn Pro Arg Asn Leu Thr Val Cys Val Glu Gly Leu Phe Asn 110 120 Asn Val Leu Tyr His Leu Val Thr Glu Asn Val Lys Leu Lys Phe 130 Leu Pro Gly Met Thr Thr Lys Gly Lys Tyr Phe Arg Asp Gly Glu 140 150 Gln Phe Ile Glu Asn Tyr Leu Met Lys Lys Ile Pro Leu Asn Val 160 Val Trp Cys Val Thr Asn Ile Asp Gly Tyr Ile Asp Thr Cys Ile 170 180 Ser Ala Thr Phe Arg Arg Gly Ala Cys His Ala Lys Lys Pro Arg 190 Ile Thr Thr Ala Ile Asn Asp Thr Ser Ser Asp Ala Gly Glu Ser 200 210 Ser Gly Thr Gly Ala Glu Val Val Pro Phe Asn Gly Lys Gly Thr 220 Lys Ala Ser Ile Lys Phe Gln Thr Met Val Asn Trp Leu C ys Glu 230 240 Asn Arg Val Phe Thr Glu Asp Lys Trp Lys Leu Val Asp Phe Asn 250 Gln Tyr Thr Leu Leu Ser Ser Ser His Ser Gly Ser Phe Gln Ile 260 270 Gln Ser Ala Leu Lys Leu Ala Ile Tyr Lys Ala Thr Asn Leu Val 280 Pro Thr Ser Thr Phe Leu Leu His Thr Asp Phe Glu Gln Val Met 290 300 Cys Ile Lys Asp Asn Lys Ile Val Lys Leu Leu Leu Cys Gln Asn 310 Tyr Asp Pro Leu Leu Val Gly Gln His Val Leu Lys Trp Ile Asp 320 330 Lys Lys Cys Gly Lys Lys Asn Thr Leu Trp Phe Tyr Gly Pro Pro 340 Ser Thr Gly Lys Thr Asn Leu Ala Met Ala Ile Ala Lys Ser Val 350 360 Pro Val Tyr Gly Met Val Asn Trp Asn Asn Glu Asn Phe Pro Phe 370 Asn Asp Val Ala Gly Lys Ser Leu Val Val Trp Asp Glu Gly Ile 380 390 Ile Lys Ser Thr Ile Val Glu Ala Ala Lys Ala Ile Leu Gly Gly 400 Gln Pro Thr Arg Val Asp Gln Lys Met Arg Gly Ser Val Ala Val 410 420 Pro Gly Val Pro Val Val Ile Thr Ser Asn Gly Asp Ile Thr Phe 430 Val Val Ser Gly Asn Thr Thr Thr Thr Val His Ala Lys Ala Leu 440 450 Lys Glu Arg Met Val Lys Leu Asn Phe Thr Val Arg Cys Ser Pro Four 60 Asp Met Gly Leu Leu Thr Glu Ala Asp Val Gln Gln Trp Leu Thr 470 480 Trp Cys Asn Ala Gln Ser Trp Asp His Tyr Glu Asn Trp Ala Ile 490 Asn Tyr Thr Phe Asp Phe Pro Gly Ile Asn Ala Asp Ala Leu His 500 510 Pro Asp Leu Gln Thr Thr Pro Ile Val Thr Asp Thr Ser Ile Ser 520 Ser Ser Gly Gly Glu Ser Ser Glu Glu Leu Ser Glu Ser Ser Phe 530 540 Phe Asn Leu Ile Thr Pro Gly Ala Trp Asn Thr Glu Thr Pro Arg 550 Ser Ser Thr Pro Ile Pro Gly Thr Ser Ser Gly Glu Ser Phe Val 560 570 Gly Ser Pro Val Ser Ser Glu Val Val Ala Ala Ser Trp Glu Glu 580 Ala Phe Tyr Thr Pro Leu Ala Asp Gln Phe Arg Glu Leu Leu Val 590 600 Gly Val Asp Tyr Val Trp Asp Gly Val Arg Gly Leu Pro Val Cys 610 Cys Val Gln His Ile Asn Asn Ser Gly Gly Gly Leu Gly Leu Cys 620 630 Pro His Cys Ile Asn Val Gly Ala Trp Tyr Asn Gly Trp Lys Phe 640 Arg Glu Phe Thr Pro Asp Leu Val Arg Cys Ser Cys His Val Gly 650 660 Ala Ser Asn Pro Phe Ser Val Leu Thr Cys Lys Lys Cys Ala Tyr 670 671 Leu Ser Gly Leu Gln Ser Phe Val Asp Tyr Glu 5. A recombinant vector containing the gene according to any one of claims 1 to 4 and capable of expressing the gene in a host cell. 6. The recombinant vector according to claim 5, wherein the gene is inserted into a baculovirus vector. 7. A transformant transformed with the recombinant vector according to claim 5. 8. The transformant according to claim 7, wherein the host of the transformant is Escherichia coli. 9. The transformant according to claim 7, wherein the host of the transformant is an insect cell. 10. The transformant according to claim 7, wherein the recombinant vector is the recombinant vector according to claim 6, and the host of the transformant is an insect cell. 11. The transformant according to claim 10, wherein the insect cells are Tn5 cells. 12. A polypeptide having the amino acid sequence represented by the formula (2). 13. A polypeptide having the amino acid sequence represented by the formula (3). 14. A polypeptide having an amino acid sequence represented by the above formula (4). 15. A virus particle-like structure of human parvovirus, which is composed of the polypeptide of claim 13 alone or the polypeptide of claim 12 and the polypeptide of claim 13. 16. A method for detecting an anti-human parvovirus antibody in a sample by an immunoassay using the polypeptide according to claim 12 or 13 or the virus particle-like structure according to claim 15 as an antigen. 17. The method according to claim 16, wherein a mixture of the polypeptide according to claim 12 and the polypeptide according to claim 13 is used as an antigen. 18. The method according to claim 17, wherein the mixing ratio of the two kinds of polypeptides is 1:10 to 10: 1. 19. A human parvovirus in a sample is prepared by preparing an antibody using the polypeptide according to claim 12 or 13 or the virus particle-like structure according to claim 15 as an immunogen, and performing immunoassay using the antibody. How to detect. 20. A gene according to claim 1 or a part thereof is amplified by PCR using a pair of oligonucleotides that hybridize with a part of the gene according to claim 1, respectively, and the amplified gene or A method for detecting human parvovirus in a sample, which comprises detecting a part thereof. 21. The oligonucleotide has the following D:
19. The detection method according to claim 18, wherein at least one type is selected from each of the NA primer group 1 and the DNA primer group 2. DNA primer group 1 D-1: GCTGCCATGTGGGAGCTTCTAATC D-4: TTCCCGCCTTATGCAAATGGGCAGC DNA primer group 2 U-2: GTGTTAGGCTGTCTTATAGGTACA U-4: CTTGTATTCATGGTCTACTAAC