JPH0488985A - Human parvovirus structural protein gene - Google Patents

Abstract

NEW MATERIAL:A human parvovirus structural protein VP-1 gene expressed by formula I to formula III (N is an undetermined base). USE:Acquisition of human parvovirus antigen, etc. PREPARATION:A human parvovirus DNA is extracted and purified from virus grains obtained from acute serum of adult patient infected with causative virus of erythema infectiosum(EI) of child. DNA synthesized based on a gene sequence of human parvovirus B 19 strain is used as a primer and human Parvovirus structural protein gene is amplified by PCR method and the resultant each DNA fragment is integrated into a cloning vector to carry out cloning.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a novel human parvovirus structural protein gene that is involved in the pathogenesis of various diseases such as miscarriage, hydrops fetalis, liver damage, hemorrhagic fever, arthritis, and rheumatism.

[Problems to be solved by conventional technology and invention]

The valpovirus family belongs to the smallest group of viruses (family Parvoviridae) that has a single-stranded, linear DNA genome. When Parvovirjdae family viruses are observed under an electron microscope, the particles are spherical, exhibit icosahedral symmetry, and have 32 capsomeres.
It is an extremely small virus with a diameter of approximately 20 to 25 nm consisting of the
tervirology, 23.6l-73 (198
5) ). Hosts of balboviruses that infect vertebrates include cows, pigs, dogs, cats, rabbits, rats, mice, mink, weasels, geese, and humans; although they are generally similar in pathogenicity, these No immunological cross-reactivity between viruses has been found. Therefore, it is important that virus antigens that can be used for human parvovirus diagnosis and vaccine development be derived from human parvovirus.

Human valpovirus was first known to exist in 1975, CLancet, i, 72-73. (1
975)], but its pathogenicity was unknown. Later, it was known to cause fulminant aplastic anemia in monks with a hereditary hemolytic disease (Lancet, i, 664-
665 (1981)), it has become certain that it is the causative virus of erythema infectiosum, which is prevalent in children [Lance et al.
t, i, 1378. (1983)), and is known to cause fever, indeterminate rash, hemorrhagic fever-like disease, liver damage, arthritis, miscarriage, and hydrops fetalis, even in healthy adults. History of medicine,
142.530-532 (1987)). It is also presumed to be involved in rheumatism [36th Japanese Society of Virology Abstracts, 328 (1988)].

As described above, human parvovirus can be said to be an extremely interesting virus clinically. However, because it is difficult to obtain viral antigens, the generalization of diagnostic systems has been slow, and blood screening and clinical tests cannot be easily performed.

The routes of human valpovirus invasion into individuals are known to be through blood transfusion and the nasal cavity, and its target cells are extremely limited erythroid progenitor cells (Nature, 302.426-429).
+983) ). When human parvovirus invades the body, the proliferation of the virus in the blood generally occurs within 6 to 12 days after infection.
Viral antigens are observed during the subclinical period, and it is difficult to detect viral antigens after symptoms appear. Therefore, human parvovirus infection is proven by detecting anti-human parvovirus antibodies in serum rather than the virus antigen itself.

The human parvovirus diagnostic system currently uses approximately 2 donated blood specimens.
Viral particles are purified from viremic serum, which is found in 1 in 10,000 cases, and used as an antigen using the immunoelectric countercurrent method [Medical History, 135.317-318 (1).
985)) and Uenf Immunoassay method [Igaku no Ayumi, 134.909-910 (1985)) and radioimmunoassay method [Journal of Infectious Diseases 58.1213
-1220 (1985)) has only been carried out within a very limited range. Moreover, the detection sensitivity of the immunoelectric countercurrent method is low, and the amount of viral antigen is insufficient in either method for more extensive testing. Under these circumstances, in order to develop a diagnostic system for human parvovirus, it is important to obtain a large amount of useful viral antigens.

Generally, methods for obtaining viral antigens for diagnostic and pharmaceutical applications include in vitro infection and propagation of viruses, or cloning of viral genes.
These methods are broadly classified into methods for expressing antigen proteins in large quantities. in
The Vitro infection system has been reported using bone marrow cells of patients with special genetic diseases [BIood, 70.384-3
91 (1987)), but the virus recovery rate is extremely low (and there are very few virus target cells in normal bone marrow, so the JN VJ TRO infection system using bone marrow cells has a limited range of use. Many cell lines had also lost their susceptibility to human parvovirus.Recently,
Infectivity was confirmed in one of the established cell lines, but virus proliferation was extremely low in this system [Clinical and Microbiology, 16, 1
77-186 (1989)).

The present inventors have previously reported an in vitro method for propagating human parvovirus using erythroblast cells derived from fetal liver tissue [JP-A-63-242166]. Although this is an effective method for propagating parvovirus, there are some difficulties in obtaining raw materials, and it has not always been sufficient for widespread use.

Human parvovirus genes are required to obtain viral antigens by genetic recombination. Currently, the complete sequence of the human parvovirus B19 strain gene derived from the serum of a sickle cell anemia patient infected with human parvovirus has been reported (J, Virol, 58.921-93
6 (1986)). Analysis of this gene product revealed that the viral structural protein present in virus particles is V.
There are only two types of proteins, P-1 (molecular weight approximately 84 KDa) and VP-2 (molecular weight approximately 58 KDa), and the reactivity between purified human parvovirus particles and patient serum indicates that a host infected with human parvovirus It has been confirmed that antigen-antibody reactions are induced by targeting these VP-1 and VP-2 (J.

Virol, 61.2627 (1987)) Furthermore, it has been confirmed that the protein coding region of the VP-2 gene is completely contained within the VP-1 gene, and therefore, it is useful as an antigen for diagnostic systems. , VP-1 and VP-2
Both antigens can be considered suitable partial antigens.

c. Means for Solving Problems] Therefore, the present inventors conducted intensive studies to obtain human parvovirus genes, and found that they have some structural differences from conventionally known human parvovirus genes at the amino acid level and DNA level. The present invention was completed by discovering a human parvovirus gene useful for the development of a novel diagnostic system.

That is, the gist of the present invention is a human parvovirus structural protein VP-1 gene having a partial base sequence represented by the following formulas (1) to (3) and a human parvovirus structural protein VP-1 gene having a partial base sequence represented by the following formulas (2) to (3). It exists in the human parvovirus structural protein VP-2 gene, which has a partial base sequence.

Formula (1) % Formula % Formula (2) AGAAGCCAGC GAGGGGGGGG GTCAAAAGCA GGGGCCACT ACTCTGTAAC TCCAGACAGT ATATGACCCA ATAAGGTGTT GCAAGTAGCT CAGTGGAAAG TTTGCACCAT T AAAGTGATGA AAAACTGTGT TGTGGAATTT TTACGGGAAC CTTATTCAAA TCΔTTATAAT ACTGGTGCAG CAGTAATCCT TGTGCAGTGATTAGTGCCA T TGTACATTT TTTTAATTCC GAGCACCATT TTCTCCCNTA GCCACAATGC GAGGCAAGG TAGTCCCATA Formula (3) % formula % (Above formula (N represents a base whose structure has not been determined.) The present invention will be explained below.

The human parvovirus structural protein genes of the present invention include the VP-1 gene with a molecular weight of 84 KDa and the VP-1 gene with a molecular weight of 58 KDa.
There are two types of VP-2 genes, VP-2 gene is vp-
Contained within the i gene region.

The VP-1 gene has the above formula (I), formula (2) and formula (3).
), the VP-2 gene contains a partial base sequence represented by
It includes the partial base sequences represented by the formulas (2) and (3) above.

Examples of materials for cloning the human parvovirus structural protein genes of the present invention include acute-phase serum from adult patients infected with the virus that causes erythema infectious disease (EI) in children.

Such virus particles in serum are precipitated and purified, for example, by saccharose density gradient centrifugation, and human valpovirus DNA is extracted from the obtained virus particles according to a conventional method.
is extracted, purified, and used for cloning.

The cloning was carried out using the gene sequence of human parvovirus strain BIQ (J, Vjrol, 58.921-936 (1
Polymerase chain reaction CPCR method using DNA synthesized based on 986)) as a primer: N
ature, fi..., 163 (1986)), the target human parvovirus structural protein gene can be amplified.

Specifically, for example, purified human parvovirus DN
Two types of DNA primers, a DNA primer consisting of a partial sequence of the gene and a DNA primer consisting of a partial sequence complementary to the gene, are annealed to A, and the DNA between the DNA primers is amplified according to a conventional method.

Each of the obtained DNA fragments is inserted into a cloning vector such as pUCI9 (manufactured by Toyobo Co., Ltd.) and cloned. Plasmid DNA is extracted from the obtained clone, and the DNA sequence incorporated into the vector is extracted by the dideoxy method or the like. By determining this, the entire base sequence of the target human parvovirus structural protein gene can be determined.

According to the present invention, the VP-1 gene has a partial base sequence represented by the formula (1), formula (2), and formula (3), and the VP-2 gene has the partial base sequence represented by the formula (1), formula (2), and formula (3). (2) and formula (3
) was found to have a partial base sequence expressed as:

Among the base sequences represented by formulas (1) to (3), 122 positions at the base level and 6 positions at the amino acid level differed from the sequence of the known human parvovirus Ble strain.

〔Effect of the invention〕

The human parvovirus structural protein gene of the present invention can be obtained by radioimmunoassay method by introducing its entire sequence or a DNA fragment obtained by PCR into a known expression vector and expressing it in a host cell transformed with the expression vector. , recombinant antigens can be obtained that can be used for diagnosis such as enzyme immunoassay.

〔Example〕

The present invention will be explained in more detail with reference to Examples below.

Example 1 [■] Preparation of human parvovirus structural protein gene Serum was collected from adults infected with the virus responsible for the epidemic of erythema infectiosum (El) in children.

Serum in which human parvoviremia was confirmed by enzyme immunoassay and DNA hybridization was selected and used as the material for the human valpovirus structural protein gene.

1 ml of the above serum with a viral DNA amount of approximately Inμg/ml
, phosphate buffered saline (PBS) pH 7.4+,
: Dilute 2 times, 12. - The supernatant obtained by centrifugation at 000 rpm for 60 minutes was layered on 2 ml of a 30% saccharose aqueous solution.

This was heated at 4℃ using a Hitachi ultracentrifuge (rotor RPS-56T).
Nite 35. The virus particles were collected as a precipitate by centrifugation at 00 Orpm for 15 hours.

This was mixed with a 75mM NaCl-25mM EDTA aqueous solution (p
After suspending in H8,0), add sodium dodecyl sulfate (SDS) to a final concentration of 1% and add 0.1 mg/ml.
Proteinase K was added and heated at 56°C for over 1 hour to thermally denature the DNA.

The denatured DNA in this suspension was treated with phenol and
Human parvovirus DNA was purified by chloroform extraction and ethanol precipitation.

This DNA was dissolved in 10 μm water.

(II) Cloning of human parvovirus structural protein gene Using 1 μm of the DNA sample obtained in (1) above, 5 aik
The method of i et al. (Nature 324.163. (19
The valpovirus gene was amplified by PCR method according to [86]. The PCR method is Takara Gene manufactured by Takara Shuzo Co., Ltd.
Amp'' DNA kit was used and the instructions were followed.

i.e. 50mM KCI, 10mM Tris-HC1pH
In a reaction solution containing 8, 3, 1, 5 mM MgCIt 0.1% (W/V) gelatin, 1 μm of human parvovirus DNA sample was injected with 6 types of synthetic NA primers shown in Figure 2 from the six types shown in Figure 1. Choose two combinations and get 10 each
0p100p was annealed and an amplification reaction was performed.

The polymerase chain reaction was carried out in a reaction solution with an allowable amount of 100 μm at 95°C for 5 minutes in the absence of Taq polymerase, and then at 95°C for 1 minute in the presence of Taq polymerase.
The amplification reaction was performed for 35 cycles at 7°C, 1 min - 72°C, 1 min.
After the cycle, the reaction was further carried out at 72°C for 7 minutes.

By the above method, four DNA fragments shown in FIG. 2 were obtained.

These DNA fragments were further made blunt-ended with T4 polymerase, and a phosphate group was introduced into the 5' end using polynucleotide kinase. They were then inserted into a pUC19 cloning vector, transformed into E. coli, and the transformants were cultured to yield approximately 100 cells each. We obtained more than one clone.

DNA was prepared from the obtained clones using a conventional method, and the DNA was prepared using a DuPont fluorescent sequencer GENESIS 200.
The partial base sequence of each DNA fragment was determined using 0. The arrangement is shown in FIG.

[Brief explanation of drawings]

Figure 1 shows the nucleotide sequences (5''→3') of the six types of synthetic NA primers (ON 1 to 0N6) used in the examples. The positions of the NA primers (ON 1 to 0N6) and the cloned DNA fragments of four human parvoviruses relative to the VP-1 and VP-2 genes are shown. In addition, ←→ in each DNA fragment indicates the DNA position of the base sequence shown in Figure 3. Figure 3 shows partial bases of the four types of DNA fragments determined in the example. The sequence is shown. In the figure, the bases that differ from the B19 strain gene are marked with a single mark, and the parts with different amino acids are marked with a single mark.

Claims (1)

[Claims] (1) A human parvovirus structural protein VP-1 gene having a partial base sequence represented by the following formulas (1) to (3). Formula (1) [There is a gene sequence. ] Formula (2) [There is a gene sequence. ] [There is a gene sequence. ] Formula (3) [There is a gene sequence. ] [There is a gene sequence. (In the above formula, N represents a base whose structure has not been determined.) (2) A human parvovirus structural protein VP-2 gene having a partial base sequence represented by the following formula (2) or formula (3). Formula (2) [There is a gene sequence. ] [There is a gene sequence. ] Formula (3) [There is a gene sequence. ] [There is a gene sequence. ] (In the above formula, N represents a base whose structure has not been determined.)