JPH0488985A - Human parvovirus structural protein gene - Google Patents
Human parvovirus structural protein geneInfo
- Publication number
- JPH0488985A JPH0488985A JP20282790A JP20282790A JPH0488985A JP H0488985 A JPH0488985 A JP H0488985A JP 20282790 A JP20282790 A JP 20282790A JP 20282790 A JP20282790 A JP 20282790A JP H0488985 A JPH0488985 A JP H0488985A
- Authority
- JP
- Japan
- Prior art keywords
- human parvovirus
- gene
- dna
- formula
- structural protein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
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- 101710172711 Structural protein Proteins 0.000 title claims abstract description 13
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- 101710106388 Structural protein VP1 Proteins 0.000 claims abstract description 4
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Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は、流産、胎児水腫、肝障害、出血熱、関節炎や
リューマチなど種々の疾患の病因に係わる新規なヒトパ
ルボウイルス構造蛋白質遺伝子に関する。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.
バルポウイルスの仲間は、−本鎖の直鎖DNAゲノムを
有する最小のウィルス群(Parvoviridae科
)に属する。Parvovirjdae科ウィルスを電
子顕微鏡で見ると、その粒子は球状で、正二十面体様対
称性を示し、32個のキャプソメア(capsomer
e )から成る0直径約20〜25nmの極めて小型の
ウィルスで、エンベローブヲ保有せず、熱、乾燥、脂質
溶剤、洗剤などに比較的耐性のウィルスである( In
tervirology、 23.6l−73(198
5) )。を椎動物に感染するバルボウイルスの宿主に
は、ウシ、ブタ、イヌ、ネコ、ウサギ、ラット、マウス
、ミンク、イタチ、ガチョウ及びヒトが含まれるが、一
般に病原性は似ているものの、これらのウィルス間で免
疫学的な交叉性は見出されていない。従って、ヒトパル
ボウイルスの診断やワクチンの開発に使用可能なウィル
ス抗原は、ヒトパルボウイルス由来のものであることが
重要な条件となる。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.
ヒトバルポウイルスは、1975年に初めてその存在が
知られたCLancet、 i 、72−73. (1
975)〕が、病原性は不明であった。その後、遺伝性
溶血性疾患を有する僧体に、劇症の再生不良性貧血を引
き起こすことが知られ(Lancet、 i、664−
665(1981)) 、小児に流行する伝染性紅斑症
の病因ウィルスとして確かなものとなった〔Lance
t、 i 、1378.(1983)) 、更に、健常
成人においても、現在までのところ、発熱、不定の発疹
症、出血熱様疾患、肝障害、関節炎、流産や胎児水腫な
どの原因となることが知られている〔医学のあゆみ、
142.530−532(1987))。またリュウー
マチへの関与も推定されている〔第36回日本ウィルス
学会抄録、 328(1988) )。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)].
以上のように、ヒトパルボウイルスは、臨床上極めて興
味深いウィルスであるといえる。しかしながら、ウィル
ス抗原の取得が困難であるため、診断系の一般化が遅れ
ており、血液のスクリーニングや臨床検査が容易に行え
ないのが現状である。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.
ヒトバルポウイルスの個体への侵入経路は、輸血と鼻腔
が知られ、その標的細胞は極く限られた赤芽球の前駆細
胞である(Nature、 302.426−429(
+983) )。ヒトパルボウイルスが体内に侵入した
場合、血中のウィルスの増殖は、一般に感染後6〜12
日の不顕性期に観察され、症状が現れてからのウィルス
抗原の検出は困難である。従って、ヒトパルボウイルス
感染の証明は、ウィルス抗原そのものではなく、血清中
に抗ヒトパルボウイルス抗体を検出することによって行
われている。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.
ヒトパルボウイルス診断系は、現在、献血検体およそ2
万例に1例の割合で発見されるウィルス血症の血清より
、ウィルス粒子を精製し、それを抗原として、免疫電気
向流法〔医学のあゆみ、 135.317−318(1
985)) 及Uエンf イムイムノアッセイ法〔医学
のあゆみ、 134.909−910(1985))や
ラジオイムノアッセイ法〔感染症学雑誌58.1213
−1220(1985))が極く限られた範囲内で行わ
れているにすぎない。しかも、免疫電気向流法は検出感
度が低く、さらに広範な検査のためには、いずれの方法
においてもウィルス抗原量が不足している。このような
状況からヒトパルボウイルスの診断系の開発のためには
、有用かつ大量のウィルス抗原の取得が重要である。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.
一般に、診断、医薬応用のためのウィルス抗原の取得方
法は、in vjtroでウィルスを感染・増殖させる
方法、もしくは、ウィルス遺伝子をクローニングして、
抗原蛋白を大量に発現させる方法に大別される。in
Vitro感染系は、特殊な遺伝的疾患患者の骨髄細胞
を用いた報告例がある[BIood、70.384−3
91(1987) )が、ウィルスの回収率は極めて低
(、また、通常の骨髄中にはウィルス標的細胞が極めて
少ないため、骨髄細胞を用いたjn vjtro感染系
はその使用範囲に制限がある。多くの株化細胞も、ヒト
パルボウイルスに対する感受性を消失していた。最近、
株化細胞の1つに感染性が確認されたが、この系ではウ
ィルスの増殖は極めて低い〔臨床と微生物、 16,1
77−186(1989))。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)).
本発明者らは、先に胎児肝組織由来の赤芽球細胞を用い
たin vitroのヒトパルボウイルス増殖方法を報
告している〔特開昭63−242166号〕この方法は
、in vitroでヒトパルボウイルスを増殖させる
のに有力な方法であるが、原料の入手の点でやや難点が
あり、広く利用するためには必ずしも十分ではなかった
。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.
遺伝子組換え法によってウィルス抗原を取得する為には
、ヒトパルボウイルス遺伝子が必要である。現在、ヒト
パルボウイルスに感染した鎌形赤血球貧血患者血清由来
のヒトパルボウイルスB19株遺伝子の全配列が報告さ
れている( J、 Virol、 58.921〜93
6(1986) )。この遺伝子産物の解析から、ウィ
ルス構造蛋白質としてウィルス粒子に存在するのは、V
P−1(分子量約84KDa)及びVP−2(分子量約
58KDa)の2種類の蛋白質のみであり、また、精製
ヒトパルボウイルス粒子と患者血清との反応性から、ヒ
トパルボウイルスに感染した宿主がこれらVP−1及び
VP−2を標的として抗原抗体反応を惹起することが確
かめられている(J。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) ) 、更に
また、VP−2遺伝子の蛋白コード領域は、VP−1遺
伝子の内部に完全に含まれていることも確認されており
、従って、診断系の抗原として、VP−1及びVP−2
抗原はどちらも好適な部分抗原であるといえる。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問題点を解決するための手段〕
そこで、本発明者らは、ヒトパルボウイルス遺伝子を得
るべく鋭意検討したところ、従来公知のヒトパルボウイ
ルス遺伝子と、アミノ酸レベル及びDNAレベルで幾つ
か構造の異なる新規な、診断系の開発に有用なヒトパル
ボウイルス遺伝子を見出し本発明を完成するに到った。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.
即ち、本発明の要旨は、下記式(1)〜式(3)で表さ
れる部分塩基配列を有するヒトパルボウイルス構造蛋白
質VP−1遺伝子及び下記式(2)〜式(3)で表され
る部分塩基配列を有するヒトパルボウイルス構造蛋白質
VP−2遺伝子に存する。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.
式(1)
%式%
式(2)
AGAAGCCAGC
GAGGGGGGGG
GTCAAAAGCA
GGGGGCCACT
ACTCTGTAAC
TCCAGACAGT
ATATGACCCA
ATAAGGTGTT
GCAAGTAGCT
CAGTGGAAAG
TTTGCACCAT
T
AAAGTGATGA
AAAACTGTGT
TGTGGAATTT
TTACGGGAAC
CTTATTCAAA
TCΔTTATAAT
ACTGGTGCAG
CAGTAATCCT
TGTGCAGTGA
TTTAGTGCCA
TTGTACATTT
TTTTAATTCC
GAGCACCATT
TTCTCCCNTA
GCCACAATGC
GAGGCAAAGG
TAGTCCCATA
式(3)
%式%
(上記式中、Nは構造未決定の塩基を表す。)以下に本
発明を説明する。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.
本発明のヒトパルボウイルス構造蛋白質遺伝子は、分子
量的84KDaのVP−1遺伝子と分子量的58KDa
のVP−2遺伝子の2種類で、VP−2遺伝子はvp−
i遺伝子の領域内に含まれている。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.
VP−1遺伝子は、前記式(I)、式(2)及び式(3
)で表される部分塩基配列を含み、VP−2遺伝子は、
前記式(2)及び式(3)で表される部分塩基配列を含
む。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.
本発明のヒトパルボウイルス構造蛋白質遺伝子をクロー
ニングするための材料としては、例えば、小児の伝染性
紅斑症(E I)の原因ウィルスに感染した成人患者急
性期血清等が挙げられる。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.
このような血清中のウィルス粒子を、例えばサッカロー
ス密度勾配遠心によって沈澱させて精製し、得られるウ
ィルス粒子から常法に従っでヒトバルポウィルスDNA
を抽出・精製して、クローニングに供する。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.
クローニングは、ヒトパルボウイルスBIQ株の遺伝子
配列(J、 Vjrol、 58.921−936(1
986) )に基づき合成したDNAをプライマーとし
て使用して、ポリメラーセ連鎖反応法CPCR法: N
ature、fi…、 163(1986) )によっ
て目的とするヒトパルボウイルス構造蛋白質遺伝子を増
幅することができる。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.
具体的には、例えば、精製したヒトパルボウイルスDN
Aに、該遺伝子の部分配列からなるDNAブライマーと
該遺伝子に相補的な部分配列からなるDNAブライマー
の2種のDNAブライマーをアニーリングさせ、常法に
従って、該DNAブライマー間のDNAを増幅させる。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.
得られた各DNA断片を、クローニングベクター、例え
ば、pUCI9(東洋紡社製)等に組み込んでクローニ
ングし、得られるクローンからプラスミドDNAを抽出
し、該ベクターに組み込まれているDNA配列をジデオ
キシ法等により決定することにより、目的とするヒトパ
ルボウイルス構造蛋白質遺伝子の全塩基配列を決めるこ
とができる。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.
本発明によれば、VP−1遺伝子は、前記式(1)、式
(2)及び式(3)で表される部分塩基配列を有してお
り、また、VP−2遺伝子は、前記式(2)及び式(3
)で表される部分塩基配列を有していることが判った。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:
これら式(1)〜式(3)で表される塩基配列中、塩基
レベルで122個所アミノ酸レベルで6個所が、公知の
ヒトパルボウイルスBle株の配列と異なっていた。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.
本発明のヒトパルボウイルス構造蛋白質遺伝子は、その
全配列或いはPCR法で得られるDNA断片を公知の発
現ベクターに導入し、該発現ベクターで形質転換した宿
主細胞内で発現させることにより、ラジオイムノアッセ
イ法、エンザイムイムノアッセイ法等の診断に使用し得
る組換え抗原を得ることができる。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.
以下に実施例を挙げて本発明を更に具体的に説明する。 The present invention will be explained in more detail with reference to Examples below.
実施例1
〔■〕ヒトパルボウイルス構造蛋白質遺伝子の調製
小児の伝染性紅斑(El)の流行剤に該原因ウィルスに
感染した成人から血清を採取した。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.
エンザイムイムノアッセイ法及びDNAハイブリダイゼ
ーション法にてヒトパルボウイルス血症を確認できた血
清を選び、ヒトバルポウィルス構造蛋白質遺伝子の材料
とした。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.
ウィルスDNA量が約Inμg/mlの上記血清1ml
を、リン酸緩衝生理食塩水(PBS) pH7,4+、
:て2倍に希釈し、12.・000rpm、60分間遠
心した上清を、30%サッカロース水溶液2ml上に重
層した。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.
これを日立超遠心機(ローターRPS−56T)で4℃
ニテ35. 00 Orpm、 15時間遠心してウ
ィルス粒子を沈澱として回収した。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.
これを75mMNaC1−25mMEDTA水溶液(p
H8,0)に縣濁後、終濃度1%となるようにドデシル
硫酸ナトリウム(SDS)を、また、0.1mg/ml
プロテイナーゼKを加えて56℃にて1時間以上加熱し
てDNAを熱変性させた。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.
この縣濁液中の変性DNAを常法に従い、フェノール・
クロロホルム抽出、エタノール沈澱によりヒトパルボウ
イルスのDNAを精製した。The denatured DNA in this suspension was treated with phenol and
Human parvovirus DNA was purified by chloroform extraction and ethanol precipitation.
このDNAは10μmの水に溶解した。This DNA was dissolved in 10 μm water.
(II)ヒトパルボウイルス構造蛋白質遺伝子のクロー
ニング
上記(1)で得たDNA試料1μmを用いて、5aik
iらの方法(Nature 324.163. (19
86)〕に準じてPCR法によってバルポウイルス遺伝
子を増幅した。PCR法は宝酒造社製タカラGene
Amp ” DNAキットを用い、その説明書に従って
行った。(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.
即ち、50mMKCI、10mMTris−HC1pH
8,3,1,5mMMgCIt0.1%(W/V)ゼラ
チンを含む反応液中で、ヒトパルボウイルスDNA試料
1μmに第1図に示した6種類の合成りNAブライマー
から第2図に示した組合せの2種を選び、それぞれ10
0p100pをアニーリングさせ、増幅反応を行った。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.
ポリメラーゼ連鎖反応は許容量100μmの反応液中で
、Taqポリメラーゼ非存在下で95℃、5分間反応後
、Taqポリメラーゼ存在下で95℃、1分−30〜3
7℃、1分−72℃、1分のサイクルで増幅反応を35
サイクル行ったのち、さらに72℃、7分間反応させた
。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.
上記方法によって、第2図に示した4個のDNA断片を
得た。By the above method, four DNA fragments shown in FIG. 2 were obtained.
これらDNA断片は、さらにT4ポリメラーゼで平滑末
端化し、ポリヌクレオチドキナーゼで5′末端にリン酸
基を導入後、pUC19クローニングベクターに組み込
み、大腸菌を形質転換し、形質転換体を培養してそれぞ
れ約100個以上のクローンを得た。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.
得られたクローンからDNAを常法により調製し、デュ
ポン社製蛍光シークエンサーGENESIS 200
0を用いて各DNA断片の部分塩基配列を決定した。そ
の配列を第3図に示した。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.
第1図は、実施例で使用した6種類の合成りNAプライ
マー(ON 1〜0N6)の塩基配列(5”→3′)を
表す。
第2図は、実施例で使用した6種類の合成りNAプライ
マー(ON 1〜0N6)及びクローニングした4種の
ヒトパルボウイルスのDNA断片のVP−1遺伝子及び
VP−2遺伝子に対する位置を示す。図中の塩基配列番
号は、ヒトパルボウイルスBlQ株の塩基配列番号に合
わせた。また、各DNA断片における←→は、第3図に
示した塩基配列のDNAの位置を示す。
第3図は、実施例で決定した4種のDNA断片の部分塩
基配列を表す。図中、B19株の遺伝子と異なる塩基に
一印を、また、アミノ酸が異なる部分に一印を付した。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)
を有するヒトパルボウイルス構造蛋白質VP−1遺伝子
。 式(1) 【遺伝子配列があります。】 式(2) 【遺伝子配列があります。】 【遺伝子配列があります。】 式(3) 【遺伝子配列があります。】 【遺伝子配列があります。】 (上記式中、Nは構造未決定の塩基を表す。)(2)下
記式(2)又は式(3)で表される部分塩基配列を有す
るヒトパルボウイルス構造蛋白質VP−2遺伝子。 式(2) 【遺伝子配列があります。】 【遺伝子配列があります。】 式(3) 【遺伝子配列があります。】 【遺伝子配列があります。】 (上記式中、Nは構造未決定の塩基を表す。)(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.)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP20282790A JPH0488985A (en) | 1990-07-31 | 1990-07-31 | Human parvovirus structural protein gene |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP20282790A JPH0488985A (en) | 1990-07-31 | 1990-07-31 | Human parvovirus structural protein gene |
Publications (1)
Publication Number | Publication Date |
---|---|
JPH0488985A true JPH0488985A (en) | 1992-03-23 |
Family
ID=16463849
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP20282790A Pending JPH0488985A (en) | 1990-07-31 | 1990-07-31 | Human parvovirus structural protein gene |
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Country | Link |
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JP (1) | JPH0488985A (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6413716B1 (en) | 1995-06-26 | 2002-07-02 | The Japanese Red Cross Society | Method for detection of human parvovirus and reagent therefor |
US7094541B2 (en) | 2001-08-31 | 2006-08-22 | Gen-Probe Incorporated | Assay for detection of human parvovirus B19 nucleic acid |
-
1990
- 1990-07-31 JP JP20282790A patent/JPH0488985A/en active Pending
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6413716B1 (en) | 1995-06-26 | 2002-07-02 | The Japanese Red Cross Society | Method for detection of human parvovirus and reagent therefor |
US7094541B2 (en) | 2001-08-31 | 2006-08-22 | Gen-Probe Incorporated | Assay for detection of human parvovirus B19 nucleic acid |
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