JPS63287483A - Modified rna replicase protein and method for protection against viral infection - Google Patents
Modified rna replicase protein and method for protection against viral infectionInfo
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- JPS63287483A JPS63287483A JP62123355A JP12335587A JPS63287483A JP S63287483 A JPS63287483 A JP S63287483A JP 62123355 A JP62123355 A JP 62123355A JP 12335587 A JP12335587 A JP 12335587A JP S63287483 A JPS63287483 A JP S63287483A
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- rna
- amino acid
- asp
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- pvar
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/52—Genes encoding for enzymes or proenzymes
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Abstract
Description
【発明の詳細な説明】
く産業上の利用分野〉
本発明は、遺伝子工学の手法によってＲＮＡウィルスの
天然型ＲＮＡ複製酵素蛋白質の特定のアミノ酸残基を人
為的に他のアミノ酸残基で置換した改変ＲＮＡ複製酵素
蛋白質を利用した新規なウィルス感染防禦方法に関する
ものであり、更に詳細には、 ＲＮＡウィルスの天然型
ＲＮＡ複製酵素蛋白質に存在する共通アミノ酸配列−Ｔ
ｙｒ−Ｘ−Ａｓｐ−Ａｓｐ−のＸ残基を人為的に操作し
て他の特定のアミノ酸残基で置換した改変ＲＮＡ複製酵
素蛋白質をコードする遺伝子を含む変異クローンを利用
した新規ウィルス感染防禦方法に関するものである。[Detailed Description of the Invention] Industrial Application Fields The present invention relates to artificially replacing specific amino acid residues of the natural RNA replication enzyme protein of RNA viruses with other amino acid residues using genetic engineering techniques. It relates to a novel method for preventing virus infection using modified RNA replication enzyme proteins, and more specifically, the common amino acid sequence-T present in the natural RNA replication enzyme proteins of RNA viruses.
Novel virus infection prevention method using a mutant clone containing a gene encoding a modified RNA replication enzyme protein in which the X residue of yr-X-Asp-Asp- is artificially replaced with another specific amino acid residue It is related to.
〈従来の技術〉
ＲＮＡを遺伝子とするウィルスは、真核生物に感染する
ウィルスにも多く認められており、従来、ウィルス感染
防禦の観点からＲＮＡウィルスの自己複製機構に関する
研究報告が数多く提示されている。<Prior art> Viruses whose genes are RNA are often found among viruses that infect eukaryotes, and many research reports have been presented on the self-replication mechanism of RNA viruses from the perspective of preventing virus infection. There is.
ＲＮＡウィルスの中でもとりわけ大腸菌ＲＮＡファージ
については、ＨａｒｕｎａとＳｐｉｅｇｅｌｍａｎによ
りＲＮＡファージＱβの感染菌からＱβＲＮ＾を複製す
るＲＮＡ複製酵素が単離されて、Ｑβレプリカーゼと命
名されたことに端を発して（Ｐｒｏｃ、Ｎａｔｌ、Ａｃ
ａｄ、Ｓｕ。Among RNA viruses, Escherichia coli RNA phage in particular has its roots in Haruna and Spiegelman's isolation of an RNA replication enzyme that replicates QβRN^ from bacteria infected with RNA phage Qβ, which was named Qβ replicase (Proc. , Natl, Ac.
ad, Su.
、ＵＳＡ、５４，５７９（１９８５） ） 、 ＲＮＡ
ライ）ｌ／ス（７）自己複製機構の解明は急速に進展
し、細菌とバクテリオファージの分子遺伝子学の段階か
ら、真核細胞とウィルスの細胞生物学の段階に移行する
に至った現在においても、 ＲＮＡウィルスは、遺伝現
象の分子レベルでの解明に多くの実証的成果を提供する
ものとして貴重な研究対象とされている。, USA, 54,579 (1985) ), RNA
(7) Elucidation of the self-replication mechanism has progressed rapidly, and we have now moved from the stage of molecular genetics of bacteria and bacteriophages to the stage of cell biology of eukaryotic cells and viruses. RNA viruses are also considered to be valuable research subjects as they provide many empirical results for the elucidation of genetic phenomena at the molecular level.
ところで、大Ｑ ｌ ＲＮＡファージは、血清学的性質
、紫外線に対する感受性、浮遊密度などの観点から、４
つのグループ（■〜ＩＶ）に大別されており、これら４
つのグループの各々代表的なファージを用いて、各々の
ＲＮＡ複製酵素（Ｎプレカーゼ）すなわち、ｆ２ （グ
ループＸ）Ｎプレカーゼ、ＧＡ （グループ■）Ｎプレ
カーゼ、Ｑβ（グループ■）Ｎプレカーゼ、及びＳＰ
（グループ■）Ｎプレカーゼなどが単離され、更に、そ
れらの構造と機能に関する基礎研究成果として、ＲＮ＆
Ｎプレカーゼ蛋白質が、α、β、ｒ、δの４つのサブユ
ニットから成っていることや、サブユニー／ トα、ｒ
、δが宿主由来の蛋白質であり、サブユニｙ トβだけ
がＲＮＡファージによってコードされる蛋白質であるこ
と、などがすでに解明されている。By the way, large Q l RNA phages are 4.
It is roughly divided into two groups (■~IV), and these four
Using representative phages from each of the two groups, we tested each RNA replication enzyme (N-precase), namely f2 (group X) N-precase, GA (group ■) N-precase, Qβ (group ■) N-precase, and SP.
(Group ■) N-precase, etc. were isolated, and furthermore, as a result of basic research on their structure and function, RN&
The N-precase protein is composed of four subunits, α, β, r, and δ.
It has already been elucidated that δ and δ are host-derived proteins, and that subunit β is the only protein encoded by RNA phage.
これらのＲＮＡＮプレカーゼに代表されるＲＮＡ依存Ｒ
ＮＡ複製酵素は、感染宿主中において、ＲＮＡから相補
的ＲＮＡを経由して直接的にＲＮＡを複製する機能を有
することから、 ＲＮＡウィルスの自己複製機構を制御
するための重要な役割を持つものとして注目される中で
、最近、多くのウィルス由来のＲＮＡ依存ＲＮＡ又はＤ
ＮＡ合成酵素（ポリメラーゼ）蛋白質の一次構造の相同
性が調査され、これらの酵素蛋白質の間に、 −Ｔ７ｒ
−Ｘ−Ａｓｐ−Ａｓｐ−のアミノ酸配列から成る共通の
領域が存在することが明らかとなり、ＲＮＡ複製酵素の
構造と機能の解明が分子レベルで展開されるようになっ
た。RNA-dependent R represented by these RNA precases
Since NA replication enzyme has the function of directly replicating RNA from RNA via complementary RNA in the infected host, it is considered to play an important role in controlling the self-replication mechanism of RNA viruses. Recently, many virus-derived RNA-dependent RNA or D
The homology of the primary structure of NA synthase (polymerase) proteins was investigated, and the -T7r
It has become clear that a common region consisting of the amino acid sequence -X-Asp-Asp- exists, and the elucidation of the structure and function of RNA replication enzymes has begun to be developed at the molecular level.
（Ｎａｔｕｒｅ、 ３０５．８２７−８２９（１９８３
）、Ｎｕｃｌｅｉｃ Ａｃ１ｄｓＲａｅａｒｃｈ、 １
２．７２９８−７２８２ （１９８４）、Ｎａｔｎｒｅ
、 ３１７．３１１８−３８８（ｌＨ５））
このように、ＲＮＡウィルスの自己複製機構の解明は、
分子生物学、疫学などの方向から分子レベルにおいて多
角的に推進されつつあるが、現在のところ、一部のワク
チンなどの開発を除いて、 ＲＮＡウィルスの感染を防
禦する有効な方法はいまだ開発されるに至っておらず、
新しいウィルス感染防禦方法の開発が強く要請されてい
るのが実情である。(Nature, 305.827-829 (1983
), Nucleic Ac1dsRaeach, 1
2.7298-7282 (1984), Natnre
, 317.3118-388 (lH5)) Thus, the elucidation of the self-replication mechanism of RNA viruses is
Although progress is being made from multiple angles at the molecular level from the directions of molecular biology and epidemiology, to date, with the exception of some vaccines, no effective method to prevent infection by RNA viruses has yet been developed. It has not yet reached the point where
The reality is that there is a strong demand for the development of new virus infection prevention methods.
〈発明が解決しようとする問題点〉
このような状況を踏え、本発明者らは、 ＲＮＡ依存Ｒ
ＮＡポリメラーゼの中でも最も基礎研究が先行している
大腸菌ＲＮＡファージを中心としてその構造と機能に関
して分子レベルでの研究を積重ねた結果、前記のＲＮＡ
依存ＲＮＡ又は口ＨＡ合或酵素の−次構造に共通に保有
されているアミノ酸配列が、４つのグループの大腸菌Ｒ
ＮＡファージ、すなわち、ＭＳ２、ＧＡ、 Ｑβ及びｓ
ｐのＲＮＡ複製酵素蛋白質中にも存在することを見出す
と共に、これらの特定のアミノ酸配列が、 ＲＮＡファ
ージの複製に重要な役割を演じていることを見出すに至
った。<Problems to be solved by the invention> Based on this situation, the present inventors have solved the problem of RNA-dependent R
As a result of extensive research at the molecular level on the structure and function of Escherichia coli RNA phage, which has the most advanced basic research among NA polymerases, we have discovered the above-mentioned RNA.
Amino acid sequences commonly held in the secondary structure of dependent RNA or oral HA synthase are found in four groups of E. coli R.
NA phages i.e. MS2, GA, Qβ and s
They found that these specific amino acid sequences also exist in the RNA replication enzyme protein of p, and also found that these specific amino acid sequences play an important role in the replication of RNA phage.
さらに具体的に説明すると、ＱβファージのＲＮＡ複製
酵素β−サブユニット蛋白質の一次構造には、他のＲＮ
Ａファージの場合と同様に、−Ｔｙｒ−Ｇ ｌｙ −Ａ
ｓｐ −Ａ ｓｐ−の共通のアミノ酸配列が存在して
いること、そして、３５７番目のＧｌ！残基を、Ａ Ｉ
ａ、Ｓ ｅｔ、 Ｐ ｒｏ、 Ｍｅｔ又はＶａｌから
選択されるアミノ酸残基で置換したクローンを作製し、
レプリカーゼ活性を調べたところ、これらの変異クロー
ンはレプリカーゼ活性を示さないこと、更に、これらの
変異クローンでクローニングした大腸菌は、Ｑβ及びＳ
Ｐファージの増殖を阻害することなどを見出すと共に、
前記変異クローンがウィルス感染の防禦に有効であると
の知見を得て本発明を完成するに至った。To explain more specifically, the primary structure of the Qβ phage RNA replication enzyme β-subunit protein contains other RNAs.
As in the case of A phage, -Tyr-G ly -A
The common amino acid sequence of sp-A sp- exists, and the 357th Gl! The residue is A I
a, Set, Pro, Met or Val substituted clones are created,
When we investigated the replicase activity, we found that these mutant clones did not show replicase activity. Furthermore, E. coli cloned with these mutant clones showed Qβ and S
In addition to discovering that it inhibits the proliferation of P phage,
The present invention was completed based on the finding that the mutant clones described above are effective in preventing viral infection.
本発明は、ＲＮＡウィルスの改変ＲＮＡ複製酵素蛋白質
を利用したウィルス感染防禦方法を提供せんとすること
を第一目的とするものであり、同時に、当該改変ＲＮＡ
複製酵素蛋白質とその製造方法、当該改変ＲＮＡ複製酵
素蛋白質をコードする遺伝子自体、及び当該遺伝子を含
む変異クローンを提供することをも目的とするものであ
る。The primary objective of the present invention is to provide a method for preventing virus infection using a modified RNA replication enzyme protein of an RNA virus, and at the same time,
Another object of the present invention is to provide a replication enzyme protein, a method for producing the same, the gene itself encoding the modified RNA replication enzyme protein, and a mutant clone containing the gene.
く問題点を解決するための手段〉
このような本発明の目的を達成するための構成は、ＲＮ
Ａウィルスの天然型ＲＮＡ複製酵素蛋白質に存在する共
通のアミノ酸配列−Ｔ ｙｒ −Ｘ −Ａ ｓｐ −Ａ
ｓｐ−のＸ残基を他のアミノ酸残基で置換した改変ＲＮ
Ａ複製酵素蛋白質をコードする遺伝子を含む変異クロー
ンにより宿主細胞をクローニングして改変ＲＮＡ複製酵
素蛋白質を産生させることを基本的要旨とするものであ
り、その具体的技術的手段について、ファージＱβのＲ
）ＩＡ複製酵素を例に挙げて以下に説明する。Means for Solving the Problems> The configuration for achieving the object of the present invention is as follows:
Common amino acid sequence present in the natural RNA replication enzyme protein of A virus -T yr -X -A sp -A
Modified RN in which the X residue of sp- is replaced with another amino acid residue
The basic gist is to produce a modified RNA replicase protein by cloning a host cell using a mutant clone containing the gene encoding the A replication enzyme protein.
) IA replication enzyme will be explained below as an example.
（１）ファージＱβのＲＮＡ複製酵素β−サブユニット
蛋白質遺伝子を含むクローンｐＲＱ ｌの作成遺伝子供
与体は、感染性を有するＺｐＱβ−３２ＤＮＡを用いた
。このＺｐＱβ−３２ＤＮＡは、 Ｔａｎｉｇｕｃｈｉ
及びＷｅｉｓｓｓａｎｎによって作成されたＱβ７アー
ジｆ） ｃＤＮＡであり（Ｎａｔｕｒｅ、 ２７土ｔ２
３（１９７Ｂ） ） 、 Ｗｅｉｓｓｓａｎｎから入手
した。また、ベクターは、プラスミドｐＢＲ３２２由来
のｐｔｌＧＢ （Ｐ、Ｌ、Ｂｉｏｃｈｅｍｉｃａ１社
（現Ｐｈａｒｍａｃｉａ社）製）を用いた。このｐＵｃ
８は、１ａｃＺ領域にマルチクローニングサイトを有し
ており、 ＩＰＴＧとＸ−ｇａ １を含むプレートで外
来遺伝子挿入の有無を判別できる特徴をもっている。
（Ｇｅｎｅ、 １９，２５ｆｌ−２８８（１９８２）
、ＰＲＯＤＵＣＴ ＲＥＦＥＲＥＮＣＥ ＧｔｌＩＤＥ
（１９８４））；−レラ（７） ＺｐＱ ／３−３２
ＤＮＡトｐＵｃ８ カら、第１図に示す工程に従って
ＲＮＡ複製酵素蛋白質遺伝子を含むクローンを作成し、
ｐＲＱｌと命名したなお、互、囲ＪＡ２２１（ｐＲＱＩ
）は、微工研菌寄第３３７２号として工業技術院微生物
工業技術研究所に寄託済である。(1) Creation of clone pRQ1 containing the RNA replication enzyme β-subunit protein gene of phage Qβ Infectious ZpQβ-32 DNA was used as the gene donor. This ZpQβ-32 DNA is
and the Qβ7agef) cDNA created by Weisssann (Nature, 27 Sat.
3 (197B)), Weisssann. Furthermore, the vector used was ptlGB (P, L, manufactured by Biochemica 1 (currently Pharmacia)) derived from plasmid pBR322. This pUc
No. 8 has a multi-cloning site in the 1acZ region, and has the characteristic that the presence or absence of foreign gene insertion can be determined using a plate containing IPTG and X-ga 1.
(Gene, 19, 25fl-288 (1982)
, PRODUCT REFERENCE GtlIDE
(1984)) ;-Lera (7) ZpQ /3-32
From the DNA pUc8, create a clone containing the RNA replication enzyme protein gene according to the steps shown in Figure 1,
In addition, it was named pRQl.
) has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as Microbiological Research Institute No. 3372.
（２）部位指定的変異作製方法によるアミノ酸置換の作
製
ＲＮＡ複製酵素蛋白質の特定のアミノ酸を他のアミノ酸
で置換した変異クローンは、 ＲＮＡ複製酵素β−サブ
ユニット蛋白質遺伝子の塩基配列の一部を部位指定的変
異作製法により改変することによって作製される。改変
する部位として、ＱβファージのＲＮＡ複製酵素β−サ
ブユニット蛋白質の３５６番目から３５９番目にある配
列−Ｔ ｙｒ −Ｇ Ｉｙ −Ａ ｓｐ −Ａ ｓｐ−
のｃｒｙ歿基を選定したのは、■この配列が、はとんど
全てのＲＮＡウィルスのＲ，ＮＡ複製酵素蛋白質に見出
される共通領域であること、しかも、■逆転写酵素蛋白
質にも保存されている（但し、Ｇｌ！ではなくＭｅｔ、
Ｖａｌ又はＬ ｅｕ）こと故、 ＲＮＡ依存型ポリメラ
ーゼがＲＮＡを転写する際に重要な機能を有すると推察
されるに至ったからである。(2) Creation of amino acid substitutions using the site-directed mutagenesis method A mutant clone in which a specific amino acid in the RNA replication enzyme protein is replaced with another amino acid is created by substituting a part of the base sequence of the RNA replication enzyme β-subunit protein gene at a site. It is produced by modification using directed mutagenesis methods. The site to be modified is the sequence from position 356 to position 359 of the RNA replication enzyme β-subunit protein of Qβ phage -T yr -G Iy -A sp -A sp-
We selected this cry base because: 1) this sequence is a common region found in the R and NA replication enzyme proteins of almost all RNA viruses, and 2) it is also conserved in the reverse transcriptase protein. (However, Met, not Gl!
This is because it has been speculated that RNA-dependent polymerase has an important function in transcribing RNA.
改変ＲＮＡ複製酵素β−サブユニー７）蛋白質遺伝子を
含むヘテロデュプレックス（ヘテロ二本鎖ＤＮＡ ）に
関しては、前記ｐＲＱＩ ＤＮＡと１７個の塩基配列か
ら成る特定のオリゴデオキシヌクレオチドを用いて、
ＩｎｏＢｅ ＆ Ｉｎａｕ７ｅの方法（１９８Ｂ）によ
り、３５７番目のＧｌ！残基をＡｌａ、Ｓｅｒ、 Ｐｒ
ｏ、 Ｍａｔ又はＶａｌ残基で置換した変異クローニン
グ製し、各々、 ｐＶＡＲ−Ａ ３５７、 ＰＶＡＲ−
Ｓ ３５７ 、 ｐＶＡＲ−Ｐ３５？、ｐＶＡＲ−Ｍ３
５７及びｐＶＡＲ−Ｖ３５７ ト命名シタ。Regarding the heteroduplex (hetero double-stranded DNA) containing the modified RNA replication enzyme β-subunit 7) protein gene, using the pRQI DNA and a specific oligodeoxynucleotide consisting of a 17 base sequence,
By the method of InoBe & Inau7e (198B), the 357th Gl! Residues are Ala, Ser, Pr
pVAR-A 357, PVAR-
S357, pVAR-P35? , pVAR-M3
57 and pVAR-V357.
（３）改変ＲＮＡ複製酵素β−サブユニット蛋白質遺伝
子を含む変異クローンによる大腸菌の形質転換
前記（２）で作製した各種変異クローンを用いて、常法
により（Ｎａｎｉａｔｉｓ Ｔ、、 Ｆｒ１ｔｓｈ Ｅ
、 Ｅ。(3) Transformation of Escherichia coli with mutant clones containing the modified RNA replication enzyme β-subunit protein gene Using the various mutant clones prepared in (2) above, transformation was performed by conventional methods (Naniatis T, Fr1tsh E).
, E.
ａｎｄ Ｓａｍｂｒｏｏｋ Ｊ、 ！ｉ Ｍｏ
１ｅｃｕｌａｒ Ｃｌｏｎｉｎｇ ” ２４９−２
５５． Ｃｏ１ｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｌａ
ｂａｒａｔｏｒｙ（１９Ｂ２））大腸菌をクローニング
して改変ＲＮＡ複製酵素蛋白質を産生させた。and Sambrook J,! i Mo
1ecular Cloning” 249-2
55. Co1d Spring Harbor La
(19B2)) Escherichia coli was cloned to produce a modified RNA replication enzyme protein.
宿主菌としては、Ｅ、 ｃｏｌｉ ＪＡ ２２１／Ｆ
’ Ｌａｃ’（ｈｓｄＲ，Ｉｅｕ ＢＪ Ｉａｃ Ｙ、
ｔｈｉ、 ｒｅｃＡ、Δｔｒｐ Ｅ５／Ｆ ’ Ｉａ
ｃ 、 ｐｒｏ ＡＨ） （Ｒｏｂｅｒｔ Ｗｏｏｄ
Ｊｏｈｎｓｏｎ Ｍｅ１ｃａｌ
Ｓｃｈｏｏｌ ａｔ Ｒｕｔｇｅｒｓ、 Ｎｅｗ Ｊｅ
ｒｓｅｙ 、弁上より入手した） 、 Ｅ、 ｃｏｌ
ｉ ５９４／Ｆ’ Ｌａｃ”（ｓｕ−、ｇａｌτ、ｉ
中
ｓｔｒ、 ｒｅｃＡ４４／ Ｆ ｌａｃ 、 Ｉａ
ｃ 、 ｐｒａＡＢ ）を用いた。なお、５９４株（ｓ
ｕ 、 ｇａｌ 、 ｓｔｒ 、 ｒｅｃＡ４４）は、
東京大学医科学研究所、池田より入手し、ｔａ、５゜４
／ Ｆ・Ｌａｃり劃よ、１，２□１／ Ｆ ’ Ｌａｃ
’株から接合によりＦ°因子５９４株に導入して作製し
た。Host bacteria include E. coli JA 221/F
'Lac'(hsdR, Ieu BJ Iac Y,
thi, recA, Δtrp E5/F'Ia
c, pro AH) (Robert Wood
Johnson Melcal School at Rutgers, New Je.
rsey, obtained from Bengami), E, col
i 594/F'Lac" (su-, galτ, i
middle str, recA44/Flac, Ia
c, praAB) was used. In addition, 594 shares (s
u, gal, str, recA44) are
Obtained from Ikeda, Institute of Medical Science, University of Tokyo, ta, 5°4
/ F. Lac, 1,2□1/ F' Lac
It was created by introducing the F° factor from the ' strain into the F° factor 594 strain by conjugation.
以上、本発明の構成について、ファージＱβのＲＮＡ複
製酵素を例として説明したが、ファージＱβのＲＮＡ複
製酵素β−サブユニット蛋白質に存在するーＴツｒ−Ｇ
１ｙ−Ａｓｐ−Ａｓｐ−のアミノ酸配列は、ＲＮＡファ
ージばかりでなく他のウィルスのＲＮＡ依存ＲＮ＾ポリ
メラーゼにも見出される普遍的な領域であることから、
各種ＲＮＡウィルスについて、ファージＱβの場合と同
様にして変異クローンを作製することが可能である。The structure of the present invention has been explained above using the RNA replication enzyme of phage Qβ as an example.
Since the amino acid sequence 1y-Asp-Asp- is a universal region found not only in RNA phages but also in RNA-dependent RN^ polymerases of other viruses,
Mutant clones can be produced for various RNA viruses in the same manner as for phage Qβ.
なお、アミノ酸残基として示したＴ７ｒは、チロシン、
Ｇｌ！はグリシン、Ａｓｐはアスパラギン酸。In addition, T7r shown as an amino acid residue is tyrosine,
Gl! is glycine and Asp is aspartic acid.
Ａｒはアラニン、Ｓｅｒはセリン、Ｐｒｏはプロリン、
Ｍｅｔはメチオニン、Ｖａｌはバリン、Ｌｅｕはロイシ
ンを表わす。Ar is alanine, Ser is serine, Pro is proline,
Met represents methionine, Val represents valine, and Leu represents leucine.
〈発明の効果〉
ＱβファージのＲＮＡ複製酵素β−サブユニット蛋白質
の３５７番目のアミノ酸置換体クローンは、宿主細胞に
おいて不活性なＲＮＡ複製酵素を産生して、Ｑβ−及び
５Ｐ−ＲＮＡファージの増殖を阻害する機能を有してお
り、９のアミノ酸置換クローンを利用することにより、
宿主細胞へのウィルス感染を高率で抑制することができ
る。<Effects of the Invention> The 357th amino acid substitution clone of the RNA replication enzyme β-subunit protein of Qβ phage produces an inactive RNA replication enzyme in host cells and promotes the proliferation of Qβ- and 5P-RNA phages. By using the nine amino acid substitution clones,
Viral infection of host cells can be suppressed at a high rate.
本発明のウィルス感染防禦方法はＱβファージに限らず
、他の同様の構造、機能をもったＲＮＡウィルスの場合
にも適用することが可能であり、特に植物ＲＮＡウィル
ス、動物ＲＮＡウィルスなどの感染防禦方法、すなわち
、植物、動物を対象とした新しい免疫法としても適宜に
応用できることから産業上の利用価値の高いものである
。The method for preventing virus infection of the present invention is applicable not only to Qβ phage but also to other RNA viruses with similar structures and functions, and is particularly applicable to preventing infection with plant RNA viruses, animal RNA viruses, etc. This method has high industrial utility value because it can be appropriately applied as a new immunization method for plants and animals.
次いで、本発明を実施例により更に詳細に説明するが、
本発明の範囲は実施例に限定されるものではない。Next, the present invention will be explained in more detail with reference to Examples.
The scope of the invention is not limited to the examples.
〈実施例〉
（１）ファージＱβのＲＮＡ複製酵素β−サブユニット
蛋白質遺伝子を含むクローンｐＱＲ１の作製り外来遺伝
子の調整
τａｎｉｇｕｃｈｉ Ｗｅｉｓｓｍａｎｎによって作製
されたＺｐＱβ−３２ＤＮＡ ４．７蒔ｇを制限酵素Ｂ
ｇｌＩで切断した後、ＢｇｌＩ切断部位を平滑末端化す
るために４種類のデオキシトリヌクレオチド（４ｄＮＴ
Ｐ）と７４ＤＮＡポリメラーゼで３７℃、２０分間反応
処理した０次いで、この反応液を７０℃、１０分間加熱
処理することによって酵素を失活させ、更に。<Example> (1) Preparation of clone pQR1 containing RNA replication enzyme β-subunit protein gene of phage Qβ Adjustment of foreign gene 4.7 g of ZpQβ-32 DNA prepared by τaniguchi Weissmann was injected with restriction enzyme B.
After cutting with glI, four types of deoxytrinucleotides (4dNT
P) and 74 DNA polymerase were reacted at 37°C for 20 minutes.Then, this reaction solution was heated at 70°C for 10 minutes to inactivate the enzyme.
Ｂｇｌ■で切断して碍られるＲＮＡ複製酵素β−サブユ
ニット蛋白質遺伝子（Ｒ）を含む２．１７８ｂｐのＤＮ
Ａ断片を低融点寒天ゲル電気泳動で分離した。A 2.178 bp DNA containing the RNA replication enzyme β-subunit protein gene (R) that is cleaved and destroyed by Bgl■
The A fragment was separated by low melting point agar gel electrophoresis.
２）クローニングベクターの調整
プラスミドｐＢＲ３２２由来のｐＵｃ８ ＤＮＡ ５０
０ｎｇを制限酵素ＢａｍＨＩと旧ｎｃＩＩ（各々ｌユニ
ット）で、３７℃、６０分間反応処理して、２．Ｈ２ｂ
ｐのｐＵｃ８ ＤＮＡ断片を調整した。2) Cloning vector adjustment pUc8 DNA derived from plasmid pBR322 50
0 ng was reacted with restriction enzymes BamHI and old ncII (1 unit each) at 37°C for 60 minutes.2. H2b
A pUc8 DNA fragment of p.p.
３）組換え体の作製と大腸菌の形質転換前記１）と２）
で調整した外来遺伝子５０ｎｇ／ １０ＩＬｌとベクタ
ーＤＮＡ Ｌｏｎｇ／ ＩＬｌを、 ５０ｍＭ Ｔｒｉ
ｓ−ＨＣｌ （ｐＨ７，４） 、 ＩＱｍＷ ＭｇＣｌ
２．１０ｓＮ ＤＴＴ （ジチオスレイトール） 、
１ｍＭスペルミジン、ｌ會Ｍ ＡＴＰ、Ｔ４ＤＮＡ
リカーゼ１ ｕｎｉｔ７１０ ｐ、 １中で１２．５℃
、−液反応させて環化することによりクローン（組換え
体）を作製した０次いで、このＤＮＡを用いて常法によ
りＥ、 ｃｏｌｉ Ｋ１２株由来のＥ−ｃｏｌｉＪＭ
１０５ （Ｎｅｕｃｌｅｉｃ Ａｕ１ｄａ Ｒｅ５ｅ
ａｒｃｈ、 ９． ３０９−３２１（１９８１） ）
ヲ形質転換し、０Ｊｌｚ＜９ ） リプトン、０．５％
酵母エキス、０．５ｚ食塩、１．５ｚ寒天、５０Ｍｇ／
■１アンピシリン（Ａ翳ｐｉｃｉｌｌｉｎ）から成る平
板寒天培地を用いて、ＡＬＰ’ 、 Ｌａｃ？を示す大
腸菌を選択した。この中からＲＮＡ複製酵素蛋白質遺伝
子を含むプラスミドを常法により抽出し、得られた４、
８４０ｂｐのクローンをｐＲｏ １ と命名した。3) Preparation of recombinant and transformation of E. coli 1) and 2) above
50 ng/10 ILl of foreign gene and vector DNA Long/ILl were mixed with 50mM Tri
s-HCl (pH 7,4), IQmW MgCl
2.10sN DTT (dithiothreitol),
1mM spermidine, 1M ATP, T4DNA
Licase 1 unit 710 p, 12.5°C in 1
A clone (recombinant) was created by reacting with E. coli K12 strain using this DNA.
105 (Neucleic Au1da Re5e
arch, 9. 309-321 (1981))
Transformed with 0Jlz<9) Lipton, 0.5%
Yeast extract, 0.5z salt, 1.5z agar, 50Mg/
■1 Using a plate agar medium consisting of ampicillin, ALP', Lac? We selected Escherichia coli that shows this. From this, a plasmid containing the RNA replication enzyme protein gene was extracted by a conventional method, and the obtained 4,
The 840 bp clone was named pRo 1 .
（２）改変ＲＮＡ複製酵素蛋白質遺伝子を含む変異クロ
ーンの作製
前記（１）により作製したｐＲＱＩ ＤＮＡを用いて、
Ｉｎｏｕｙｅ＆ Ｉｎｏｕｙｅ７）部位指定的変異作
製法に従って、目的とするＲＮＡ複製酵素β−サブユニ
ット蛋白質の３５７番目のｃｔｙ残基を、Ａｌａ、Ｓｅ
ｒ、 Ｐｒｏ、 Ｍｅｔ又はＶａｌ残基で置換した改変
ＲＮＡ複製酵素蛋白質遺伝子を含む変異クローン（組換
え体）を作製した。(2) Preparation of mutant clone containing modified RNA replication enzyme protein gene Using the pRQI DNA prepared in (1) above,
Inouye & Inouye7) According to the site-directed mutagenesis method, the 357th cty residue of the target RNA replication enzyme β-subunit protein was replaced with Ala, Se.
Mutant clones (recombinants) containing modified RNA replication enzyme protein genes substituted with r, Pro, Met, or Val residues were produced.
１） ｐＲＱＩ ＤＮＡ由来のＤＮＡ断片ＩｐＲＱＩ
ＤＮＡ ５０ｎｇ／ ＩＬ １ を、 ２０ｓＮ
ＫＣｌ、 ｌＯｍＮＴｒｉｓ−ＨＣＩ（ｐＨ８，
０） 、 ＩＱｍＷ ＭｇＣｌ２．１ｍＭジチオスレイ
トールを含緩衝液中で制限酵素Ｓ＠ａＩＯ。1) DNA fragment IpRQI derived from pRQI DNA
DNA 50ng/IL1, 20sN
KCl, lOmNTris-HCI (pH 8,
0), IQmW MgCl2. Restriction enzyme S@aIO in buffer containing 1mM dithiothreitol.
５ｕｎｉｔｓ／ １ｂ Ｉ と３７℃、２時間反応させ
て得られる４ 、 ５ａｏｂｐの口ＨＡ断片Ｉを稀釈し
てＤＮＡ濃度１５０ｎｇ／ＩＬ＋とした。The oral HA fragment I of 4 and 5 aobp obtained by reacting with 5 units/1b I at 37°C for 2 hours was diluted to a DNA concentration of 150 ng/IL+.
２） ｐＲＱＩ ＤＮＡ由来のＤＮＡ断片■ｐＲＱＩ
ＤＮＡ ５０ｎｇ／ ＩＬｌを、５（ｌｓＭＫＧｌ 、
ｌＯｍＮＴｒｉｓ−ＨＣＩ（ｐＨ７，５）、１ｏｓＮ
ＭｇＣｌ２ 、１ｍＭジチオスレイトールを含む緩
衝液中で制限酵素ＸｈｏｌＯ，５ｕｎｉｔｓ ／ ＩＬ
ｌ 、 ＰｓｔＩ Ｏ，５ｕｎｉｔｓ ／ ４１と３７
℃、２時間反応させた後、改変しようとする領域を含ま
ない大きいＤＮＡ断片を５駕アクリルアミドゲル電気泳
動で分離して、４，０４６ｂｐのＤＮＡ断片ＩＩを得た
。2) DNA fragment derived from pRQI DNA ■pRQI
DNA 50ng/ILl, 5(lsMKGl,
1OmNTris-HCI (pH 7,5), 1osN
Restriction enzyme XholO, 5 units/IL in buffer containing MgCl2, 1 mM dithiothreitol
l, PstI O, 5 units/41 and 37
After reacting for 2 hours at 0.degree. C., a large DNA fragment not containing the region to be modified was separated by 5-pan acrylamide gel electrophoresis to obtain a 4,046 bp DNA fragment II.
３）オリゴデオキシヌクレオチドの合成改変しようとす
るアミノ酸残基を中心にした１７個の゛塩基配列から成
るオリゴデオキシヌクレオチドをＤＮＡ合成９３８０Ｂ
型（Ａｐｐｌｉｅｄ Ｂｉ。3) Synthesis of oligodeoxynucleotides Oligodeoxynucleotides consisting of a 17 base sequence centered on the amino acid residue to be modified are synthesized using DNA synthesis 9380B.
Type (Applied Bi.
Ｓ７Ｓ７５ｔｅ社製）で合成し、その５′一端をリン酸
化して成る下記のオリゴデオキシヌクレオチドを作製し
た。(manufactured by S7S75te) and phosphorylated its 5' end to produce the following oligodeoxynucleotide.
■ ＣＴ−ＧＴＴ−ＴＡＣ−ＧｉＧＡ−ＣＡＣ−ＯＡ
？■ ＣＴ−ＧＴＴ−ＴＡＣ−ＧＣＧ−ＧＡＣ−ＧＪ
ｋＴ■ ＣＴ−ＧＴＴ−ＴＡ（ニーＴＣＴ−ＧＡＣ−
ＯＡ？■ ＣＴ−ＧＴＴ−丁ＡＣ−ＣＣＴ−ＧＡＧ−
ＧＡＴ■ ＣＴ−ＧＴＴ−ＴＡＣ−ＡＴＧ−ＧＡＣ−
ＧＡＴ（Φ ＣＴ−ＧＴＴ−ＴＡＣ−０７丁−〇ＡＣ
−ＧＡ↑■は天然型、■４＊ ｐＶＡＲ−Ａ３５７、■
−はｐＶＡＲ−５３３７、＠はｐＶＡＲ−Ｐ２Ｓ５ 、
■（＊ ｐＶＡＲ−Ｍ３５７ 、■ハｐＮＡＲ−Ｖ３５
７に各々対応する。■ CT-GTT-TAC-GiGA-CAC-OA
? ■ CT-GTT-TAC-GCG-GAC-GJ
kT■ CT-GTT-TA (knee TCT-GAC-
OA? ■ CT-GTT-Ding AC-CCT-GAG-
GAT■ CT-GTT-TAC-ATG-GAC-
GAT(Φ CT-GTT-TAC-07-AC
-GA↑■ is natural type, ■4* pVAR-A357, ■
- is pVAR-5337, @ is pVAR-P2S5,
■(* pVAR-M357, ■ HapNAR-V35
7 respectively.
０ヘテロデユプレツクス（ヘテロ二本ｊｌｌＤＮＡ）の
作製
前記り〜３）で作製した各ＤＮＡ断片を混合し。Preparation of 0 heteroduplex (heteroduplex jll DNA) The DNA fragments prepared in steps 3) and 3) above were mixed.
３分間沸湯水に浸漬した後、３０℃に３０分間、４℃に
３０分間、氷水中に１０分間保持して段階的に冷却処理
することにより、沸湯水に浸漬して一本鎖になったＤＮ
Ａ断片に再生化を施した。続いて、口ＨＡポリメラーゼ
（Ｋｌｅｎｏｗ Ｅｎｚｙｍｅ）で修飾し、 Ｔ４ＤＮ
Ａ リガーゼで処理してＤＮＡの環化を行った。After immersing it in boiling water for 3 minutes, it was cooled stepwise by holding it at 30℃ for 30 minutes, at 4℃ for 30 minutes, and in ice water for 10 minutes, so that it became a single strand by immersing it in boiling water. D.N.
Fragment A was regenerated. Subsequently, T4DN was modified with HA polymerase (Klenow Enzyme).
DNA was circularized by treatment with A ligase.
５）変異クローンによる大腸菌の形質転換このようにし
て作製した変異クローン（組換え体）を□い工、ユ旦Ｊ
Ａ ２□１ ／Ｆ・Ｌ６−８形質転換・し、変異クロー
ンの作製に際して用いたオリゴデオキシヌクレオチドを
プローブとして変異クローンの選択を行った。ここで得
られた変１４りＴ：１−７ｔｔ各ｈ、ｐＶＡＲ−Ａ３５
７ 、 ｐＶＡＲ−９３５７、ｐＶＡＲ−Ｐ２Ｓ５ 、
ｐＶＡＲ−Ｎ３５？ 、 ｐＶＡＲ−Ｖ３５？ ト命
名した。これらは、ＱββファージＲＮＡ複製酵素−サ
ブユニット蛋白質遺伝子の３５７番目のＧｌ！残基を、
各々、Ａ１５１．５ｅｒ、 Ｐｒｏ、Ｍｒｔ、Ｖａｌ残
基で置換した改変蛋白質遺伝子を含んでいる。5) Transformation of E. coli with mutant clones The mutant clones (recombinants) thus created were transformed into
A2□1/F.L6-8 was transformed, and mutant clones were selected using oligodeoxynucleotides used in the production of mutant clones as probes. Variant 14 T obtained here: 1-7tt each h, pVAR-A35
7, pVAR-9357, pVAR-P2S5,
pVAR-N35? , pVAR-V35? It was named. These are the 357th Gl! of the Qββ phage RNA replication enzyme subunit protein gene! the residue,
Each contains a modified protein gene substituted with A151.5er, Pro, Mrt, and Val residues.
（３）ファージＱβのＲＮＡ複製酵素の構造と機能の検
討
ｌ）遺伝子機能の発現
宿主細胞内での遺伝子機能の発現を検討すべく、大腸菌
ＲＮＡファージの各種突然変異株を大腸菌に感染させ、
そこで産生される子ファージの数を調べた結果を末尾第
１表に示す。(3) Examination of the structure and function of the RNA replication enzyme of phage Qβ l) Expression of gene function In order to examine the expression of gene function within host cells, E. coli was infected with various mutant strains of E. coli RNA phage.
The results of investigating the number of child phages produced are shown in Table 1 at the end.
プラスミドｐＲ９１を保有する大腸菌細胞では、Ｑβフ
ァージの成熟蛋白質遺伝子（Ｍ）、外被蛋白質遺伝子（
Ｃ）の変異株は増殖できず、ＲＮＡ複製酵素蛋白質遺伝
子（Ｒ）の変異株だけが増殖できた。従って、このクロ
ーンでは、活性あるＲＮＡ複製酵素蛋白質が産生されて
いることが証明された。In E. coli cells carrying plasmid pR91, the mature protein gene (M) and coat protein gene (
The mutant strain C) was unable to proliferate, and only the mutant strain of the RNA replication enzyme protein gene (R) was able to proliferate. Therefore, it was demonstrated that active RNA replication enzyme protein was produced in this clone.
２）変異クローン（改変プラスミド）を保持している大
腸菌を指示間としてＲＮＡファージを感染させた場合の
感染効率
前記（２）で作製した各種の変異クローンを保持してい
る大ｍ＠ＣＥ、 凹ＪＡ ２２１ ／Ｆ ’ Ｌ ａ
ｃ’）を、酵母エキス５ｇ／Ｉ、バクトドリブトン８ｇ
／ｌＮａＣ１５ｇ／Ｉから成るＹＴ培地中で３７℃に保
温しながら振盪培養し、対数増殖期中期の菌を一定量の
大引１ｉＲＮＡフアージｆ２、ＧＡ、 Ｑβ又はｓｐと
混合し、更に、２．５１の０．８＄の寒天を含むＹ丁培
地（軟寒天培地）を加えて、ベトリ＠（直径９０ｍｍ×
深さ１５Ｉ）内に予め作製しておいた１、５％の寒天を
含むＹＴ培地から成る寒天平板培地上に重層した。寒天
が固まったのを確めた上で３７℃、−夜培養し、そこに
現われた溶菌環の数を数えた、感染効率は、対象とした
プラスミドｐｕｃｓを保有している大ａ菌に感染したフ
ァージ数を１とした場合の各大腸菌に感染したファージ
の数として表わした。末尾表２に示す結果から明らかな
ように、Ｑβ及びＳＰのプラーク形成能が、３５７番目
のアミノ酸置換体クローンで、著しく低下していること
から、Ｑβ及びＳＰファージの増殖が抑制されること、
抑制の程度は置換されるアミノ酸の種類にも依存するこ
とが判明した、なお、ｆ２及びＧＡのプラーク形成能は
、いずれのクローンでも等しく、増殖抑制の程度も余り
高くないが、これは、 ＲＮＡファージのＲＮＡ複製酵
素の鋳型特異性によるものと推察される。また、末尾表
２に示したｐＶＡＲ−Ａ３９Ｇは、３３０番目のＧｌｙ
残基をＡｌａ残基で置換した変異クローンを意味する。2) Infection efficiency when RNA phage is infected with Escherichia coli carrying a mutant clone (modified plasmid) as an indicator. JA 221 /F' La
c'), yeast extract 5g/I, Bactodributon 8g
The bacteria in the mid-logarithmic growth phase were cultured in YT medium consisting of 15 g/l NaC with shaking while keeping the temperature at 37°C. Add Y-dong medium (soft agar medium) containing 0.8$ of agar to Vetri@(diameter 90mm x
The cells were layered on an agar plate medium made of YT medium containing 1.5% agar and prepared in advance at a depth of 15 I). After confirming that the agar had solidified, it was cultured at 37°C overnight, and the number of lytic rings that appeared there was counted. The infection efficiency was determined by infecting E. a. It is expressed as the number of phages that infected each E. coli, with the number of phages infected as 1. As is clear from the results shown in Table 2 at the end, the plaque forming ability of Qβ and SP is significantly reduced in the 357th amino acid substitution clone, which indicates that the proliferation of Qβ and SP phages is suppressed.
It was found that the degree of inhibition also depended on the type of amino acid substituted.The plaque forming ability of f2 and GA was the same in all clones, and the degree of growth inhibition was not very high. This is presumably due to the template specificity of the phage's RNA replication enzyme. In addition, pVAR-A39G shown in Table 2 at the end has the 330th Gly
It refers to a mutant clone in which a residue is replaced with an Ala residue.
３）変異クローン（改変プラスミド）を保持している大
ａｍ内でのＱβファージの一段階増殖実験
各種変異クローンを保持している大腸菌（Ｌｃｏｌｉ
５９４／Ｆ ’ Ｌ ａｃ’ｇ ）を１０ｍにの塩化カ
ルシウムを含む酵母エキス５ｇ／ ｌ、バクトドリブト
ン８ｇ／ ｌ 、 Ｈａ（：ｌ ５ｇ ／ ｌから成る
Ｙ丁培地中で、３７℃に保温しながら振盪培養し、菌数
が１０ 個／ｍ見にまで増殖した時点で、Ｑβファー
ジを１０’ＰＦＩＪ／ ｍ文（感染多重度０．１）で感
染させた、菌に感染しないファージを除くために、感染
後１０分目に遠心分離してファージ感染大腸菌を沈澱さ
せた０次いで、この大腸菌を当量の新しいＹＴ培地に菌
懸濁し、更に、１ｍＭのイソプロピル−β−Ｄ−チオグ
ラクトシド（ＩＰＴＧ）を含む培地で稀釈した後、３０
℃で振盪培養を続けた。感染後、第２図に示した時点で
一定量を採取し、これを十分量の指示間Ｅ、 ｃｏｌ
ｉ Ａ／入及び２．５■見の０．６ｚの寒天を含むＹＴ
培地と混合し、ペトリ皿（直径９０１層×深さ１５廁謬
）内に予め作製しておいた１、５ｚの寒天を含むＹ↑培
地から成る寒天平板培地上に重層した。寒天が固まった
ことを確認した後、このペトリ皿を３７℃で一夜保温し
た。3) One-step multiplication experiment of Qβ phage in large am carrying mutant clones (modified plasmids) Escherichia coli (Lcoli) carrying various mutant clones
594/F'Lac'g) was shaken at 37°C in 10ml of Y-Ding medium consisting of 5g/l of yeast extract containing calcium chloride, 8g/l of Bactodributone, and 5g/l of Ha(:l). After culturing, when the number of bacteria grew to 10 cells/m, the cells were infected with Qβ phage at 10' PFIJ/m (multiplicity of infection 0.1) to remove phages that did not infect the bacteria. 10 minutes after infection, the phage-infected E. coli was precipitated by centrifugation.Next, this E. coli was suspended in an equivalent amount of fresh YT medium, and further added to a medium containing 1 mM isopropyl-β-D-thioglycoside (IPTG). After diluting with 30
The shaking culture was continued at °C. After infection, a certain amount is collected at the time shown in Figure 2, and a sufficient amount of E, col
i A/YT containing 0.6z agar of 2.5cm and 2.5cm
The mixture was mixed with a medium and layered on an agar plate medium consisting of Y↑ medium containing 1 and 5z agar prepared in advance in a Petri dish (901 layers in diameter x 15 layers in depth). After confirming that the agar had solidified, the Petri dish was kept at 37°C overnight.
この平板培地上に現われた溶菌班の数から大腸菌内で増
殖したファージの数を算出した・第２図にその結果を示
す。The number of phages proliferating within E. coli was calculated from the number of lytic plaques appearing on this plate medium. The results are shown in Figure 2.
なお、採取した試料を直ちに指示菌と共に接種した場合
は、０、Δ、口で示し、また、採取した試料をクロロホ
ルム処理して菌体を破壊した後、指示菌と共に接種した
場合は、拳、＾、騰で示した。In addition, if the collected sample was immediately inoculated with the indicator bacteria, it is indicated by 0, Δ, or mouth, and if the collected sample was treated with chloroform to destroy the bacterial cells and then inoculated with the indicator bacteria, it is indicated by fist, Δ, or mouth. ^, indicated by rising.
更に、０は、ｐＲＱｌ 、 Δｔ＊、ｐＶＡＲ−Ｐ３５
？、ＯはｐＶＡＲ−Ａ３５７をそれぞれ保持している大
腸菌中でのＱβファージの増殖を意味する。Furthermore, 0 is pRQl, Δt*, pVAR-P35
? , O means the growth of Qβ phage in E. coli harboring pVAR-A357, respectively.
第２図のＱβ野生株の一段増殖実験結果の比較から、不
活性なＲＮＡ ′！ｌｉ製酵素蛋白質を産生している細
胞でのファージ感染中心数（白抜き）及び産生される全
子孫ファージ（黒塗り）は、いずれも活性なＲＮＡ複製
酵素産生細胞に比べて著しく減少していることが判明し
た。From the comparison of the results of the one-step growth experiment of the Qβ wild strain shown in Figure 2, it was found that inactive RNA ′! The number of phage infection centers (white) and the total number of progeny phages produced (black) in cells producing li-produced enzyme proteins are both significantly reduced compared to cells producing active RNA replication enzymes. It has been found.
示す。show.
ファージＱβのＲＮＡ複製酵素の構造と機能に関する以
上の知見から、３５７番目のアミノ酸置換による不活性
なＱβＲＮＡ複製酵素蛋白質により、ＱβＲＮＡ及びＳ
Ｐ ＤＮＡの合成が著しく抑制されることが確認された
が、ＲＮＡウィルスのＲＮＡ依存ＲＮＡ複製酵素蛋白質
に存在する共通のアミノ酸配列−Ｔｙｒ −Ｘ −Ａ
ｓｐ −Ａ ｓｐ−のＸ残基を他のアミノ酸残基で置換
した改変ＲＮＡ依存ＲＮＡ複製酵素蛋白質をコードする
遺伝子を含む変異クローンを利用した本発明のＲＮＡウ
ィルスの感染防禦方法は、ＲＮＡファージのみならず、
同様の植物ウィルス、動物ウィルスの新しい感染防禦方
法、すなわち従来の免疫法とは異る新しい免疫手法とし
て多角的に利用し得る普遍的な技術として位置づけられ
るものであり、その産業上の利用価値は極めて高い。Based on the above knowledge regarding the structure and function of the RNA replication enzyme of phage Qβ, it is clear that the inactive Qβ RNA replication enzyme protein resulting from the 357th amino acid substitution allows QβRNA and S
Although it was confirmed that P DNA synthesis was significantly suppressed, the common amino acid sequence -Tyr-X-A present in the RNA-dependent RNA replication enzyme protein of RNA viruses
The method for preventing infection of RNA viruses of the present invention using a mutant clone containing a gene encoding a modified RNA-dependent RNA replication enzyme protein in which the Not,
It is positioned as a universal technology that can be used from multiple angles as a new method for preventing infection with similar plant viruses and animal viruses, that is, a new immunization method different from conventional immunization methods, and its industrial value is Extremely high.
第１ｒＩ４は、ＱβファージのＲｊｌＡ複製酵素蛋白質
遺伝子のクローンの作製工程を示し、第２図は、Ｑβフ
ァージ野生株による一段増殖実験の結果を出 願 人
株式会社 ヤクルト本社井 口 義
夫
第２図
Ｔｉｍｅ ａｆｔｅｒ １ｎｆｅｃｔｉｏｎ （ｍｉｎ
）手続補正書（方式）
％式％
２、発明の名称
４、代理人Figure 1rI4 shows the cloning process of the RjlA replication enzyme protein gene of Qβ phage, and Figure 2 shows the results of a one-step proliferation experiment using a wild strain of Qβ phage.
Yakult Honsha Co., Ltd. Yoshi Iguchi
Husband 2nd figure Time after 1nfection (min
) Procedural amendment (method) % formula % 2, title of invention 4, agent
Claims (6)
る共通アミノ酸配列−Ｔｙｒ−Ｘ−Ａｓｐ−Ａｓｐ−の
Ｘ残基を他のアミノ酸残基で置換した改変ＲＮＡ複製酵
素蛋白質。(1) A modified RNA replication enzyme protein in which the X residue of the common amino acid sequence -Tyr-X-Asp-Asp- present in the RNA replication enzyme proteins of RNA viruses is replaced with another amino acid residue.
製酵素β−サブユニット蛋白質の３５６番目から３５９
番目のアミノ酸配列−Ｔｙｒ−Ｇｌｙ−Ａｓｐ−Ａｓｐ
−のＧｌｙ残基をＡｌａ、Ｓｅｒ、Ｐｒｏ、Ｍｅｔ又は
Ｖａｌから選択されるアミノ酸残基で置換した特許請求
の範囲第１項記載の改変ＲＮＡ複製酵素蛋白質。(2) The RNA virus is Qβ phage, which contains RNA replication enzyme β-subunit protein from positions 356 to 359.
amino acid sequence-Tyr-Gly-Asp-Asp
2. The modified RNA replication enzyme protein according to claim 1, wherein the Gly residue of - is replaced with an amino acid residue selected from Ala, Ser, Pro, Met, or Val.
る共通アミノ酸配列−Ｔｙｒ−Ｘ−Ａｓｐ−Ａｓｐ−の
Ｘ残基を他のアミノ酸残基で置換した改変ＲＮＡ複製酵
素蛋白質をコードする遺伝子を含む変異クローンにより
クローニングした宿主菌を培養することを特徴とする改
変ＲＮＡ複製酵素蛋白質の製造方法。(3) A mutation involving a gene encoding a modified RNA replicase protein in which the X residue of the common amino acid sequence -Tyr-X-Asp-Asp- present in the RNA replication enzyme protein of RNA viruses is replaced with another amino acid residue. A method for producing a modified RNA replication enzyme protein, which comprises culturing a cloned host bacterium.
蛋白質の３５６番目から３５９番目のアミノ酸配列−Ｔ
ｙｒ−Ｇｌｙ−Ａｓｐ−Ａｓｐ−のＧｌｙ残基をＡｌａ
、Ｓｅｒ、Ｐｒｏ、Ｍｅｔ又はＶａｌから選択されるア
ミノ酸残基で置換した改変ＲＮＡ複製酵素蛋白質をコー
ドする遺伝子又は当該遺伝子を含む変異クローン。(4) Amino acid sequence from position 356 to position 359 of RNA replication enzyme β-subunit protein of Qβ phage-T
The Gly residue of yr-Gly-Asp-Asp- is replaced with Ala
A gene encoding a modified RNA replication enzyme protein substituted with an amino acid residue selected from , Ser, Pro, Met, or Val, or a mutant clone containing the gene.
選択される特許請求の範囲第４項記載の変異クローン。(5) The mutant clone according to claim 4, which is selected from pVAR-A357 or pVAR-S357.
る共通アミノ酸配列−Ｔｙｒ−Ｘ−Ａｓｐ−Ａｓｐ−の
Ｘ残基を他アミノ酸残基で置換した改変ＲＮＡ複製酵素
蛋白質をコードする遺伝子を含む変異クローンにより宿
主細胞をクローニングして改変ＲＮＡ複製酵素蛋白質を
産生させることを特徴とする宿主細胞のウィルス感染防
禦方法。(6) A mutant clone containing a gene encoding a modified RNA replicase protein in which the X residue of the common amino acid sequence -Tyr-X-Asp-Asp- present in the RMA replicase protein of RNA viruses is replaced with another amino acid residue. 1. A method for preventing virus infection of a host cell, which comprises cloning the host cell to produce a modified RNA replication enzyme protein.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62123355A JPH084498B2 (en) | 1987-05-20 | 1987-05-20 | Modified RNA replication enzyme protein and method for preventing virus infection |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62123355A JPH084498B2 (en) | 1987-05-20 | 1987-05-20 | Modified RNA replication enzyme protein and method for preventing virus infection |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS63287483A true JPS63287483A (en) | 1988-11-24 |
| JPH084498B2 JPH084498B2 (en) | 1996-01-24 |
Family
ID=14858526
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP62123355A Expired - Lifetime JPH084498B2 (en) | 1987-05-20 | 1987-05-20 | Modified RNA replication enzyme protein and method for preventing virus infection |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH084498B2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996028562A3 (en) * | 1995-03-09 | 1997-03-13 | Hoechst Schering Agrevo Gmbh | Method of checking undesired multiplication of viruses and of preparing virus-resistant organisms |
-
1987
- 1987-05-20 JP JP62123355A patent/JPH084498B2/en not_active Expired - Lifetime
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996028562A3 (en) * | 1995-03-09 | 1997-03-13 | Hoechst Schering Agrevo Gmbh | Method of checking undesired multiplication of viruses and of preparing virus-resistant organisms |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH084498B2 (en) | 1996-01-24 |
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