JP3002724B2 - DNA promoting translation activity into protein and method for efficiently synthesizing protein from protein gene using said DNA - Google Patents
DNA promoting translation activity into protein and method for efficiently synthesizing protein from protein gene using said DNAInfo
- Publication number
- JP3002724B2 JP3002724B2 JP10114428A JP11442898A JP3002724B2 JP 3002724 B2 JP3002724 B2 JP 3002724B2 JP 10114428 A JP10114428 A JP 10114428A JP 11442898 A JP11442898 A JP 11442898A JP 3002724 B2 JP3002724 B2 JP 3002724B2
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- Japan
- Prior art keywords
- protein
- dna
- gene
- plasmid
- translation
- 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.)
- Expired - Lifetime
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Description
【０００１】[0001]
【発明が属する技術分野】本発明は、新規な翻訳活性化
塩基配列を有するＤＮＡに関する。本発明はまた、該Ｄ
ＮＡ塩基配列の下流に有効な蛋白質を合成させる目的遺
伝子を有するプラスミド又は組換えウイルス、及び該プ
ラスミドを含む形質転換体又は該組換えウイルスに感染
した培養細胞に関する。更に本発明は、昆虫ウイルスの
翻訳活性を促進する塩基配列に対応するＤＮＡをプラス
ミド中に取り込むことにより，その下流に位置する蛋白
質遺伝子から効率よく蛋白質を合成させる方法に関す
る。TECHNICAL FIELD The present invention relates to a DNA having a novel translation-activating nucleotide sequence. The present invention also relates to the D
The present invention relates to a plasmid or a recombinant virus having a target gene for synthesizing an effective protein downstream of an NA base sequence, and a transformant containing the plasmid or a cultured cell infected with the recombinant virus. Furthermore, the present invention relates to a method for efficiently synthesizing a protein from a protein gene located downstream thereof by incorporating into a plasmid a DNA corresponding to a nucleotide sequence that promotes the translation activity of an insect virus.
【０００２】[0002]
【従来技術】ベクターに組み込まれた外来遺伝子を培養
細胞や昆虫体内で発現させる手法は、遺伝子工学を利用
して蛋白質を生産する場合に必要不可欠である。しか
し、組み込まれた遺伝子によっては，必要量の発現蛋白
質を得られない場合も多い。ＵＳＰ４９３７１９０にお
いて、脊椎動物のＲＮＡウイルス，encephalomyocardit
is virus（ＥＭＣＶ）のゲノム５´側の非翻訳領域に翻
訳活性を促進する塩基配列が記載されている。しかしな
がらＥＭＣＶは細胞内でウイルスにとって少量しか合成
する必要のない非構造蛋白質とキャプシド蛋白質を一つ
のポリ蛋白質として合成するために、十分な翻訳促進活
性は得られない。さらに、ＥＭＣＶは脊椎動物を宿主と
しているために、外来遺伝子発現法として近年多く利用
されているバキュロウイルス発現系の宿主細胞である昆
虫細胞ではほとんど機能しないため、実用上問題がある
（Polkinghorne, I., and Roy, P. (1995) Nucleic Aci
ds Research Vol. 23, 188-191）。2. Description of the Related Art A technique for expressing a foreign gene incorporated in a vector in a cultured cell or an insect body is indispensable when producing a protein using genetic engineering. However, in many cases, a required amount of expressed protein cannot be obtained depending on the integrated gene. In USP 4,937,190, a vertebrate RNA virus, encephalomyocardit
The nucleotide sequence that promotes translation activity is described in the untranslated region on the 5 'side of the genome of is virus (EMCV). However, since EMCV synthesizes a non-structural protein and a capsid protein, which need to be synthesized only in small amounts in a cell, as a single polyprotein, sufficient translation promoting activity cannot be obtained. Furthermore, since EMCV uses a vertebrate as a host, it hardly functions in an insect cell which is a host cell of a baculovirus expression system which has been widely used in recent years as a foreign gene expression method, and thus has a practical problem (Polkinghorne, I ., and Roy, P. (1995) Nucleic Aci
ds Research Vol. 23, 188-191).
【０００３】[0003]
【発明が解決しようとする課題】従って、昆虫細胞で機
能する翻訳活性を促進する塩基配列の発見が強く望まれ
ている。即ち、本発明の課題は、必要量の発現蛋白質の
合成を促進し、かつ外来遺伝子発現法として近年多く利
用されているバキュロウィルス発現系の宿主細胞である
昆虫細胞で機能し得る翻訳活性塩基を見出し、目的とす
る蛋白質を効率よく生産することにある。Accordingly, there is a strong demand for the discovery of a nucleotide sequence that functions in insect cells and that promotes translational activity. That is, an object of the present invention is to provide a translationally active base capable of promoting the synthesis of a required amount of an expressed protein and capable of functioning in an insect cell which is a host cell of a baculovirus expression system which has recently been frequently used as a foreign gene expression method. It is an object of the present invention to produce a target protein efficiently.
【０００４】[0004]
【課題を解決するための手段】本発明者は、前記の課題
を解決するため、昆虫細胞で機能する可能性が高い、昆
虫ウイルスの遺伝子塩基配列中から、翻訳活性を促進す
る塩基配列を見出すべく鋭意研究を重ねる中で、新規昆
虫ウイルスである、チャバネアオカメムシ（学名：Plau
tia stali）から分離されたＰＳＩＶ（Plautia stali i
ntestine virus）に由来する遺伝子塩基配列中から翻訳
活性を促進する塩基配列を見出し、本発明を完成した。Means for Solving the Problems In order to solve the above-mentioned problems, the present inventor finds a nucleotide sequence that promotes translational activity from insect virus gene nucleotide sequences that are likely to function in insect cells. In the course of our intensive research, we have found a new insect virus, Chabanea stink bug (scientific name: Plau
tia stali) has been separated from PSIV (Plautia stali i
The present inventors have found a nucleotide sequence that promotes translation activity from the nucleotide sequence of a gene derived from ntestine virus) and completed the present invention.
【０００５】本発明によるＤＮＡは蛋白質の翻訳時に必
要なリボソームをＲＮＡ上に効率的に取り込むことによ
り，その下流に位置する遺伝子から効率よく蛋白質を合
成させる機能を持つ。しかも本発明によるＤＮＡによ
り、ＲＮＡにキャップ構造がついていない場合にも効率
よく蛋白質を合成できることが判明した。即ち、本発明
は、キャプシド蛋白質遺伝子、とくにチャバネアオカメ
ムシ（学名：Plautia stali）から分離されたＰＳＩＶ
（Plautia stali intestine virus）ウイルスに由来す
るキャプシド蛋白質遺伝子の上流の配列に含まれる塩基
配列に対応する新規な翻訳活性化ＤＮＡに関する。[0005] The DNA according to the present invention has a function of efficiently synthesizing a protein from a gene located downstream thereof by efficiently incorporating ribosomes required for translation of the protein into RNA. In addition, it has been found that the DNA according to the present invention can efficiently synthesize proteins even when RNA has no cap structure. That is, the present invention, the capsid protein gene, particularly Plautia crossota stali (scientific name: Plautia stali) separated from PSIV
(Plautia stali intestine virus) The present invention relates to a novel translation-activating DNA corresponding to a base sequence contained in a sequence upstream of a capsid protein gene derived from a virus.
【０００６】また本発明は、該ＤＮＡ塩基配列の下流に
有効な蛋白質を合成させる目的遺伝子を有するプラスミ
ド及び組換えウイルスに関する。更に本発明は、該プラ
スミドを含む形質転換体及び該組換えウイルスに感染し
た培養細胞にも関する。[0006] The present invention also relates to a plasmid and a recombinant virus having a target gene for synthesizing an effective protein downstream of the DNA base sequence. Further, the present invention also relates to a transformant containing the plasmid and cultured cells infected with the recombinant virus.
【０００７】また本発明は、昆虫ウイルス、とくに前記
ＰＳＩＶの遺伝子塩基配列中の翻訳活性を促進する塩基
配列に対応するＤＮＡをプラスミド又は組換えウイルス
中に取り込むことにより，その下流に位置する蛋白質遺
伝子から効率よく蛋白質を合成させる方法に関する。[0007] The present invention also relates to a protein gene located downstream of an insect virus, particularly a DNA corresponding to a nucleotide sequence that promotes translation activity in the nucleotide sequence of the PSIV gene, by incorporating the DNA into a plasmid or a recombinant virus. And a method for efficiently synthesizing a protein from the protein.
【０００８】本発明のＰＳＩＶ由来の塩基配列は、その
ゲノム構造が前記ＥＭＣＶのものとは異なり、ウイルス
増殖時に多量に合成する必要があるキャプシド蛋白質を
非構造蛋白質とは独立して合成するための専用の翻訳促
進配列を持っている。従って、ＰＳＩＶのものはＥＭＣ
Ｖのものよりも強い翻訳促進活性を持っていると期待で
きる。[0008] The base sequence derived from PSIV of the present invention has a genomic structure different from that of the above-mentioned EMCV, and is used for synthesizing a capsid protein which needs to be synthesized in large amounts at the time of virus propagation independently of a non-structural protein. Has a dedicated translation promoting sequence. Therefore, the one of PSIV is EMC
V can be expected to have a stronger translation promoting activity than that of V.
【０００９】本発明によるＤＮＡを、発現ベクターから
ＲＮＡを転写する際にＲＮＡポリメラーゼによって認識
されるプロモーター配列と目的遺伝子の翻訳開始点との
間に挿入しておくと、翻訳活性化塩基配列を５´側に持
つｍＲＮＡが合成され、それから蛋白質を合成する際に
リボソームを効率よく利用することができ、結果として
目的とする遺伝子産物をより多く発現させることができ
る。なお、本発明による翻訳活性化塩基配列を有するＤ
ＮＡは、該ＤＮＡを外来遺伝子として組み込んだプラス
ミドが導入された大腸菌（寄託番号、ＦＥＲＭ Ｐ−１
６７１５）から容易に入手することができる。When the DNA according to the present invention is inserted between a promoter sequence recognized by RNA polymerase when RNA is transcribed from an expression vector and the translation initiation site of a target gene, the translation-activating nucleotide sequence becomes 5%. The mRNA on the 'side is synthesized, and the ribosome can be used efficiently when synthesizing a protein therefrom. As a result, the target gene product can be expressed more. It should be noted that D having the translation-activating base sequence according to the present invention
NA refers to Escherichia coli (deposit number, FERM P-1) into which a plasmid having the DNA incorporated as a foreign gene has been introduced.
6715).
【００１０】[0010]
【実施例】大腸菌のファージに由来するＴ７ＲＮＡポリ
メラーゼが認識するプロモータ配列の下流にクロラムフ
エニコールアセチルトランスフエラーゼ（ＣＡＴ）遺伝
子を挿入し、さらにその下流にＰＳＩＶの翻訳促進配列
とキャプシド蛋白遺伝子を接続したベクターを構築した
（図１）。このプラスミドからＴ７ＲＮＡポリメラーゼ
でＲＮＡを合成し、そのＲＮＡを用いてウサギの網状赤
血球ライセートでinvitro translationを行った。その
翻訳産物をＳＤＳ−ポリアクリルアミドゲル電気泳動に
よって分離した（図２，レーン１）。対照実験のＣＡＴ
遺伝子の（レーン２）の場合と比較すると、ＣＡＴ（２
５ ｋＤａ）は殆ど合成されず，翻訳活性化配列の下流
にあるキャプシド蛋白質遺伝子から大量の蛋白質（３９
ｋＤａ）が合成されている。EXAMPLE A chloramphenicol acetyltransferase (CAT) gene was inserted downstream of a promoter sequence recognized by T7 RNA polymerase derived from Escherichia coli phage, and a PSIV translation promoting sequence and a capsid protein gene were inserted downstream thereof. A connected vector was constructed (FIG. 1). RNA was synthesized from this plasmid using T7 RNA polymerase, and the RNA was used for in vitro translation using a rabbit reticulocyte lysate. The translation products were separated by SDS-polyacrylamide gel electrophoresis (FIG. 2, lane 1). CAT of control experiment
Compared to the case of the gene (lane 2), CAT (2
5 kDa) is hardly synthesized, and a large amount of protein (39 kDa) is derived from the capsid protein gene downstream of the translation activating sequence.
kDa) has been synthesized.
【００１１】[0011]
配列の数：１ 配列番号：１ 配列の長さ：４３０ 配列の型：核酸 起源：ＰＳＩＶ ゲノム内での位置：５′側から５７７１−６２００ 配列 GTATTCTAGA TTGTATGAAT TGGCAAAGAT CTGGAGAGGA TGAAGGATTG AATGCTCAAG CAAACGTTAG CTTTGCTTTA AAGGAATTAT CTCTCCACCC CGAGGATGTG TGGGACCAGT GGTTTCCCCT CATTCTCAGA GCATGTAATA AACACGGTGT CGAAGTAGAA TTTCTATCTC GACACGCGGC CTTCCAAGCA GTTAGGGAAA CCGACTTCTT TGAAGAAGAA AGCTGACTAT GTGATCTTAT TAAAATTAGG TTAAATTTCG AGGTTAAAAA TAGTTTTAAT ATTGCTATAG TCTTAGAGGT CTTGTATATT TATACTTACC ACACAAGATG GACCGGAGCA GCCCTCCAAT ATCTAGTGTA CCCTCGTGCT CGCTCAAACA TTAAGTGGTG TTGTGCGAAA AGAATCTCAC TTCAAGAAAA Number of sequences: 1 SEQ ID NO: 1 Sequence length: 430 Sequence type: Nucleic acid Origin: Position in PSIV genome: 5771-6200 from the 5 'side CATTCTCAGA GCATGTAATA AACACGGTGT CGAAGTAGAA TTTCTATCTC GACACGCGGC CTTCCAAGCA GTTAGGGAAA CCGACTTCTT TGAAGAAGAA AGCTGACTAT GTGATCTTAT TAAAATTAGG TTAAATTTCG AGGTTAAAAA TAGTTTTAAT ATTGCTATAG TCTTAGAGGT CTTGTATATT TATACTTACC ACACAAGATG GACCGGAGCA GCCCTCCAAT ATCTAGTGTA CCCTCGTGCT CGCTCAAACA TTAAGTGGTG TTGTGCGAAA AGAATCTCAC TTCAAGAAAA
【図１】翻訳活性の検出に使用したＤＮＡ鋳型の模式
図。FIG. 1 is a schematic diagram of a DNA template used for detecting translation activity.
【図２】それらから転写したＲＮＡを用いたin vitro t
ranslationの結果（写真）。[Figure 2] in vitro t using were transferred from their RNA
ranslation results (photo).
フロントページの続き (51)Int.Cl.7 識別記号 ＦＩ Ｃ１２Ｒ 1:91） (Ｃ１２Ｎ 7/00 Ｃ１２Ｒ 1:92） (Ｃ１２Ｎ 15/09 ＺＮＡ Ｃ１２Ｒ 1:92） (Ｃ１２Ｐ 21/02 Ｃ１２Ｒ 1:91） Continued on the front page (51) Int.Cl. 7 Identification symbol FI C12R 1:91) (C12N 7/00 C12R 1:92) (C12N 15/09 ZNA C12R 1:92) (C12P 21/02 C12R 1:91 )
Claims (9)
るＤＮＡ。 GTATTCTAGA TTGTATGAAT TGGCAAAGAT CTGGAGAGGA TGAAGGATTG AATGCTCAAG CAAACGTTAG CTTTGCTTTA AAGGAATTAT CTCTCCACCC CGAGGATGTG TGGGACCAGT GGTTTCCCCT CATTCTCAGA GCATGTAATA AACACGGTGT CGAAGTAGAA TTTCTATCTC GACACGCGGC CTTCCAAGCA GTTAGGGAAA CCGACTTCTT TGAAGAAGAA AGCTGACTAT GTGATCTTAT TAAAATTAGG TTAAATTTCG AGGTTAAAAA TAGTTTTAAT ATTGCTATAG TCTTAGAGGT CTTGTATATT TATACTTACC ACACAAGATG GACCGGAGCA GCCCTCCAAT ATCTAGTGTA CCCTCGTGCT CGCTCAAACA TTAAGTGGTG TTGTGCGAAA AGAATCTCAC TTCAAGAAAA1. A DNA having a translation-activating nucleotide sequence shown below. GTATTCTAGA TTGTATGAAT TGGCAAAGAT CTGGAGAGGA TGAAGGATTG AATGCTCAAG CAAACGTTAG CTTTGCTTTA AAGGAATTAT CTCTCCACCC CGAGGATGTG TGGGACCAGT GGTTTCCCCT CATTCTCAGA GCATGTAATA AACACGGTGT CGAAGTAGAA TTTCTATCTC GACACGCGGC CTTCCAAGCA GTTAGGGAAA CCGACTTCTT TGAAGAAGAA AGCTGACTAT GTGATCTTAT TAAAATTAGG TTAAATTTCG AGGTTAAAAA TAGTTTTAAT ATTGCTATAG TCTTAGAGGT CTTGTATATT TATACTTACC ACACAAGATG GACCGGAGCA GCCCTCCAAT ATCTAGTGTA CCCTCGTGCT CGCTCAAACA TTAAGTGGTG TTGTGCGAAA AGAATCTCAC TTCAAGAAAA
に、目的の蛋白質を合成させる遺伝子を有することを特
徴とするプラスミド。2. A plasmid comprising a gene for synthesizing a target protein downstream of the DNA base sequence according to claim 1.
が、プロモーター配列と目的遺伝子の翻訳開始点との間
に挿入されていることを特徴とするプラスミド。3. A plasmid, wherein the DNA base sequence according to claim 1 or 2 is inserted between a promoter sequence and a translation initiation site of a target gene.
細胞に導入された形質転換体。4. A transformant in which the plasmid according to claim 2 or 3 has been introduced into a host cell.
に、目的の蛋白質を合成させる遺伝子を有することを特
徴とする組換えバキュロウイルス。5. A recombinant baculovirus comprising a gene for synthesizing a target protein downstream of the DNA base sequence according to claim 1.
モーター配列と目的遺伝子の翻訳開始点との間に挿入さ
れていることを特徴とする組換えバキュロウイルス。6. A recombinant baculovirus wherein the DNA base sequence according to claim 1 is inserted between a promoter sequence and a translation initiation site of a target gene.
イルスに感染した培養細胞。7. A cultured cell infected with the recombinant baculovirus according to claim 5 or 6.
細胞であることを特徴とする、請求項７に記載のウイル
ス感染細胞。8. The virus-infected cell according to claim 7, wherein the cultured cell is an insect cell of a baculovirus expression system.
ミド中に取り込むことにより、その下流に位置する蛋白
質遺伝子から効率よく蛋白質を合成させる方法。9. A method for efficiently synthesizing a protein from a protein gene located downstream thereof by incorporating the DNA base sequence of claim 1 into a plasmid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP10114428A JP3002724B2 (en) | 1998-04-10 | 1998-04-10 | DNA promoting translation activity into protein and method for efficiently synthesizing protein from protein gene using said DNA |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP10114428A JP3002724B2 (en) | 1998-04-10 | 1998-04-10 | DNA promoting translation activity into protein and method for efficiently synthesizing protein from protein gene using said DNA |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH11290084A JPH11290084A (en) | 1999-10-26 |
| JP3002724B2 true JP3002724B2 (en) | 2000-01-24 |
Family
ID=14637480
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP10114428A Expired - Lifetime JP3002724B2 (en) | 1998-04-10 | 1998-04-10 | DNA promoting translation activity into protein and method for efficiently synthesizing protein from protein gene using said DNA |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP3002724B2 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101161622B1 (en) * | 2009-08-31 | 2012-07-04 | 헬릭스 주식회사 | DNA fragment to promote translation efficiency and recombinant vectors containing the same |
-
1998
- 1998-04-10 JP JP10114428A patent/JP3002724B2/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| JPH11290084A (en) | 1999-10-26 |
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