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NUCLEIC ACID FOR CONTROLLING GENE EXPRESSION UPDATE_EN meetings

Foreign code F190009827
Posted date Jun 27, 2019
Country WIPO
International application number 2019JP014846
International publication number WO 2019194236
Date of international filing Apr 3, 2019
Date of international publication Oct 10, 2019
Priority data
  • P2018-073806 (Apr 6, 2018) JP
Title NUCLEIC ACID FOR CONTROLLING GENE EXPRESSION UPDATE_EN meetings
Abstract The present invention provides a nucleic acid with which the expression amount of a target gene can be controlled. The nucleic acid according to the present invention comprises (i) a base sequence represented by 5'-[T(G/A)ACATG(A/C)T]n-3' (in the base acid sequence, n is an integer of 1-20) or (ii) a base sequence obtained by substituting, adding or deleting at least one base in the aforementioned base sequence. The expression amount of a gene positioned downstream on the base sequence can be adjusted.
Outline of related art and contending technology BACKGROUND ART
The material utilized in the production processes, in order to produce the product for the purpose of, using a technique such as recombinant DNA, the gene of interest (group) such as plants and animals or microorganisms suitable in many cases expressed in a host. Interest product in order to increase the amount of production, per individual one of the host organism to the increased expression of a gene of interest is preferably performed. For this purpose as the existing method, for example a strong promoter upstream of the gene of interest or to add a sequence, a copy of a large number of expression vectors may be used.
Here, various techniques have been constructed in an existing gene expression system, a particular DNA sequence is inserted into the only additional step, in addition to increased expression of the gene of interest if possible, using the biological material is very useful in the production. In addition, recombinant DNA techniques and hosts utilized in material production processes, a plurality of genes is used. For example, catalyzes the oxidation reaction of various cytochrome P450 enzyme substrate is, not exhibit enzymatic reactions alone, and an external electron donor (NADPH) through electron transfer component such as a-electron transfer is required. In addition, the enzyme is a multi-component composite, the catalytic reaction of the electron transfer than its own course, in other words electron transfer component is often controlled by the lack of the enzyme (non-patent document 1 and 2). Therefore, an electron transfer component only enzyme expression as long as it can increase, it becomes possible to produce efficient material is, for existing gene expression systems, the presence or absence of expression of a gene of interest (on, off) of the center of the control system development. Therefore, the amount of gene expression for fine adjustment in the expected development of the control system.
Here, a eukaryotic organism, particularly animals and humans have evolved in the genomes of many, often the same sequence is a repeated sequence of consecutive (repeating sequence) is seen. This iterative sequence is almost the function of the unknown, which is typically a human disease gene Huntington's disease to reveal some of the functions in the center. Genome analysis in recent years by the development of technologies, many repeated sequences of prokaryotic microorganisms have been found, still many unknown function. But recently, the center portion of the pathogen, repeats and the relationship between the pathogenic and the repetition number has been revealed (non-patent document 3). For example, N. meningitidis Neisseria meningitidis nadA of iterative sequence is present upstream of the gene (TAAA) by n number of iterations by changing the amount of production of protein NadA, the toxicity of strain was shown to be different (non-patent document 4). In addition, in the respiratory infections Moraxella catarrhalis, located on the upstream side is the number of repetitions n (AGAT) providing a change to the amount of expression by UspA2, pathogenic difference has been confirmed (non-patent document 5).
Scope of claims (In Japanese)[請求項1]
 目的遺伝子の発現量を調節するための核酸であって、下記(i)または(ii)に記載の塩基配列からなる核酸:
(i)5’-[T(G/A)ACATG(A/C)T]n-3’
 (上記塩基配列において、nは1~20の整数である。)、または、
(ii)上記(i)に示される塩基配列において1または数個の塩基が置換、付加、または、欠失した塩基配列からなり、かつ、前記塩基配列の下流に位置する遺伝子の発現量を調節することのできる塩基配列。

[請求項2]
 請求項1に記載の核酸であって、
前記(i)5’-[T(G/A)ACATG(A/C)T]n-3’で示される塩基配列におけるnが1~7の整数である、核酸。

[請求項3]
 請求項1または2に記載の核酸を含むベクター。

[請求項4]
 請求項3に記載のベクターであって、
 前記ベクターは発現量を調整する目的遺伝子をさらに含み、前記核酸が前記目的遺伝子の発現量を調節可能なように上流に配置されている、ベクター。

[請求項5]
 請求項3または4に記載のベクターを含む宿主細胞。

[請求項6]
 請求項5に記載のベクターを含む宿主細胞であって、
 前記目的遺伝子が宿主細胞に対する外来遺伝子である、宿主細胞。

[請求項7]
 目的遺伝子を発現させる方法であって、
 請求項1または2に記載の核酸が上流に連結された前記目的遺伝子を発現させる工程を含む、発現方法。

[請求項8]
 請求項7に記載の発現方法であって、
 前記核酸が、前記目的遺伝子の発現量が所望の量となるように選択された反復数を有する塩基配列からなる核酸である、発現方法。

[請求項9]
 請求項7または8に記載の発現方法であって、
 前記目的遺伝子を発現させる工程が、請求項4に記載のベクターに含まれる前記目的遺伝子を細胞系または無細胞系において発現させる工程である、発現方法。

[請求項10]
 請求項7または8に記載の目的遺伝子を発現させる方法であって、
 前記目的遺伝子が細胞内の内在遺伝子であり、
 前記目的遺伝子を発現させる工程の前に、請求項1または2に記載の核酸を前記細胞の染色体上に存在する前記目的遺伝子の上流に導入する工程をさらに含む、発現方法。

[請求項11]
 宿主細胞を用いたタンパク質の産生方法であって、
 前記宿主細胞において前記タンパク質をコードする遺伝子の発現を促し、前記タンパク質を産生させる工程であって、前記宿主細胞は前記タンパク質をコードする遺伝子の上流に請求項1に記載の核酸を有する工程を含む、
タンパク質の産生方法。

[請求項12]
 請求項11に記載のタンパク質の産生方法であって、
 前記タンパク質を産生される工程において、前記宿主細胞が請求項3に記載のベクターを含むものである、
タンパク質の産生方法。

[請求項13]
 請求項11または12に記載のタンパク質の産生方法であって、
 前記核酸が、前記目的遺伝子の発現量が所望の量となるように、選択された反復数の塩基配列からなる核酸である、タンパク質の産生方法。
  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • NATIONAL INSTITUTE OF ADVANCED INDUSTRIAL SCIENCE AND TECHNOLOGY
  • Inventor
  • SUENAGA Hikaru
  • MATSUZAWA Tomohiko
  • SAHARA Takehiko
IPC(International Patent Classification)
Specified countries National States: AE AG AL AM AO AT AU AZ BA BB BG BH BN BR BW BY BZ CA CH CL CN CO CR CU CZ DE DJ DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IR IS JO JP KE KG KH KN KP KR KW KZ LA LC LK LR LS LU LY MA MD ME MG MK MN MW MX MY MZ NA NG NI NO NZ OM PA PE PG PH PL PT QA RO RS RU RW SA SC SD SE SG SK SL SM ST SV SY TH TJ TM TN TR TT TZ UA UG US UZ VC VN ZA ZM ZW
ARIPO: BW GH GM KE LR LS MW MZ NA RW SD SL ST SZ TZ UG ZM ZW
EAPO: AM AZ BY KG KZ RU TJ TM
EPO: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
OAPI: BF BJ CF CG CI CM GA GN GQ GW KM ML MR NE SN TD TG
Reference ( R and D project ) Leading-edge Biotechnology Research Group,AIST

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