CN106480024B - 受维甲酸调控的具有浓度依赖效应的重组启动子及其使用 - Google Patents

受维甲酸调控的具有浓度依赖效应的重组启动子及其使用 Download PDF

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CN106480024B
CN106480024B CN201510539568.5A CN201510539568A CN106480024B CN 106480024 B CN106480024 B CN 106480024B CN 201510539568 A CN201510539568 A CN 201510539568A CN 106480024 B CN106480024 B CN 106480024B
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李小彦
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Abstract

受维甲酸调控的具有浓度依赖效应的重组启动子及其使用。本发明公开了一种重组启动子的碱基序列。该重组启动子受到细胞外液中维甲酸的调控，其调控的目的蛋白表达量具有维甲酸浓度依赖效应，在维甲酸浓度低于10‑7M的条件下，目的蛋白完全不表达。该重组启动子序列全长297bp，由调控盒A和调控盒B组成。调控盒A包含有10个DR5元件（5’‑GTTCAC‑3’）串联组成，调控盒B为115bp人工设计的协助启动元件。

Description

受维甲酸调控的具有浓度依赖效应的重组启动子及其使用
技术领域
本发明属于医学生物技术研究领域，涉及一种受维甲酸调控的具有浓度依赖效应的重组启动子，具体为该启动子由调控盒A和调控盒B组成，调控盒A包含有10个DR5元件（5’-GTTCAC-3’）串联组成，调控盒B为115bp人工设计的协助启动元件。该重组启动子链接的报告基因能够用于显示及检测细胞外液或环境中维甲酸类药物的浓度水平；该重组启动子能够用于体外细胞内或动物中调控目的基因的表达；另外，该重组启动子还能够用于人体基因治疗中调控治疗基因的表达。
背景技术
自然界生物的基因组中存在一种维甲酸反应元件（retinoic acid responseelements ，RARE），该维甲酸反应元件一般以2～4个重复排列的形式存在于基因调控启动子的上游序列中。该维甲酸反应元件会联合其它转录因子调控元件共同调控下游基因的转录表达。
维甲酸反应元件的碱基序列组成一般有多个类型，分别命名为DR1、DR2、DR3、DR4和DR5元件。细胞内转录因子RAR和RXR形成异源二聚体后，会识别基因组上的维甲酸反应元件与之结合，当细胞摄入维甲酸类药物后，维甲酸进入细胞核内与RAR/RXR结合，从而启动下游基因的表达。
然而，在自然界中并不存在单一的受维甲酸反应元件调控的启动子，也就是说，维甲酸-RAR/RXR需要协同其它转录因子（如NF-κB或STAT）共同作用，才能启动下游基因的表达。
另外，自然界中也不存在具有浓度依赖效应的维甲酸调控启动子。以往的科研工作者试图构建具有浓度依赖效应的维甲酸调控启动子，但是都很不完美，如Jason Aoto等1构建了一种维甲酸调控启动子，它是由6个DR5元件和一个胸苷激酶启动子（ThymidineKinase，TK promoter，750bp）组成，然而在该作者发表文章的Figure 3C中，可以清楚地看出，在没有维甲酸存在的情况下，EGFP仍然有一个很明显的本底表达。这是因为该作者使用了胸苷激酶启动子做为协同启动子元件，胸苷激酶启动子本身就具有独立的转录活性，在维甲酸-RAR/RXR不存在的情况下，依旧能启动下游基因的表达。
因此，发明一个具有浓度依赖效应的维甲酸调控启动子（即在不加入维甲酸时，下游基因不表达，而随着维甲酸浓度的增加，下游基因逐步提高表达水平），不仅对于体外实验研究是一个很好的基因调控工具，而且对于体内基因治疗来说，也是一个新的治疗基因的调控手段。
1．Aoto J, Nam CI, Poon MM, Ting P, Chen L. Synaptic signaling by all-trans retinoic acid in homeostatic synaptic plasticity. Neuron. 2008 Oct 23;60(2):308-20. doi: 10.1016/j.neuron.2008.08.012。
发明内容
本发明是基于通过对DR5元件的重新组合，串联使用了数量为10个的DR5元件，组成了调控盒A，其碱基序列如下：
cccagtgcaa gtgcaggtgc cagaacattt gtctgtaggg ttcaccgaaa gttcactcgg 60
ggtagggttc accgaaagtt cactcggggt agggttcacc gaaagttcac tcggggtagg 120
gttcaccgaa agttcactcg ggttgcggcc gctgcagcag acacggttca ccgaaagttc 180
ac 182
然而，这10个DR5元件组成的调控盒A是无法高效启动下游基因的表达。调控盒A需要协助启动元件调控盒B这一顺式反应元件才能高效地启动下游基因的表达。调控盒B为一个长度为115bp人工设计的协助启动元件，该元件的碱基序列如下：
tcgcatagag ggtatataat ggaagctcga cttccagctt ggcaattctg tgtgacagtc 60
cggtatgttg gtaaagcgga tccgaattcg agctccgtcg acaagcttcg ccacc 115
调控盒A和调控盒B共同组成了受维甲酸调控的具有浓度依赖效应的重组启动子，该启动子的完整碱基序列如下：
cccagtgcaa gtgcaggtgc cagaacattt gtctgtaggg ttcaccgaaa gttcactcgg 60
ggtagggttc accgaaagtt cactcggggt agggttcacc gaaagttcac tcggggtagg 120
gttcaccgaa agttcactcg ggttgcggcc gctgcagcag acacggttca ccgaaagttc 180
actcgcatag agggtatata atggaagctc gacttccagc ttggcaattc tgtgtgacag 240
tccggtatgt tggtaaagcg gatccgaatt cgagctccgt cgacaagctt cgccacc 297
长度为297bp的受维甲酸调控的具有浓度依赖效应的重组启动子，在297碱基位置后直接链接下游基因ORF的起始密码子ATG，在维甲酸-RAR/RXR存在的情况下，就能够启动目的基因的表达。
本发明还公开了受维甲酸调控的具有浓度依赖效应的重组启动子的应用方法。该启动子链接的绿色荧光蛋白或荧光素酶等报告基因，具有显示及检测细胞外液或环境中维甲酸类药物的浓度水平，是一种检测维甲酸类药物的方法。
该启动子链接的目的基因进入细胞核后，加入维甲酸类药物（如全反式维甲酸），以此来调控目的基因的表达，是一种在体外细胞内或动物中用于调控目的基因表达的方法。
该启动子链接的治疗基因整合入宿主基因组后，静脉或口服维甲酸类药物（如全反式维甲酸）来调控治疗基因的表达，是一种在人体基因治疗中用于调控治疗基因表达的方法。
本发明还指出该启动子可以用于调控T细胞嵌合抗原受体（CAR-T）基因的表达，避免细胞因子风暴的形成。
附图说明
图1.在huh7细胞中转染重组启动子链接的绿色荧光蛋白报告基因，在培养液中加入全反式维甲酸（浓度为10-8 M），24小时后用荧光显微镜（Olympus IX-71）拍摄，huh7细胞中无绿色荧光蛋白表达。
图2. 在huh7细胞中转染重组启动子链接的绿色荧光蛋白报告基因，在培养液中加入全反式维甲酸（浓度为10-7 M），24小时后用荧光显微镜（Olympus IX-71）拍摄，huh7细胞中见少量绿色荧光蛋白表达。
图3. 在huh7细胞中转染重组启动子链接的绿色荧光蛋白报告基因，在培养液中加入全反式维甲酸（浓度为10-6 M），24小时后用荧光显微镜（Olympus IX-71）拍摄，huh7细胞中见大量绿色荧光蛋白表达。
图4. 在huh7细胞中转染重组启动子链接的荧光素酶报告基因，在培养液中加入不同浓度的全反式维甲酸，24小时后裂解细胞检测荧光素酶的表达。
<110> 李小彦
<120> 受维甲酸调控的具有浓度依赖效应的重组启动子及其使用
<130> RARE
<141> 2015-8-30
<160> 3
<210> 1
<211> 182
<212> PRT
<213> UNKNOWN
<223> DR5元件
<400>
cccagtgcaa gtgcaggtgc cagaacattt gtctgtaggg ttcaccgaaa gttcactcgg 60
ggtagggttc accgaaagtt cactcggggt agggttcacc gaaagttcac tcggggtagg 120
gttcaccgaa agttcactcg ggttgcggcc gctgcagcag acacggttca ccgaaagttc 180
ac 182
<210> 2
<211> 115
<212> PRT
<213> UNKNOWN
<223> 人工协助启动元件
<400>
tcgcatagag ggtatataat ggaagctcga cttccagctt ggcaattctg tgtgacagtc 60
cggtatgttg gtaaagcgga tccgaattcg agctccgtcg acaagcttcg ccacc 115
<210> 3
<211> 297
<212> PRT
<213> UNKNOWN
<223> 受维甲酸调控的具有浓度依赖效应的重组启动子
<400>
cccagtgcaa gtgcaggtgc cagaacattt gtctgtaggg ttcaccgaaa gttcactcgg 60
ggtagggttc accgaaagtt cactcggggt agggttcacc gaaagttcac tcggggtagg 120
gttcaccgaa agttcactcg ggttgcggcc gctgcagcag acacggttca ccgaaagttc 180
actcgcatag agggtatata atggaagctc gacttccagc ttggcaattc tgtgtgacag 240
tccggtatgt tggtaaagcg gatccgaatt cgagctccgt cgacaagctt cgccacc 297

Claims (4)

1.一种受维甲酸调控的具有浓度依赖效应的重组启动子，其序列如序列3所示(SEQ IDNO：3)。
2.一种检测维甲酸类药物的方法，由权利要求1所述启动子链接的绿色荧光蛋白或荧光素酶报告基因，具有显示及检测细胞外液或环境中维甲酸类药物的浓度水平。
3.维甲酸类药物在制备调控宿主目的基因表达药物中的应用，所述宿主整合由权利要求1所述启动子链接的目的基因。
4.根据权利要求3所述的应用，如权利要求1所述的启动子可用于调控T细胞嵌合抗原受体（CAR-T）基因的表达，避免基因表达过强而导致的细胞因子风暴。
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5091518A (en) * 1989-11-16 1992-02-25 The Salk Institute For Biological Studies Beta retinoic acid response elements compositions and assays
WO1996029400A1 (fr) * 1995-03-23 1996-09-26 Institut National De La Recherche Agronomique (Inra) Procede de regulation de l'expression d'un gene dans un baculovirus, par un site de fixation d'un recepteur de l'acide retinoique, et vecteur pour la mise en ×uvre dudit procede
US5922596A (en) * 1990-12-10 1999-07-13 Bristol-Myers Squibb Co. Promoter of the retinoic acid receptor gene for directing gene expression
WO2001016368A1 (en) * 1999-09-01 2001-03-08 Bristol-Myers Squibb Company In vitro transcription systems and uses

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH06508507A (ja) * 1991-03-18 1994-09-29 ザ　ソールク　インスチチュート　フォア　バイオロジカル　スタディズ 受容体認識配列組成物およびこれを用いた分析法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5091518A (en) * 1989-11-16 1992-02-25 The Salk Institute For Biological Studies Beta retinoic acid response elements compositions and assays
US5922596A (en) * 1990-12-10 1999-07-13 Bristol-Myers Squibb Co. Promoter of the retinoic acid receptor gene for directing gene expression
WO1996029400A1 (fr) * 1995-03-23 1996-09-26 Institut National De La Recherche Agronomique (Inra) Procede de regulation de l'expression d'un gene dans un baculovirus, par un site de fixation d'un recepteur de l'acide retinoique, et vecteur pour la mise en ×uvre dudit procede
WO2001016368A1 (en) * 1999-09-01 2001-03-08 Bristol-Myers Squibb Company In vitro transcription systems and uses

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
Hugues de The等.Identification of a retinoic acid responsive element in the retinoic acid receptor & beta gene.《Nature》.1990,第343卷(第6254期),第177-180页. *
Jason Aoto等.Synaptic signaling by all-trans retinoic acid in homeostatic synaptic plasticity.《Neuron》.2008,第60卷(第2期),第308-320页. *
Swanson,B.等.登录号：DQ904457.1.《GeneBank》.2006, *
Synaptic signaling by all-trans retinoic acid in homeostatic synaptic plasticity;Jason Aoto等;《Neuron》;20081023;第60卷(第2期);第308-320页，补充材料 *
登录号：DQ904457.1;Swanson,B.等;《GeneBank》;20060926 *
筛选维甲酸样内分泌干扰物报告基因方法的建立;陈刚等;《环境与职业医学》;20091025;第26卷(第5期);第468-470页 *

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