CN1218511A - 启动子 - Google Patents
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Abstract
本发明提供具Tec酪氨酸激酶启动子活性的DNA,以及在其中已整合了该启动子,使得某一外源基因在诸如造血干细胞和肝细胞中能高水平表达的载体。
Description
技术领域
本发明涉及基因工程领域,尤其是基因治疗领域。
技术背景
基因治疗试图通过用正常的基因来取代或补充有缺陷的基因来治疗先天或获得性的所谓基因疾病的遗传缺陷。虽然研究过多种基因治疗的方法,但至今为止仅有极其有限的方法获得了成功,包括腺苷脱氨酶(ADA)缺陷的治疗。这主要是由于在靶细胞中有效导入治疗基因的方法以及在细胞中表达转导的基因的方法还未建立起来的缘故。到目前为止,脂质体、HVJ-脂质体、反转录病毒,诸如此类都曾经用作载体把治疗基因导入到靶细胞中。但是在基因转录效率方面,上述方法没有一种是令人满意的。通过例如,改进启动子已经作了许多尝试以提高转导基因的表达效率。然而,在任何一种情况下,所期望的基因的表达效率仍然很差,而且基因产物的量不足以担负基因治疗。因此,在基因治疗领域,已经开始寻找能在不同靶细胞中高水平表达治疗基因的载体。
在血液学领域,据信参与造血干细胞增殖的一种蛋白质,Tec酪氨酸激酶在小鼠肝脏中高水平表达,而且在肾脏、心脏和卵巢中也有表达(致癌基因(oncogene),5,1781-1786(1990))。在人体内,Tec酪氨酸激酶高度表达于大范围的血细胞和淋巴细胞中(白血病(LEUKEMIA),8,1663-1672(1994))。
发明的公开
本发明的目的之一是提供一种已在其中插入了在广范围的血液和淋巴细胞以及如肝脏的器官的细胞中有效作用的启动子的载体,从而提供用于血液和淋巴细胞的一种基因治疗技术。
本发明人注意到据信参与造血干细胞增殖的一种蛋白顶,Tec酪氨酸激酶在大范围的血液和淋巴细胞以及如肝脏的器官的细胞中高度表达。本发明人从小鼠基因组DNA中分离了Tec酪氨酸激酶的启动子,构建了一个载体,其中插入了该启动子,并在紧邻启动子的下游连接了一个外源基因,并尝试把该外源基因在细胞中进行表达。结果发现该外源基因确实在细胞内以高水平表达了,于是就完成了本发明。因而,本发明涉及:
(1)至少包含序列号:1的核苷酸序列的一部分并具启动子活性的DNA;
(2)包含(1)的DNA的表达载体;以及
(3)携帝(2)的表达载体的细胞。
本发明中,“具有启动子活性的DNA”指有诱导与该DNA邻接的DNA区段转录的活性的DNA。本发明包括含有序列号:1的核苷酸序列的一部分,并且除具序列号:1的核苷酸序列外还要具有启动子活性的DNA。优选地,本发明的DNA一般长度至少约为50bp,更优选地至少约为100bp,而更更优选地是至少约200bp,最优选地是至少约300bp。
根据本发明,任何用于基因转导的载体基本上都可以用作在其中插入具启动子活性DNA的“载体”。尤其在基因治疗中,应当使用病毒载体,例如逆转录病毒、腺病毒、或腺伴随病毒载体,以及非病毒载体,例如脂质体。
任何细胞都可包括在本发明的“携带表达载体的细胞”中。应使用已证实确实可以表达Tec酪氨酸激酶基因的细胞,包括血细胞和淋巴细胞,例如造血干细胞、骨髓细胞、B细胞和T细胞,以及内部器官如肝脏、肾脏、心脏以及卵巢的细胞。
附图简要说明
图1显示本发明的鼠Tec基因的5'侧翼区的核苷酸序列。
图2显示BA/F3及NIH3T3细胞的荧光素酶活性,在该细胞中已转入了pUC00Luc,而该pUC00Luc中插入了鼠Tec基因的5'侧翼区片段及与其相邻的荧光素酶基因。一个不含5'侧翼区片段的被用作对照pUC00Luc。
发明的最优实施方式
以下的实施例将对本发明作具体描述,但不应认为是对本发明范围的限定。
实施例1鼠基因组文库的构建
提取BA/F3细胞中的高分子量基因组DNA。用Sau3AI(Takara Shuzo)进行部分酶切DNA,并用细菌碱性磷酸酶(BAP;Takara Shuzo)脱磷酸化。把所得的DNA片断插入列用BamHⅠ消化的EMBL3载体中(Stratagene),并用“Gigapack Gold extracts”(Stratagene)进行体外包装。如此得到的重组噬菌体用于感染大肠杆菌LE392菌株。
实施例2鼠Tec启动子的筛选
为了从鼠Tec基因中获得启动子区域,如下以PCR制备筛选探针。首先,合成相应于TeccDNA(序列号:2;致癌基因(Oncogene),8,417-424(1993)从第15到第32个核苷酸的一个引物,以及从第122到第141个核苷酸的另一个引物,并用以扩增鼠Tec cDNA的5'区域。进行PCR扩增时,用10纳克鼠Tec cDNA作为模板。纯化PCR产物(约127bp),用32P进行标记,并作为探针筛选基因组文库,而其是通过在含有5×SSC(1×SSC:150mM NaCl,15mM柠檬酸钠),5×Denhardt’s溶液(1mg/ml聚乙烯吡咯烷酮,1mg/ml牛血清白蛋白,1mg/ml菲可(Ficoll),0.5%SDS,100ng/ml鲑鱼精DNA,以及32P标记的PCR片断的溶液中65℃杂交过夜进行。滤膜用2×SSC/0.1%SDS在55℃下20分钟洗涤两次,并用0.2×SSC/0.1%SDS在55℃下20分钟洗涤两次。把滤膜在“Kodak XAR Films”(Kodak)上用增感屏在-80℃下曝光24到72小时。结果得到13个阳性克隆。以同样方式用放射性标记的相应于该Tec DNA位置第15到39的探针第二次筛选这些阳性克隆,其通过在与上述相同的条件下杂交而进行,只是杂交温度为55℃,洗涤在2×SSC/0.1%SDS中于55℃下每次20分钟洗涤两次。随后得到两个阳性克隆。
实施例3鼠Tec基因转录起始位点分析
为了分析鼠Tec基因的转录起始位点,如下进行RACE-PCR。首先,从BA/F3细胞中提取5μg的mRNA,并用寡核苷酸,5'-TTAGCATCATGAACAAC-3'(引物1)作为此mRNA的反义引物进行退火,该寡核苷酸相应于该Tec cDNA位置第358到第374的核苷酸(下文中使用与序列号:2中相同的核苷酸位置)。退火产物进行cDNA合成和d(A)加尾。第一次PCR用上述引物作为模板,以寡核苷酸,5'-CCTTACCCTCATAGTAGCTCA-3'(引物2),以及寡核苷酸,5'-GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT-3'(引物3)用作引物进行,其中的引物2相应于Tec cDNA位置第227到247的核苷酸位置。第一次PCR为94℃为94℃40秒,55℃2分钟,72℃3分钟,循环40次。此外,第二次PCR和第一次PCR条件相同,除了用相应于Tec cDNA第122到141位置上的核苷酸的寡核苷酸5'-TCAACACTATCCTAGAAGAG-3'(引物4)和寡核苷酸5'-GACTCGAGTCGACATCG-3代替引物2和引物3作为引物。作为本实验的阴性对照,RACE-PCR中不使用反转录酶。然后,PCR产物在琼脂糖上电泳,用溴化乙锭(EtBr)染色。结果仅在反转录酶存在下进行RACE-PCR检测到约250bp的PCR产物。为了验证已扩增了所研究的区域,把PCR产物进行琼脂糖电泳后转移到硝酸纤维滤膜上,然后用相应于Tec cDNA第15到39位置核苷酸的寡核苷酸5'-GCAGTTTGGACGTCGCTCTGTCTTG-3'与此膜杂交。结果显示PCR产物包含Tec cDNA的大部分5'区域,即RACE-PCR准确地扩增了Tec mRNA。
确定了所得DNA片断的5'端序列之后,在8个克隆的7个中的多聚T(Poly T)序列之后发现了相同的序列(5'-CGCAGTTTGG…),因此,此序列的5'末端“C”就被认作转录起始位点。图1中转录起始位点用箭头指出。
实施例4 鼠Tec基因的5'侧翼区分析
用双脱氧法确定鼠Tec基因的5'侧翼区的核苷酸序列。所得序列如图1及序列号1所示。结果说明在5'侧翼区无可明确区分为TATA盒或CAAT盒的序列。相反地,却有GATA位点和SP-1因子结合位点的共有序列(图1)。
实施例5鼠Tec基因5'侧翼区启动子活性的分析
把由鼠Tec基因5'侧翼区的一部分及外显子1(从-409到+22的区域)组成的一个片断插入到不含启动子的表达载体,pUC00Luc中,并导入BA/F3细胞中,该细胞高效表达Tec mRNA,还导入到NIH3T3细胞中,它不表达Tec mRNA,方法如下。首先,用磷酸缓冲盐溶液(PBS)洗涤1×107个处于生长期的细胞,并用500μg DEAE-葡聚糖(Pharmacia)和10μg报告质粒DNA在室温下温育25分钟。然后把细胞在含100μM氯喹的培养基上在37℃下培养1小时。培养在5%二氧化碳下进行。细胞用PBS洗涤,在培养基上温育48小时。收获后用于用“Luciferase AssaySystem”(Promega)的荧光素酶活性测定。作为上述实验的对照,使用已在其中导入了不含5'-区的pUC00Luc的细胞。按照一般方法(按照“荧光酶测定系统(Luciferase assay System”(Promega)手册上的方法)进行荧光素酶活性测定。
结果表明,当其中导入含5'区片段的pUC00Luc时,BA/F3细胞的荧光素酶活性比NIH3T3细胞高10倍(图2)。与此相反,在对照实验中,任何细胞都未检测出荧光素酶活性(图2)。因此,表明该鼠Tec基因的5'侧翼区有启动子活性。
按照本实施例所进的方法,可以相信本领域的熟练技术人员可以通过用例如ExoⅢ和Bal31这样的外切核酸酶或限制酶消化而制备得的具序列号1的核苷酸序列的更短的启动子并检测该DNA片段的启动子活性来制备和鉴定更短的DNA片段。
工业适用性
本发明中,分离了在许多血细胞、淋巴细胞,以及如肝脏等的器官的细胞中高效表达的Tec酪氨酸激酶的启动子,并阐明了其结构。而且,本发明使得在其中插入了该启动子载体的生产和在造血干细胞、肝细胞及类似细胞中高水平表达的外源基因成为可能。尤其在血细胞或如肝脏等器官的细胞的基因治疗领域期望会有大的突破。
序列表序列号:1序列长度:480序列类型:核酸链型:双链拓朴结构:线性分子类型:基因组DNA序列描述:AGCTTGTCAG TAAGCCACCA TTCTTCTATC ACCCCAGAGC ACAGCATCAT CGGTTTTCAC 60CCGCGAGGGG CTAAGCGGAA GTGGAGGTCG GTTCTTAGCC ACCCACAAGT GCTATTGCTA 120CGTCCTCCGA GCCGGGGATC GAAGGAGCAT TTTTCTGGAC GGTTCTCTTA GGATGGGAAG 180TCCGGACTTA GAGAGACCCC ACGCCGCGTC TGTCTGGATA AGAGACGCTC CCTGGAACTT 240CGGCCGCAGG ACCGAGAGCT CCGATTCTTC CCTTTGGCTT TGAAATCGCG GAAGGAAGGT 300GGGACACTGG CGCTCTGGGC ACGAGGCAGA GCGACGCGAG GGCGGGCCAG GAGAGCCGGG 360CGGTGGGCGT GGCGATGGGT TTGGTCAGCG CTTGCCGAGC TCCGGGCTCC GCAGTTTGGA 420CGTCGCTCTG TCTTGGCTTG TCTCGGCACG CGCTCCGTCA AGGTAAGAAC CAAGGGACTC 480序列号:2:序列长度:2574序列类型:核酸链型:双链拓朴结构:线性分子类型:cDNA序列描述:GAGCTCCGGC CTCCGCAGTT TGGACGTCGC TCTGTCTTGG CTTGTCTCGG CACGCGCTCC 60GTCAAGAATC CGGAGATCGT CAATGGCTGG AGAAAGAGCA ACCAGAAGAC CGAGATGAAT 120TTCAACACTA TCCTAGAAGA GATTCTTATT AAAAGGTCCC AGCAGAAAAA GAAGACATCA 180CTCTTAAACT ACAAAGAGAG ACTTTGTGTA CTTCCAAAAT CCGTGTTGAG CTACTATGAG 240GGTCGAGCGG AGAAGAAATA CAGAAAGGGC GTCATTGATA TTTCCAAAAT CAAGTGTGTG 300GAGATAGTGA AGAACGATGA TGGTGTCATT CCCTGTCAAA ATAAATTTCC ATTCCAGGTT 360GTTCATGATG CTAATACACT TTATATTTTT GCACCTAGTC CACAAAGCAG GGACCGATGG 420GTGAAGAAGT TAAAAGAAGA AATAAAGAAC AACAATAATA TCATGATTAA ATACCATCCT 480AAATTCTGGG CAGATGGGAG TTACCAGTGT TGTAGACAAA CAGAAAAACT AGCACCCGGA 540TGTGAGAAGT ACAATCTTTT TGAGAGTAGT ATAAGAAAGA CCCTGCCTCC CGCGCCAGAA 600ATAAAGAAGA GAAGGCCTCC TCCACCAATT CCCCCAGAGG AAGAAAATAC TGAAGAAATC 660GTTGTAGCGA TGTATGACTT CCAAGCGACG GAAGCACATG ACCTCAGGTT AGAGAGAGGC 720CAAGAGTATA TCATCCTGGA AAAGAATGAC CTCCATTGGT GGAGAGCGAG AGATAAGTAT 780GGGAGTGAAG GATATATCCC AAGTAATTAC GTCACAGGGA AGAAATCCAA CAACTTAGAT 840CAATATGAGT GGTACTGCAG AAATACCAAC AGAAGCAAAG CAGAACAGCT CCTCAGAACG 900GAAGATAAAG AAGGTGGTTT TATGGTGAGA GACTCCAGTC AACCAGGCTT GTACACTGTC 960TCCCTTTACA CAAAGTTTGG GGGAGAAGGC TCATCAGGTT TCAGGCATTA TCACATAAAG 1020GAAACAGCAA CATCCCCAAA GAAGTATTAC CTGGCAGAGA AGCATGCTTT CGGGTCCATT 1080CCTGAGATCA TTGAATATCA CAAGCACAAT GCGGCAGGGC TTGTCACCAG GCTGCGGTAC 1140CCGGTCAGTA CAAAGGGGAA GAACGCTCCC ACTACTGCGG CCTTCAGCTA TGATAAGTGG 1200GAGATTAACC CATCAGAGCT GACCTTTATG AGAGAGTTGG GGAGCGGACT GTTTGGAGTG 1260GTGAGGCTTG GCAAGTGGCG GGCCCAGTAC AAAGTGGCCA TCAAAGCTAT CCGGGAAGGC 1320GCCATGTGTG AAGAGGATTT CATAGAGGAA GCTAAAGTCA TGATGAAGCT GACACACCCC 1380AAGCTGGTAC AGCTCTATGG TGTATGCACC CAGCAGAAGC CCATCTACAT CGTTACCGAG 1440TTCATGGAAC GGGGCTGCCT TCTGAATTTC CTCCGGCAGA GACAAGGCCA TTTCAGCAGA 1500GACATGCTGC TAAGCATGTG TCAAGATGTC TGTGAAGGGA TGGAGTACCT GGAGAGAAAC 1560TTCTTCATCC ACAGAGACCT GGCTGCCAGA AATTGTCTAG TGAATGAAGC AGGAGTTGTC 1620AAAGTATCTG ATTTTGGAAT GGCCAGGTAC GTTCTGGATG ATCAGTACAC AAGTTCTTCT 1680TGCGCCAAGT TCCCTGTGAA GTGGTGTCCC CCAGAAGTGT TTAATTACAG CCGCTTTAGC 1740AGCAAGTCAG ACGTCTGGTC GTTTGGTGTG CTAATGTGGG AAATATTCAC AGAAGGCAGG 1800ATGCCCTTTG AGAAGAACAC CAATTACGAA GTGGTAACCA TGGTGACTCG TGGCCACCGC 1860CTCCACCGGC CAAAGCTGGC TTCCAAATAT TTGTATGAGG TGATGCTGAG ATGCTGGCAA 1920GAGAGACCAG AGGGAAGGCC TTCCTTTGAA GACTTGCTGC GTACGATAGA TGAACTAGTT 1980GAATGTGAAG AAACTTTTGG AAGATGAATG GTGGTCCCAG TTTCCAAGGC AAGAGGAAGA 2040AATGGTGTGC CATCGGAACG CAATTCTCTT GGCACCTGGG AGTATAGACT GCTCTGCTTA 2100CAACACGGTA GCCCCAGCTC ATCTGCTGCT GATCCAGCCT GAGCTCAGTC CCTGCTTTGC 2160CGGCTGCACA GATGGTCTCT CAGAGCTGGT GACGTGAAGC ACTGATTTTG CTCATTTCTT 2220CAAGGGTTTG AGTGCCAGCC ATGTATACCA GGCTCTGTGC CCAGGCCTCA GGAGATGAAC 2280ATGGGACTAT GCTAGCTGAT GCTAGCGGAA AGCCAGGGTG GTTGTGATGG GGACGAGTCA 2340TGTCCCAGCG TCTCTTCCAT GCCCTTTGGC TATTACATAA ACCTGGGCCT GGAGTGTTGT 2400CTACCACTGA GTTCTAGGAA AAGCAGGAAC CCACCTGGAT ACGTAGTAAT CCTCTGTTTT 2460GGAAACATCT CTTTCCAAAC TTGTTCTTAG TAGTATGCTT AAAAATTTGT ATATTGTATA 2520TATTGTAAAT ACATATAATA TATAAAGTTA TATATTTATA AGTAAAAAAA AAAA 2574
Claims (3)
1.一种至少含有一部分序列1的核苷酸并具启动子活性的DNA。
2.含有权利要求1的DNA的表达载体。
3.携带权利要求2的表达载体的细胞。
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JP5429496 | 1996-03-12 | ||
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JP54294/96 | 1996-03-12 |
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CN1218511A true CN1218511A (zh) | 1999-06-02 |
CN1187452C CN1187452C (zh) | 2005-02-02 |
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CNB971945888A Expired - Fee Related CN1187452C (zh) | 1996-03-12 | 1997-03-10 | 启动子 |
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US (2) | US6225459B1 (zh) |
EP (1) | EP0894866B1 (zh) |
JP (1) | JP3162726B2 (zh) |
KR (1) | KR100490818B1 (zh) |
CN (1) | CN1187452C (zh) |
AT (1) | ATE277192T1 (zh) |
AU (1) | AU2233297A (zh) |
CA (1) | CA2248541A1 (zh) |
DE (1) | DE69730825T2 (zh) |
DK (1) | DK0894866T3 (zh) |
HK (1) | HK1020583A1 (zh) |
WO (1) | WO1997034007A1 (zh) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
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CN114427000A (zh) * | 2022-01-21 | 2022-05-03 | 信阳师范学院 | 一种特定基因转录起始位点的鉴定方法 |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
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US6500943B2 (en) * | 1996-03-12 | 2002-12-31 | Dnavec Research Inc. | Promoter |
US7199107B2 (en) | 2002-05-23 | 2007-04-03 | Isis Pharmaceuticals, Inc. | Antisense modulation of kinesin-like 1 expression |
CA2715080C (en) | 2007-09-28 | 2021-09-28 | Intrexon Corporation | Therapeutic gene-switch constructs and bioreactors for the expression of biotherapeutic molecules, and uses thereof |
CN108220312A (zh) * | 2017-12-20 | 2018-06-29 | 武汉伯远生物科技有限公司 | 用于分析启动子活性的载体以及基于rna计数的启动子活性分析方法 |
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1997
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- 1997-03-10 AU AU22332/97A patent/AU2233297A/en not_active Abandoned
- 1997-03-10 JP JP53242797A patent/JP3162726B2/ja not_active Expired - Fee Related
- 1997-03-10 EP EP97906864A patent/EP0894866B1/en not_active Expired - Lifetime
- 1997-03-10 DE DE69730825T patent/DE69730825T2/de not_active Expired - Fee Related
- 1997-03-10 AT AT97906864T patent/ATE277192T1/de not_active IP Right Cessation
- 1997-03-10 DK DK97906864T patent/DK0894866T3/da active
- 1997-03-10 KR KR10-1998-0707175A patent/KR100490818B1/ko not_active IP Right Cessation
- 1997-03-10 WO PCT/JP1997/000741 patent/WO1997034007A1/ja active IP Right Grant
- 1997-03-10 CN CNB971945888A patent/CN1187452C/zh not_active Expired - Fee Related
- 1997-03-12 US US09/142,529 patent/US6225459B1/en not_active Expired - Fee Related
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1999
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2000
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
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CN114427000A (zh) * | 2022-01-21 | 2022-05-03 | 信阳师范学院 | 一种特定基因转录起始位点的鉴定方法 |
Also Published As
Publication number | Publication date |
---|---|
ATE277192T1 (de) | 2004-10-15 |
HK1020583A1 (en) | 2000-05-12 |
EP0894866A4 (en) | 2000-12-20 |
CA2248541A1 (en) | 1997-09-18 |
EP0894866B1 (en) | 2004-09-22 |
DK0894866T3 (da) | 2004-12-06 |
KR19990087708A (ko) | 1999-12-27 |
WO1997034007A1 (fr) | 1997-09-18 |
AU2233297A (en) | 1997-10-01 |
DE69730825D1 (de) | 2004-10-28 |
EP0894866A1 (en) | 1999-02-03 |
KR100490818B1 (ko) | 2005-08-04 |
CN1187452C (zh) | 2005-02-02 |
US6225459B1 (en) | 2001-05-01 |
DE69730825T2 (de) | 2005-09-29 |
JP3162726B2 (ja) | 2001-05-08 |
US20010005589A1 (en) | 2001-06-28 |
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