CN101072878A - 含有mj1基因的重组载体和使用其的重组方法 - Google Patents
含有mj1基因的重组载体和使用其的重组方法 Download PDFInfo
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- CN101072878A CN101072878A CNA2005800416834A CN200580041683A CN101072878A CN 101072878 A CN101072878 A CN 101072878A CN A2005800416834 A CNA2005800416834 A CN A2005800416834A CN 200580041683 A CN200580041683 A CN 200580041683A CN 101072878 A CN101072878 A CN 101072878A
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Abstract
本发明涉及一种含有MJ1基因的重组载体及使用该重组载体的重组方法,其中所述的MJ1基因编码一种源自肠球菌(Enterococcus)温和噬菌体φFC1的整合酶。更具体而言,本发明涉及一种含有MJ1基因的重组载体,所述基因可独立地在人细胞中产生位点特异性重组而不需其它因子并且不造成切除。因此,本发明对于哺乳动物的基因治疗非常有用。
Description
技术领域
本发明涉及一种含有MJ1基因的重组载体及其使用该重组载体的重组(site-specific integration)方法,其中所述的MJ1基因编码一种源自肠球菌(Enterococcus)温和噬菌体FC1的整合酶。更具体而言,本发明涉及一种含有MJ1基因的重组载体,所述基因可独立地在人体细胞中产生位点特异性重组而不需其它因子并且不造成切除,本发明还涉及使用所述载体的重组方法。
背景技术
粪肠球菌(Enterococcus faecalis)是一种革兰氏阳性厌氧菌,其通常存在于从蟑螂到人类的大部分动物的肠内。在发明人的实验室中经过紫外照射诱导后(Kim et al.,(1994)Mol.Cell.,4,155-158)首次从溶源性菌株KBL703的培养物中分离到E.faecalis KBL703菌株和其温和噬菌体FC1。细菌噬菌体FC1具有约为40.5Kbp的双链DNA基因组,头部为20面体和尾部为非收缩尾鞘。噬菌体FC1通过位点特异机制整合到宿主染色体中。已鉴定出MJ1基因位于attP位点上游,该基因编码一种位点特异性整合酶。MJ1编码一段465个氨基酸的多肽,并在其N-端结构域和位点特异性整合酶相似。在attP区域附近对DNA序列的分析鉴定出两个预测的细菌-噬菌体接合区域(attL和attR)。从这些区域的序列中推断出相应的细菌附着位点(attB)(Kim et al.,(1996)Biochem.Mol.Biol,29,448-45412)。
整合酶MJ1介导两个DNA识别序列之间的单向位点特异性重组,所述的两个序列是噬菌体附着位点attP和细菌附着位点attB。为了完成整合,温和噬菌体FC1编码一种整合酶MJ1,MJ1介导attP和attB之间的整合。在290bp的attB序列中,attB位点有3bp的保守核心序列,其与730bp的attP的3bp保守核心序列重叠。围绕核心序列,attP和attB各占其序列的一半,从而形成attL、attR和重复3bp核心序列(Yang et al.,(2002)J.Bacteriol.,184,1859-1864)。
在基因治疗中,载体是用于将目的基因转移到靶细胞中的工具。问题在于,并没有一种“良好的通用载体”,目前可以得到的所有载体既有优点也有不足。例如,一种病毒载体可能可以非常高效地进入靶细胞,但是一旦进入就产生强烈的免疫应答,导致该细胞被免疫系统杀死,从而,首先对宿主安全造成了威胁。因此,很显然原来在病毒和非病毒载体之间清晰的界限变得更加模糊。由于这些原因,开发安全的整合系统以获得基因是非常重要的。然而,普遍的用于将外源基因放置到基因组中的原始方法,现有技术通常是随机整合。对于引入DNA的位置缺乏控制,导致不可预知的基因表达和重要基因潜在的不期望的突变。一个好的解决方法是可以在靶基因组中制造高效位点特异性整合入安全位点。在这些情况下,保守位点特异性重组在基因工程策略上是很重要的。
位点特异性重组酶也具有高度专一性,另外,其作用效率更高。一些重组酶不需要辅助因子,允许其在外源细胞环境中具有活性。重组酶例如Cre和FLP在同一靶位点既可以整合也可以切除(Sauer,B.(1994)Curr.Opin.Biotechnol.,5,521-527)。因此尽管这些重组酶可高效地实现在哺乳动物细胞的整合,但由于其切除反应,它们所介导的净整合频率很低。为了在特异位点稳定并且高效的表达目的基因,必须要解决例如低整合效率和稳定性的问题。
本发明鉴定出源自粪肠球菌FC1的整合酶MJ1在效率和稳定性上是很好的实例。
发明内容
(技术问题)
本发明提供了一种含有MJ1基因的重组载体,所述基因编码具有氨基酸序列SEQ ID No.2的整合酶,其中该整合酶在动物细胞中介导位点特异性重组而不介导切除。
根据本发明权利要求1的重组载体,MJ1基因可以具有任何能编码具有氨基酸序列SEQ ID No.2的整合酶的碱基序列。优选地,MJ1基因具有SEQ ID No.1的碱基序列和具有如图3所示的质粒图谱的重组载体。
本发明提供一种在动物细胞中进行位点特异性重组的方法,所述方法包括用一含有编码具有氨基酸序列SEQ ID No.2的整合酶的MJ1基因的重组载体,一含有attB基因的重组载体和一含有attP基因的重组载体共转染动物细胞。
在本发明的方法中,重组载体可以是任何具有MJ1基因,attB基因或者attP基因的重组载体。优选地,MJ1基因具有序列SEQ ID No.1的碱基序列和含有MJ1基因的重组载体具有如图3所示的质粒图谱。
(技术方案)
在本发明中,显示源自粪肠球菌的FC1的整合酶MJ1在人细胞以及大肠杆菌(E.coli.)中也发挥作用。在染色体外载体系统中,构建了含有attP,attB和整合酶MJ1编码序列的每个质粒并共转染到人细胞系HEK293T(图1~7)。在MJ1存在或不存在时,通过绿色荧光蛋白(GFP)作为报告标记对位点特异性重组和反向切除反应进行监测。带有attB(pcB)基因和整合酶MJ1编码基因(pcMJ1)的质粒具有细胞巨化病毒(CMV)启动子。然而,同时带有attP和GFP的质粒(pGP(-))由于没有CMV启动子而不能表达GFP,因此不能构建重组质粒(pREC-I)。由此,当attP和attB被attL和attR代替时可观察到位点特异性切除反应。
此外,已经确认在染色体外载体系统中,MJ1整合酶在人体细胞中没有任何辅助因子下起作用,以及整合酶MJ1的效率由于无切除而更高。而且att位点的几百碱基对可以减少到大约50bp。
在本发明中,attP指在细菌噬菌体的附着位点(739bp)和attB指在细菌的附着位点(290bp)。以及,attL和attR指由在attP和attB之间位点特异性重组所产生的左侧区域(404bp)和右侧区域(624bp)。
为了克隆attB,使用细菌噬菌体FC指示菌菌株肠球菌KBL707(保藏号KFCC 12177)的基因组DNA作为PCR的模板,所述菌种由韩国微生物保藏中心(KCCM)保藏。至于attL和attR,使用KBL703菌株(Kim et al.Mol.Cells4;155-158)的基因组DNA作为PCR模板,其染色体和细菌噬菌体FC整合。对于attP,使用插入了7.7kbFC1的DNA的pFE1作为PCR模板(Kim andY.W.1999.Genetic studies of bacteriophage FC1 from Enterococcus faecalis.PH.D.Thesis.Korea University.Korea)。
在本发明中,SEQ ID No.1显示了MJ1基因的碱基序列而SEQ ID No.2显示了MJ1整合酶的氨基酸序列。此外,SEQ ID No.3显示了attP基因的碱基序列和SEQ ID No.4显示了attB的碱基序列。
对attP和attB位点最小区域的检测对于利用高效策略进行基因治疗是非常有价值的。利用已经报道的739bp的attP和290bp的attB全长对于有效的基因治疗太长了。因此进行基于MJ1结合位点在att处的attP和attB套式缺失以找到att位点的最小区域。这些结果证明attP和attB的有效最小区域是54bp(SEQID No.3中166-233碱基)和48bp(SEQ ID No.4中66-113碱基),以及这个区域大小对于MJ1介导的整合已经足够发挥作用。
在本发明的具体实施例中,对细菌和细菌噬菌体进行培养,构建含有MJ1基因,attP或attB位点的质粒,使用所述质粒共转染人胎儿肾细胞系293,测定GFP报告基因的荧光活性,在动物细胞中进行MJ1基因的RT-PCR,进行FACS(流式细胞仪)分析,并确定attP和attB的整合以及attL和attR的切除。
附图说明
通过具体实施方式的描述并参考附图,本发明的上述和其他特性及优点将更加显而易见,其中:
图1是含有290bp attB基因和CMV启动子的pcB载体的质粒图谱。
图2是含有739bp attP基因和不包括CMV启动子但有GFP报告基因的pGP(-)载体的质粒图谱。
图3是含有本发明的MJ1基因的pcMJ1载体的质粒图谱。
图4是含有624bp attL基因和CMV启动子的pcL载体的质粒图谱。
图5是含有404bp attR基因和不包括CMV启动子但有GFP报告基因的pGR(-)载体的质粒图谱。
图6是由于和PcB,pGP(-)以及pcMJ1共转染整合形成的pREC-I载体的质粒图谱。
图7是由于和PcL,pGR(-)以及pcMJ1共转染整合形成的pREC-E载体的质粒图谱。
图8是MJ1在293T细胞中表达的图像。
图9是共聚焦显微镜下显示的在293T细胞中整合结果的图像。
图10是显示没有MJ1的GFP表达的FACS数据。(Y轴是细胞数而X轴是GFP表达水平)
图11是显示有MJ1的GFP表达的FACS数据。(Y轴是细胞数而X轴是GFP表达水平)
图12是显示图10和11的量化数值的图表。
图13是共聚焦显微镜下显示的在293T细胞中切除结果的图像。
图14是显示attP和attB的套式缺失的模式图。
图15是由于attP和attB位点区域大小得到的整合效率的图表。
具体实施方式
通过下述具体实施例将更加具体地描述本发明。下述具体实施例是为了解释说明发明目的,而并不限制本发明的范围。
实施例1:细菌和细菌噬菌体的培养
在Todd Hewitt肉汤培养基中(THB:Difco Co.U.S.A),37℃静止培养粪肠球菌KBL703菌株和KBL707菌株(保藏号KFCC 12177)。E.Coli(DH5)在LB培养基于37℃振荡培养。使用紫外照射从粪肠球菌KBL703中诱导温和细菌噬菌体FC1并纯化。本发明所用细菌菌株和质粒如表1所示。
【表1】
菌株或质粒 | 特征 | |
Enterococcus faccalis(粪肠球菌) | KBL703 | FC1噬菌体的溶源性菌株 |
KBL707 | FC1噬菌体指示菌株 | |
Escherichia coli(大肠杆菌) | DH5á | supE44,lacU169,hsdR17,recA1,endA1,gyrA96,thi-1,relA1 |
质粒 | pcDNA3 | 哺乳动物细胞表达载体,Ampr,Neor,Col1的复制起点,CMV的启动子,T7,Sp6,SV40 |
pEGFP-N1 | 哺乳动物细胞表达载体,Kanr,Neor,pUC的复制起点,CMV的启动子,HSV,TK,SV40e,GEP exp | |
pG(-) | 缺失PCMV的pEGFP-N1 | |
pGP(+) | 带有739bp attP片段的pEGFP-N1 | |
pGP(-) | 带有739bp attP片段的pG(-) | |
pcB | 带有290bp attB片段的pcDNA3 | |
pGR(-) | 带有404bp attR片段的pG(-) |
pGR(+) | 带有404bp attR片段pEGFP-N1 | |
pcL | 带有624bp attL片段的pcDNA3 | |
pcMJ1 | 带有mj1的pcDNA3 |
实施例2:用于转染到动物细胞中的质粒的构建
为了合成大约1500bp的MJ1整合酶基因,290bp的attB基因和347bp的attL基因,使用细菌噬菌体FC1的DNA和KBL707以及KBL703的基因组DNA作为模板和如表2所示的引物进行PCR反应。在pcDNA3(Invitrogen Carsbad,CA)的BamHI和EcoRI位点或者HindIII和BamHI位点对PCR产物进行亚克隆而构建pcMJ1(图3),pcB(图1)和pcL(图4)。
通过从pEGFP-N1(ClonTech,Palo Alto,CA)上切除其上小的NdeI-BglII片段并使用klenow处理进行平末端自连接构建质粒pG(-)。通过对使用表2引物扩增的attP和attR的PCR产物进行亚克隆得到质粒pGP(-)(图2)和pGR(-)(图5)。本发明用作引物的合成寡核苷酸如表2所示。
【表2】
正义链 | 反义链 | |
MJ1 | CGGGATCCATGAAACGTGCAGCATTG | CGGAATTCACCGAATGCATGTTCGTA |
attP | CGGAATTCACCGAATGCATGTTCGTA | GCGTTAACTGCCAATATAGC |
attB | CGGCCATTGAATTAGGGTGTCGAAT | CGGATTGCCAGATGGATGAT |
attL | CGCAAGCTTGAAACGTTAAAAACTTTTAAT | CGGGATCCCGGCCATTGAATTAGGGTGTC |
attR | CGCAAGCTTCGGATTGCCAGATGGATGATT | CGGGATCCGAAGATCTTGTTCTCGAGCAT |
MJ1RT | GGAAATCAATTAGGGCTTTT | TCGTTATGCTTCTTGGAAAT |
GAPDH | CATCTCTGCCCCCTCTGCTG | CGACGCCTGCTTCACCACCT |
实施例3:转染和荧光显微分析
在追加了10%热-灭活胎牛血清(FBS)和100U/ml青霉素的Dulbecco改良的Eagle培养基(DMEM)100mm的平板中培养人胚肾细胞293T(American TypeCulture Collection,Manassas,Va)。细胞被分开到8个六孔培养板或60mm的平板上并生长直至约50%发生融合。在实施例2中制备的携带attP,attB和MJ1编码序列的质粒通过磷酸钙方法等量转染(6μg或3μg总DNA)。
转染了的细胞在盖玻片上生长并置于6孔培养板上。培育72h后,在共聚焦荧光显微镜下(Zeiss)检测细胞的GFP表达。转染效率由在被转染细胞中的荧光细胞百分数测定。
实施例4:反转录-PCR(RT-PCR)
使用TrIzol试剂(Invitrogen)从pcMJ1转染的293T细胞中提取总RNA。使用SUPERSCRIPT II RNase-反转录酶(Invitrogen)和pfu DNA聚合酶(Promega)进行反转录和PCR扩增。GAPDH基因表达水平作为内部对照和表2描述了引物的序列。PCR反应进行25个循环,条件是94℃5min,94℃20s,52℃30s和68℃40s。用1.5%琼脂糖凝胶对PCR产物进行分析和用溴化乙锭染色以显影。如图8所示,和内部对照基因GAPDH相比,MJ1的RNA表达水平显著提高。PCR产生了带有MJ1编码区域的特异引物对的320bp产物(图8)。RT-PCR显示整合酶MJ1在人细胞中异位表达。
实施例5:荧光激活细胞分选(FACS)分析
对GFP的表达进行FACS分析。在转染后72h,收集细胞,用3ml 5mM的EDTA/PBS重悬并冲洗两次,然后固定到300μl 0.1%BSA和0.05%NaN3/PBS中。用使用CellQuset程序(Becton Dickinson)的FACScan分析仪(Becton DickinsonImmunocytometry Systems)进行FACS分析。
下面,将对证明了上述提及的本发明实施例结果的实验结果进行公开。
实验例1:位点特异性重组或切除
不同结构可以控制整合反应(attP×attB)或者切除(attL×attR)。为了测定MJ1在整合反应中的作用,293T细胞和质粒pcB和pGP(-),以及和pcMJ1或者不和pcMJ1一起进行共转染。72小时后,用共聚焦显微镜观察细胞。只在由MJ1-介导整合产生的质粒pREC-I(图6)中,发生GFP的表达(图9A)。作为对比,在没有MJ1的共转染细胞中如预料一般未观察到GFP的表达(图9B)。为了显示每种情况都不是因为启动子,也分别对在有CMV启动子和没有CMV启动子下,和质粒pGP(+)或pGP(-)转染的细胞进行了共聚焦显微镜观察(图13)。如预期,在只用pGP(-)转染的细胞中没有观察到GFP的表达。为了调查整合效率,用质粒pcB和pGP(-)以及和MJ1表达质粒pcMJ1或者不和pcMJ1一起转染293T细胞并且在转染72小时后对细胞进行FACS分析。表明了在MJ1存在或不存在情况下转染细胞的荧光性的柱状图如图10和图11所示。在没有MJ1的情况下,和带有att位点的质粒一起转染的细胞显示396个荧光细胞(图10)。相反,在MJ1存在时,整合反应中的荧光细胞数为1237,所以如图12所示其差距达到大约4倍。
对于切除,带有attR(pGR(-))和attL(pGL)而不是pGP(-)的质粒和pcB共转染到293T细胞中。其结果是,与整合形成对比,没有观察到GFP表达(图13A)。为了证明此结果不是由启动子造成,用共聚焦显微镜分别对和带有或者不带有CMV启动子的质粒pGR(+)或者pGR(-)一起转染的细胞进行观察。pGR(+),而不是pGR(-)显示出GFP活性(图13B和13C)。这一结果表明推定的重组质粒pREC-E(图7)不是由MJ1产生。结果显示MJ1催化的切除不在attL和attR之间发生。
已证明在人细胞染色体外载体基础上,MJ1高效介导attP和attB之间位点特异性整合,但是MJ1-催化的切除在attR和attL之间并不发生。
实验例2:attB和attP之间最小区域的测定
在本发明之前,没有关于对MJ1起作用的attB和attP之间最小区域的报道。为了最小化这些att位点的作用区域,使用attB和attP的套式缺失克隆(图14)进行上述位点特异性整合分析系统的测定。在已知的MJ1结合位点周围通过套式缺失得到每个带有3bp保守核心序列的缺失位点的质粒。通过单链寡核苷酸退火得到含有不同长度的att位点的短的双链接头分子。使用这些缺失位点以代替pGP(-)和pcB质粒中的全长att位点,质粒和pcMJ1或不和pcMJ1共转染以及通过FACS分析测定整合效率。
结果是,图15显示了由于attP和attB位点区域大小得到的整合效率。和质粒pcMJ1一起或者不和其一起转染的细胞的差异由个数值%表示。整合效率的不随attP以及attB位点的减小而降低。然而,整合效率由54bp attP和48bp attB之间的整合效率逐渐降低。在最终的分析中,在50bp attP和44bp attB之间几乎所有整合作用都没有。这些结果证明attP和attB之间有效的最小区域是54bp(SEQ ID No.3中166-233碱基)和48bp(SEQ ID No.4中66-113碱基),和这一大小足够MJ1在整合中起作用。
(工业实用性)
整合酶MJ1在构建基因转移系统中比其它病毒载体或存在的基因转移系统有更多的优点。
第一,MJ1不需要任何粪肠球菌-特异性辅助因子进行位点特异性重组。相反,λ噬菌体在两种类型的重组都需要IHF,并且许多类型的重组都需要IHF,包括噬菌体HK022和HP1(Dorgai et al.,(1998)J.Mol.Biol.,277,1059-1070)。
本发明证明发生在人细胞attP和attB之间的位点特异性重组如观测的GFP表达一样,只需要整合酶MJ1而不需要任何辅助因子。
第二,不像其它来自噬菌体的同时介导整合和切除的整合酶(重组酶),整合酶MJ1只显示出整合的作用。有报道来自噬菌体HK022的整合酶(重组酶)可以在鼠NIH3T3细胞中进行整合(attP/attB)和切除(attL/attR)重组(kolot et al.,(1999)Mol.Biol.Rep.,26,207-213)。在本发明中,MJ1只介导整合,并且被整合的基因不被从宿主基因组中切除,所以期望的基因可以被连续表达。
第三,已显示attB和attP位点的最小区域48bp和54bp使得MJ1介导整合。似乎可以在与原始att序列(Thyagarajan et al.,(2000)Gene,244)高度相似的真核基因组自然发生的假-att位点完成这一整合。与att位点良好匹配的预期的稀有度可以限制整合到小量染色体假-att位点,这可以在染色体内部位置产生可用的整合频率。
除了这些优点,MJ1-介导的位点特异性整合系统对于基因转移系统的改进是有益的。由于这一系统使得插入的基因整合到宿主基因组DNA假位点而使得影响必须基因的表达或/和干扰宿主细胞机制的可能性必然降低。另外来自噬菌体的整合酶可以转移大于10kb的插入基因到靶位点,使得许多数十kb-大小的噬菌体聚集,由于噬菌体可以通过其本来的整合酶转移性将其全噬菌体基因组整合到细菌基因组。
序列表
11:38 2007-4-5<110>高丽大学校产学协力团
<120>含有MJ1基因的重组载体和使用该重组载体的位点特异性整合方法
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Claims (4)
1.一种含有编码整合酶的MJ1基因的重组载体,所述整合酶具有SED IDNo.2所示氨基酸序列,其中该整合酶介导在动物细胞中的位点特异性重组,而不介导切除。
2.根据权利要求1所述的重组载体,其中MJ1基因具有SED ID No.1的碱基序列和重组载体具有图3所示的质粒图谱。
3.一种在动物细胞中进行位点特异性重组的方法,包括用一含有根据权利要求1的MJ1基因的重组载体、一含有attB基因的重组载体以及一含有attP基因的重组载体共转染该动物细胞。
4.根据权利要求3的方法,其中所述重组载体是根据权利要求2的重组载体。
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR1020040107077 | 2004-12-16 | ||
KR1020040107077A KR100777025B1 (ko) | 2004-12-16 | 2004-12-16 | Mj1 유전자를 포함하는 재조합 벡터 및 이를 이용한부위 특이적 인테그레이션 방법 |
KR10-2004-0107077 | 2004-12-16 | ||
PCT/KR2005/004351 WO2006065103A1 (en) | 2004-12-16 | 2005-12-16 | Recombiant vector containing mj1 gene and method of site-specific integration using the same |
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CN101072878A true CN101072878A (zh) | 2007-11-14 |
CN101072878B CN101072878B (zh) | 2011-02-16 |
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JP (1) | JP2006166903A (zh) |
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CN110129279A (zh) * | 2019-04-24 | 2019-08-16 | 昆明理工大学 | 一种粪肠球菌噬菌体及其分离、纯化、富集和应用 |
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US6746870B1 (en) * | 1999-07-23 | 2004-06-08 | The Regents Of The University Of California | DNA recombination in eukaryotic cells by the bacteriophage PHIC31 recombination system |
CN1390934A (zh) * | 2001-06-08 | 2003-01-15 | 桂林华诺威基因药业有限公司 | 二类内含子靶向整合真核细胞染色体基因组上的特定序列 |
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KR20060068401A (ko) | 2006-06-21 |
US20060134788A1 (en) | 2006-06-22 |
CN101072878B (zh) | 2011-02-16 |
US7273757B2 (en) | 2007-09-25 |
KR100777025B1 (ko) | 2007-11-28 |
JP2006166903A (ja) | 2006-06-29 |
WO2006065103A1 (en) | 2006-06-22 |
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