CN111518785B - 一种高活性piggyBac转座酶及其应用 - Google Patents
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Abstract
本发明的目的在于提供一种高活性piggyBac转座酶及其应用。一种piggyBac转座酶，其特征在于：氨基酸位点165位的甘氨酸改变为半胱氨酸，或氨基酸位点282位的蛋氨酸改变为苏氨酸，或这两个位点同时发生上述改变。本发明的高活性piggyBac转座酶，提高了piggyBac转座子系统的转座效率。
Description
技术领域
本发明涉及一种高活性piggyBac转座酶，本发明还涉及piggyBac转座酶的应用，属于生物技术领域。
背景技术
基因转移技术在基因治疗和非人转基因动物建立中被广泛使用。常用的介导稳定转基因的媒介包括：病毒载体，如：反转录病毒、慢病毒和腺相关病毒；和非病毒载体，如：PhiC31整合酶和各类转座子系统。基于病毒载体的转基因技术在实际操作中涉及病毒包装，对操作者和操作环境有较高的技术与安全要求。此外，在哺乳动物体内使用病毒载体介导转基因可能导致免疫反应，存在安全和排斥问题。非病毒载体是一种相对安全和简便的转基因媒介。但现有的非病毒载体普遍存在效率低下的问题。
piggyBac转座子系统是现有在动物转基因应用中效率最高的非病毒载体。piggyBac转座子早先于粉斑夜蛾Trichoplusia ni的基因组中被发现，大小为2472bp，两端包含反向末端重复序列ITR，中间包含编码594个氨基酸的转座酶。piggyBac转座子类属于DNA转座子，通过剪切和粘贴机制可以由位于外源DNA或内源基因组的起始位点切离，并随机插入基因组中的另一位点。piggyBac转座子插入靶位点的序列为四核苷酸的TTAA。piggyBac转座子在切离TTAA时一般不会改变原位点及原位点的附近序列，即所谓无痕转座。2005年，复旦大学研究人员发现，piggyBac转座子能在体外培养哺乳动物细胞及小鼠基因组中转座，并能携带至多10kb的外源基因而不影响其转座效率。基于piggyBac转座子能介导随机插入，无痕转座，携带大片段外源基因，以及在哺乳动物细胞中转座的特点，其在基因治疗、非人转基因哺乳动物建立和随机插入突变引入等邻域中的潜在应用有待被进一步开发，而提高piggyBac转座子系统的效率在这其中至关重要。
发明内容
本发明的目的在于提供一种高活性piggyBac转座酶及其应用。
本发明采用了如下技术方案：
一种piggyBac转座酶，其特征在于：氨基酸位点165位的甘氨酸改变为半胱氨酸，或氨基酸位点282位的蛋氨酸改变为苏氨酸，或这两个位点同时发生上述改变。
本发明还提供上述piggyBac转座酶，在制备介导缺陷piggyBac转座子转座插入HEK293细胞基因组的试剂中的应用。
本发明还提供上述piggyBac转座酶在制备基因整合试剂中的应用。
本发明还提供上述piggyBac转座酶，在制备无痕基因修复的试剂中的应用，其特征在于：piggyBac转座酶为PBase(G165C/M282T)。
本发明还提供上述piggyBac转座酶，在建立转基因非人动物中的应用，其特征在于：包括将编码新型高活性piggyBac转座酶PBase(G165C/M282T)和携带Katshka远红外荧光蛋白编码序列的piggyBac转座子同时显微注射入小鼠受精卵内的步骤。
本发明还提供上述piggyBac转座酶，在制备建立转基因非人动物的试剂中的应用。
进一步，piggyBac转座酶，在制备建立转基因非人动物的试剂中的应用，其中，试剂中还包含携带Katshka远红外荧光蛋白编码序列的piggyBac转座子。
本发明还提供上述高活性piggyBac转座酶在制备基因代偿治疗所需试剂中的应用，其特征在于：包括制备携带新型高活性piggyBac转座酶与携带凝血因子IX的缺陷piggyBac转座子的裸DNA的步骤。
发明的有益效果
本发明的高活性piggyBac转座酶，提高了piggyBac转座子系统的转座效率，从而使得piggyBac转座子能更高效地被用于基因治疗、稳定转基因细胞系和转基因非人动物建立、以及在培养细胞和非人动物基因组中引入插入突变。
附图说明
图1是本发明高活性piggyBac转座酶PBase的氨基酸序列示意图。
图2是筛选获得高活性piggyBac转座酶的技术方案设计图。
图3是本发明高活性piggyBac转座酶的一个实施例。
图4是本发明高活性piggyBac转座酶应用于基因治疗的一个实施例。
图5是本发明高活性piggyBac转座酶应用于基因治疗的另一个实施例。
图6是本发明高活性piggyBac转座酶应用于建立转基因非人动物的一个实施例。
具体实施方式
以下通过具体实施方式来详细说明本发明的技术方案。
本发明提高piggyBac转座酶转座效率所采用的实施方式是利用易错聚合酶链式反应error-prone PCR对野生型piggyBac转座酶的编码序列进行随机突变，获得大量携带不同氨基酸非保守改变的piggyBac转座酶突变体。
获得piggyBac转座酶突变体的具体步骤是：
1)利用引物PBaseF序列见SEQ ID No.1和PBaseR序列见SEQ ID No.2对野生型piggyBac转座酶序列运用易错聚合酶链式反应进行扩增，反应体系如表1所示。反应条件为：变性95℃，1分钟；退火60℃，1分钟；延伸72℃，3分钟；循环数为5。
2)将扩增得到的产物用EcoRI酶进行酶切，并装入pCAG-EGFP质粒(购自Addgene)的两个EcoRI位点间，从而获得携带piggyBac转座酶突变体的表达载体。
表1
上述实施方式中所涉及的质粒抽提，质粒转化，大肠杆菌培养，酶切和连接为本领域人员所熟知的实验。常规实验条件可参照M.R.格林和J.萨姆布鲁克编著的《分子克隆实验指南》第四版。
1.筛选高活性piggyBac转座酶
在一优选实施例中，将携带非保守氨基酸改变的piggyBac转座酶与携带Blasticidine(Blast)抗药筛选标记的缺陷piggyBac转座子的核酸各1μg与10μLLipofectamine 2000脂质体(购自Invitrogen)混合于250μLOpti-MEM培养基中。将核酸脂质体混合液加入含有4x105单层培养的HEK293细胞的培养皿内。
在一些备选的实施例中，携带非保守氨基酸改变的piggyBac转座酶与携带抗药筛选标记的缺陷piggyBac转座子的核酸可以通过磷酸钙转染、聚乙二醇-聚乙烯亚胺共聚物转染、或电穿孔转染导入体外培养的动物细胞。
在另一些备选的实施例中，携带非保守氨基酸改变的piggyBac转座酶与携带抗药筛选标记的缺陷piggyBac转座子的核酸可以被装入病毒载体。这里的病毒载体包括但不限于腺病毒、腺相关病毒，反转录病毒，慢病毒和单纯疱疹病毒。随后，对携带转座子系统的病毒进行包装，并感染导入靶细胞。
将成功被导入转座子的HEK293细胞用胰酶处理收集，并做1：10稀释培养，同时在培养基中加入5μg/ml Blasticidine(购自Invitrogen)，药物筛选两周，得到携带稳定缺陷piggyBac转座子插入的细胞克隆。用10％中性缓冲福尔马林固定液固定细胞，并用台盼蓝染色对细胞克隆进行计数。筛选获得高活性piggyBac转座酶的技术方案设计图如图2所示，转座酶的活性决定了细胞克隆数。
如图1所示，本实施方式筛选得到的新型高活性piggyBac转座酶为氨基酸位点165位的甘氨酸改变为半胱氨酸，G165C，GGT变为TGT，见SEQ ID No.3，或氨基酸位点282位的蛋氨酸改变为苏氨酸，M282T，ATG变为TTG，见SEQ ID No.4，或这两个位点同时发生上述改变G165C/M282T，见SEQ ID No.5。
如图3所示，本发明的新型高活性piggyBac转座酶PBase(G165C)、PBase(M282T)和PBase(G165C/M282T)的转座效率高于野生型piggyBac转座酶，介导缺陷piggyBac转座子高效转座插入HEK293细胞基因组，产生更多携带稳定缺陷piggyBac转座子插入的细胞克隆。
上述高活性piggyBac转座酶PBase，G165C/M282T的编码序列可以根据特定动物细胞类型选择优选密码子。
在一个针对小鼠细胞的优选例中，新型高活性piggyBac转座酶的编码序列为SEQID No.6。
在一个针对大鼠细胞的优选例中，新型高活性piggyBac转座酶的编码序列为SEQID No.7。
在一个针对人细胞的优选例中，新型高活性piggyBac转座酶的编码序列为SEQ IDNo.8。
2.高活性piggyBac转座酶的应用
本实施方式中高活性piggyBac转座酶能与缺陷piggyBac转座子协同使用。
2.1高活性piggyBac转座酶在制备基因代偿治疗所需试剂中的应用。
本发明高活性piggyBac转座酶在基因代偿的试剂应用的一个实施例中，将携带新型高活性piggyBac转座酶与携带凝血因子IX的缺陷piggyBac转座子的裸DNA各25μg与3mL乳酸林格氏缓冲液混合。随后将混合液通过液压尾静脉注射在7秒内快速注射入小鼠体内，转染肝脏。在注射后5天、30天和60天，分别通过尾静脉采取血样，加入肝素抗凝获得血浆样品。血浆中凝血因子IX水平由酶联免疫吸附试验ELISA(购自Abcam)测定，具体的实验条件为厂商所建议的条件。如图4所示，本发明的新型高活性piggyBac的转座酶相较于野生型piggyBac转座酶能更高效地介导治疗基因整合，持久稳定地提高血浆中凝血因子IX水平。
在另一些实施方式中，新型高活性piggyBac转座酶与携带治疗基因的缺陷piggyBac转座子的核酸通过纳米颗粒导入被治疗对象体内。
在另一些实施方式中，新型高活性piggyBac转座酶与携带治疗基因的缺陷piggyBac转座子的核酸可以被装入病毒载体。这里的病毒载体包括但不限于腺病毒、腺相关病毒，反转录病毒，慢病毒和单纯疱疹病毒。随后，对携带转座子系统的病毒进行包装，并感染导入被治疗对象体内。
在另一些实施方式中，新型高活性piggyBac转座酶与携带治疗基因的缺陷piggyBac转座子的核酸可以通过上呼吸道，肌肉注射，静脉注射导入被治疗对象体内。
在另一些实施方式中，新型高活性piggyBac转座酶与携带治疗基因的缺陷piggyBac转座子的核酸可以先行导入离体的成纤维细胞或血液细胞，包括：造血干细胞或各祖细胞，随后将细胞植入被治疗对象体内。
在另一些实施方式中，缺陷piggyBac转座子可携带的核酸片段为对治疗有价值的基因，包括但不限于腺苷脱氨酶，凝血因子VIII，β-球蛋白，血红蛋白，抗肌萎缩蛋白，α-抗胰蛋白酶，低密度蛋白受体，囊性纤维化跨膜通道调节因子，嵌合抗原受体，各类细胞因子，自杀基因和反义序列。
2.2基因无痕修复
piggyBac转座子插入靶位点的序列为四核苷酸的TTAA。piggyBac转座子在切离TTAA时一般不会改变原位点及原位点的附近序列，即所谓无痕转座。利用这一特点，piggyBac转座子系统可以与精准基因编辑工具，包括：锌指核酸酶ZFN，转录激活因子样效应物核酸酶TALEN和成簇常间隔短回文重复序列及相关核酸酶CRISPR/Cas9，相结合运用于致病基因突变无痕修复。
本发明高活性piggyBac转座酶应用于基因代偿治疗的一个实施例中，如图5所示，将编码精准基因编辑工具系统的核酸和携带缺陷piggyBac转座子及基因修复供体的模板核酸同时导入带致病基因突变的靶细胞。上面piggyBac转座子被置于修复供体基因组序列旁的TTAA位点内，并携带正向药物筛选标记，如:puromycin抗性基因，和反向药物筛选标记，如：TK，thymidine kinase基因。携带缺陷piggyBac转座子及基因修复供体的核酸通过同源重组被靶向导入基因组内，原致病基因突变被置换，正向药物筛选得到靶基因修复的细胞克隆。随后，导入新型高活性piggyBac转座酶将缺陷piggyBac转座子从TTAA处无痕切离，反向药物筛选得到致病基因突变无痕修复的细胞克隆。
3.转基因非人动物建立
本发明高活性piggyBac转座酶应用于转基因非人动物建立的一个实施例中，转基因小鼠可由携带转座子插入突变的受精卵产生获得，如图6所示，具体的步骤是：将编码新型高活性piggyBac转座酶和携带Katshka远红外荧光蛋白编码序列的缺陷piggyBac转座子的核酸，按摩尔比1：1的比例混合。取混合DNA样品5ng/μL，通过原核显微注射同时导入离体的小鼠受精卵的基因组内。用100mg/kg ketamine和10mg/kg xylazine麻醉假孕母鼠，手术打开腹腔，暴露输卵管。在输卵管内，植入10-15枚被注射的受精卵，随后进行手术缝合。在哺育得到的后代中，获得携带缺陷转座子插入的阳性小鼠。在绿色光照射下，阳性小鼠体表产生远红外荧光，可在荧光解剖镜下观察到。
在本发明应用于转基因非人动物建立的另一些实施例中，编码新型高活性piggyBac转座酶的核酸DNA可先在体外被反转录成信使RNA，随后与缺陷piggyBac转座子的核酸同时被显微注射入受精卵。
在本发明应用于转基因非人动物建立的另一些实施例中，转基因小鼠可由携带转座子插入突变的精原干细胞或胚胎干细胞产生获得。
在本发明应用于转基因非人动物建立的另一些实施例中，缺陷piggyBac转座子可携带的核酸片段包括但不限于启动子，调控元件，报告基因，基因编码序列，聚腺苷酸转录终止信号，基因敲低构建，基因捕获构建。
Claims (8)
1.一种piggyBac转座酶，其特征在于：
氨基酸位点165位的甘氨酸改变为半胱氨酸，序列如SEQ IDNo.3所示；
或氨基酸位点282位的蛋氨酸改变为苏氨酸，序列如SEQ IDNo.4所示；
或这两个位点同时发生上述改变，序列如SEQ ID No.5所示。
2.如权利要求1所述的piggyBac转座酶，在制备介导缺陷piggyBac转座子转座插入HEK293细胞基因组的试剂中的应用。
3.如权利要求1所述的piggyBac转座酶在制备基因整合试剂中的应用。
4.如权利要求1所述的piggyBac转座酶，在制备无痕基因修复的试剂中的应用，其特征在于：
piggyBac转座酶为PBase，其序列如SEQ ID No.6，或SEQ IDNo.7，或SEQ ID No.8所示。
5.如权利要求4所述的piggyBac转座酶，在建立转基因小鼠中的应用，其特征在于：
包括将编码新型高活性piggyBac转座酶PBase和携带Katshka远红外荧光蛋白编码序列的piggyBac转座子同时显微注射入小鼠受精卵内的步骤。
6.如权利要求1所述的piggyBac转座酶，在制备建立转基因小鼠的试剂中的应用。
7.如权利要求6所述的应用，其特征在于：所述试剂中还包含携带Katshka远红外荧光蛋白编码序列的piggyBac转座子。
8.如权利要求4所述的高活性piggyBac转座酶在制备基因代偿治疗所需试剂中的应用，其特征在于：包括制备携带新型高活性piggyBac转座酶与携带凝血因子IX的缺陷piggyBac转座子的裸DNA的步骤。
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