CN114591987A - 一种用于检测活细胞中mTORC1活性的遗传编码荧光生物传感器及其构建方法 - Google Patents
一种用于检测活细胞中mTORC1活性的遗传编码荧光生物传感器及其构建方法 Download PDFInfo
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Abstract
本发明公开了一种用于检测活细胞中mTORC1活性的遗传编码荧光生物传感器及其构建方法,其包括:融合体基因结构A、融合体基因结构B、用于连接融合体基因结构A和融合体基因结构B的连接部。融合体基因结构A是由mTORC1磷酸化底物的编码基因、标记蛋白基因和寡聚化标签基因组成的融合基因;融合体基因结构B是由所述mTORC1磷酸化底物的结合蛋白编码基因和寡聚化标签基因组成的融合基因;连接部为自剪切多肽的编码基因。该传感器可对抑制态mTORC1进行快速有效地活细胞检测,并可广泛用于mTORC1活性抑制剂及调控基因的筛选。
Description
技术领域
本发明属于生物传感器领域,具体涉及一种用于检测活细胞中mTORC1活性的遗传编码荧光生物传感器及其构建方法。
背景技术
哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)是真核生物中一种关键的代谢调节激酶,具有丝氨酸和苏氨酸激酶活性(Ser/Thr kinase)。mTOR通过整合营养物质、生长因子和能量代谢等细胞内外信号参与基因转录、蛋白质翻译和核糖体合成等多种生物学功能调控,从而进一步调节细胞的代谢生长、凋亡和自噬等核心生理过程,因此,也被称为细胞的“大脑”。mTOR及其介导的信号通路紊乱与人类代谢、寿命、癌症及癫痫等重大疾病的形成和发展密切相关。
介导mTOR生物学效应的分子分两种:一种是哺乳动物雷帕霉素靶标蛋白复合物1(The mammalian Target of Rapamycin complex 1,mTORC1),另一种是哺乳动物雷帕霉素靶标蛋白复合物2(mTORC2)。关于mTORC1信号的生理病理研究相对广泛,mTORC1复合物包含激酶mTOR和调控蛋白RICTOR及支架蛋白mLST8等功能,因此,mTORC1活性的变化是相关疾病生理病理变化的重要判断依据。
相关技术中,大多数mTORC1检测技术主要是利用特异性抗体通过蛋白印迹和免疫组织化学手段检测体外提取蛋白或固定组织细胞中mTORC1底物磷酸化水平(如磷酸化S6K或磷酸化4EBP1)来判断mTORC1的活性,但普遍存在检测耗时长、无法实现高通量快速检测、检测成本高、受制于抗体特异性或对于检测人员的操作要求高等问题。
因此,开发一种能够克服上述缺点,并可以实现在活细胞中通过荧光显微镜快速、有效、实时检测mTORC1活性的检测方法及生物传感器,对于细胞水平上的高通量mTORC1抑制药物或调控物的筛选和鉴定具有极为重要的意义。
发明内容
本发明旨在至少解决上述现有技术中存在的技术问题之一。为此,本发明提出一种用于检测活细胞中mTORC1活性的遗传编码荧光生物传感器及其构建方法,该传感器可以作为检测活细胞mTORC1活性的传感器,其在mTORC1失活条件下从弥散态形成聚集点是其响应mTORC1活性的显著特征,从而可进一步用于mTORC1抑制剂的筛选以及mTORC1活性调控基因的筛查。
本发明的第一个方面,提供一种生物传感器,该生物传感器包括:
融合体基因结构A、融合体基因结构B、用于连接融合体基因结构A和融合体基因结构B的连接部;
所述融合体基因结构A是由mTORC1磷酸化底物的编码基因、标记蛋白基因和寡聚化标签基因组成的融合基因;
所述融合体基因结构B是由所述mTORC1磷酸化底物的结合蛋白编码基因和寡聚化标签基因组成的融合基因;
所述连接部为自剪切多肽的编码基因。
根据本发明的第一个方面,在本发明的一些实施方式中,所述mTORC1磷酸化底物为4EBP1。
4EBP1(真核细胞翻译起始因子4E结合蛋白1)是mTOR的下游分子之一,主要其调控蛋白质的翻译功能。Gene ID:1978(人源)或13685(人源)。
根据本发明的第一个方面,在本发明的一些实施方式中,所述mTORC1磷酸化底物的结合蛋白为EIF4E。
EIF4E(真核翻译起始因子4E)是一种蛋白质编码基因,可别信使RNA 5'末端的7-甲基鸟苷帽结构。Gene ID:1977(人源)或13684(人源)。
在本发明的一些实施方式中,所用的4EBP1和EIF4E可采用任意物种来源的的4EBP1和EIF4E。在本发明的一些优选实施方式中,所述4EBP1和EIF4E为人源或鼠源的4EBP1和EIF4E。在本发明的一些更优选实施方式中,所述4EBP1和EIF4E为人源4EBP1和EIF4E。
根据本发明的第一个方面,在本发明的一些实施方式中,所述寡聚化标签包括六聚体标签(HoTag3)、四聚体标签(HoTag6)中的至少一种。当然,本领域技术人员可以根据实际使用需求,合理使用其他的寡聚化蛋白标签进行替代,如HoTag2、HoTag7等。
根据本发明的第一个方面,在本发明的一些实施方式中,所述标记蛋白基因包括荧光蛋白。
在本发明的一些实施方式中,所述荧光蛋白为单体荧光蛋白。所述单体荧光蛋白包括但不限于AcGFP、mNG、mBFP和mCherry。
根据本发明的第一个方面,在本发明的一些实施方式中,所述自剪切多肽包括任意具有“自我剪切”的多肽(P2A),包括但不限于T2A、E2A和F2A。或使用内部核糖体进入位点序列(IRES)来替代,从而实现能使一个表达载体表达一条转录本,产生多种蛋白的功能。
在本发明的一些优选实施方式中,所述生物传感器具体由3部分组成:融合体基因结构A、融合体基因结构B和用于连接结构A和结构B的连接部。其中,融合体基因结构A是由4EBP1编码基因、单体荧光蛋白编码基因mFP和六聚体标签基因HOTag3组成的融合基因。融合体基因结构B是由4EBP1结合蛋白EIF4E基因和四聚体标签基因HOTag6组成的融合基因。连接部为自剪切多肽P2A序列的编码基因。
在本发明的一些实施方式中,所述生物传感器的氨基酸序列如SEQ ID NO:1所示。
本发明中的生物传感器经试验验证,可在活细胞中通过荧光显微镜快速、有效、实时检测mTORC1活性受抑制的情况,并将其应用于细胞水平较高通量mTORC1抑制药物或调控物的筛选和鉴定。
在相关技术中,基于活细胞激酶活性的遗传编码报告***仅存在TORCAR和AIMTOR***,但上述两种***中存在信噪比低等诸多缺陷,因此,发明人基于上述生物传感器开发得到活细胞遗传编码的mTORC1活性报告***(也可称mTORC1失活报告***,mTOR inactivereporter,mTIR)。其作为一种荧光生物传感器用于mTORC1在活细胞内的实时活性检测,可应用于细胞水平高通量mTORC1信号调控物和相关疾病先导化合物的筛选和鉴定,是目前首个可应用于高通量活细胞mTORC1活性抑制药物筛选的生物传感器。
本发明的第二个方面,提供编码本发明第一个方面所述生物传感器的核酸分子。
在本发明的一些实施方式中,所述核酸分子的核苷酸序列如SEQ ID NO:2所示。
在本发明的一些实施方式中,所述生物传感器为具有红色荧光的mTIR传感器,其红色荧光来自其中的荧光蛋白mCherry编码基因。
所述生物传感器的检测原理为:将该mTIR传感器的基因序列克隆至细胞表达载体后,通过病毒转导或基因转染的方式将载体送入细胞中,使其开始表达。该mTIR传感器能报道细胞内mTORC1活性的动态变化,当mTORC1失活时,mTORC1在细胞内蛋白磷酸酶的作用下被mTIR传感器表达出的4EBP1去磷酸化,而带有寡聚体标签的非磷酸化4EBP1荧光融合蛋白和其结合蛋白EIF4E通过蛋白质多价相互作用,形成高分子量蛋白聚集体,在荧光显微镜下用荧光蛋白相应的激发光激发,会在一定的发射光光谱范围内可接收到明亮的荧光聚集体信号。而当细胞内mTORC1被激活时,4EBP1中的多个磷酸化位点会被mTORC1磷酸化,从而使得其不能跟EIF4E相互作用,不能形成高分子量蛋白聚集体,此时,使用荧光显微镜检测到的是呈弥散状的荧光信号。因此,可以通过聚集体的荧光信号呈弥散或聚集状的状态来判断细胞内的mTORC1的活性情况,继而发挥传感器报告基因的功能。
本发明的第三个方面,提供一种用于检测mTORC1活性的产品。
所述产品包括如下(1)~(6)中的至少一种:
(1)含有SEQ ID NO:1所示核酸分子的表达盒;
(2)含有SEQ ID NO:1所示核酸分子的载体;
(3)含有(1)中所述表达盒的载体;
(4)含有SEQ ID NO:1所示核酸分子的重组微生物;
(5)含有(1)中所述表达盒的重组微生物;
(6)含有(2)中所述载体的重组微生物。
本发明的第四个方面,提供本发明第一个方面所述的生物传感器在制备活细胞mTORC1活性检测产品中的应用。
在本发明实施例中,发明人使用mTORC1药物或生理抑制、以及遗传操控失活两种试验进行概念确证。发现无论是生理刺激、药物处理还是基因调控操作,上述mTIR传感器都能作为检测活细胞mTORC1活性的传感器,其在mTORC1失活条件下从弥散态形成聚集点是其响应mTORC1活性的显著特征,这一特征使得上述mTIR传感器可作为mTORC1活性传感器的证据。
本发明的第五个方面,提供本发明第一个方面所述的生物传感器在mTORC1活性抑制剂筛选中的应用。
在本发明实施例中,发明人使用上述mTIR传感器能够很好的判断出药物是否具有mTORC1抑制效果,阴性对照与阳性对照也符合理论要求,没有出现检测失败的问题。而且发明人使用该mTIR传感器对2000多个FDA批准药物进行初步筛选,获得了至少15个能在活细胞水平引起mTIR响应的药物,进一步使用免疫印迹检测也确认了这些药物能抑制mTORC1的活性,说明上述mTIR传感器能作为一个药物筛选工具应用于跟mTORC1相关疾病的药物筛选。
本发明的第六个方面,提供本发明第一个方面所述的生物传感器在制备mTORC1活性调控基因筛选平台中的应用。
在本发明实施例中,发明人以Raptor和RheB基因敲低为例,发现上述mTIR传感器的检测结果准确,其能感应调控mTORC1活性的基因下调或缺失,可在细胞水平上对其药物效果或抑制效果进行很好的确证,可大大减少通过免疫印迹来验证的工作量并提高其检测的实时性和准确性。
本发明的第七个方面,提供本发明第一个方面所述的生物传感器的使用方法,包括如下步骤:
将本发明第一个方面所述的生物传感器转入待测细胞中,使用荧光显微镜观测荧光情况,若荧光呈现聚集状,说明细胞内的mTORC1失活或受到抑制。若荧光呈现弥散状,说明细胞内的mTORC1处于激活状态或被激活
本发明的有益效果是:
(1)本发明中的mTIR传感器通过结合4EBP1-mCherry-HOTag3和EIF4E-HOTag6两个融合体基因结构,通过特定的组合次序和基因融合,可对抑制态mTORC1进行快速有效地活细胞检测。
(2)本发明中的mTIR传感器具有较好的灵活性,可以通过更换荧光蛋白来实现不能情况下的检测,而且,该mTIR传感器经过试验验证能够有效用于活细胞中,并可以进一步用于筛选抑制mTORC1活性的药物或者相关mTORC1活性调控基因,检测难度和要求低,具有极高的应用价值。
附图说明
图1为本发明实施例中的mTIR传感器结构示意图。
图2为本发明实施例中的mTIR传感器检测原理图。
图3为本发明实施例中的mTIR传感器的表达载体图谱。
图4为使用荧光显微镜观测转入mTIR传感器的HEK293细胞、Hela细胞和U2OS细胞的荧光图像(A)和免疫印迹图(B)。
图5为本发明实施例中的对照组、血清饥饿组、雷帕霉素组以及必需氨基酸饥饿组的检测结果。
图6为Torin1处理后的含有mTIR传感器的HEK293细胞、Hela细胞和U2OS细胞的检测结果。
图7为mTIR传感器对mTOR基因敲降的响应,其中,包括:sh-mTOR敲低与对照组的免疫印迹对比(A)以及荧光成像情况(B)。
图8为mTIR传感器对不同加药组的荧光成像情况,包括:阴性对照(A)、阳性对照(B)和候选mTORC1抑制剂的代表性结果(C)。
图9为不同敲低组的荧光图像。
图10为不同敲低组的免疫印迹图(A)和mTIR阳性细胞百分比统计图(B)。
具体实施方式
为了使本发明的发明目的、技术方案及其技术效果更加清晰,以下结合具体实施方式,对本发明进行进一步详细说明。应当理解的是,本说明书中描述的具体实施方式仅仅是为了解释本发明,并非为了限定本发明。
所使用的实验材料和试剂,若无特别说明,均为常规可从商业途径所获得的耗材和试剂。
一种遗传编码荧光生物传感器(mTIR传感器)
如图1所示,本发明实施例中的mTIR传感器主要由3部分组成:融合体基因结构A(图1中的A部分)、融合体基因结构B(图1中的B部分)和用于连接结构A和结构B的连接部(图1中的连接部)。
其中,融合体基因结构A是由4EBP1编码基因、单体荧光蛋白编码基因mFP和六聚体标签基因HOTag3组成的融合基因。
融合体基因结构B是由4EBP1结合蛋白EIF4E基因和四聚体标签基因HOTag6组成的融合基因。
连接部为自剪切多肽P2A序列的编码基因。
最终得到的遗传编码荧光生物传感器(mTIR传感器)的氨基酸序列具体为:
MYPYDVPDYAMSGGSSCSQTPSRAIPATRRVVLGDGVQLPPGDYSTTPGGTLFSTTPGGTRIIYDRKFLMECRNSPVTKTPPRDLPTIPGVTSPSSDEPPMEASQSHLRNSPEDKRAGGEESQFEMDIGGSGSGGGTPVATMVS KGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVK HPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERM YPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGM DELYKSGLRSGSGSAGGSAGGSAGGSAGGSAGGSAGGSAGGSRGEIAKSLKEIAKSLKEIAWSLKEIAKSLKGGTEGRGSLLTCGDVEENPGPKLDYKDDDDKMATVEPETTPTPNPPTTEEEKTESNQEVANPEHYIKHPLQNRWALWFFKNDKSKTWQANLRLISKFDTVEDFWALYNHIQLSSNLMPGCDYSLFKDGIEPMWEDEKNKRGGRWLITLNKQQRRSDLDRFWLETLLCLIGESFDDYSDDVCGAVVNVRAKGDKIAIWTTECENREAVTHIGRVYKERLGLPPKIVIGYQSHADTATKSGSTTKNRFVVVDGSGSAGGSAGGSAGGSAGGSAGGSAGGSAGGSRTLREIEELLRKIIEDSVRSVAELEDIEKWLKKI(SEQ ID NO:1)。
其对应的核苷酸序列具体为:
5’-ATGTACCCTTATGATGTGCCAGATTATGCCATGTCCGGGGGCAGCAGCTGCAGCCAGACCCCAAGCCGGGCCATCCCCGCCACTCGCCGGGTGGTGCTCGGCGACGGCGTGCAGCTCCCGCCCGGGGACTACAGCACGACCCCCGGCGGCACGCTCTTCAGCACCACCCCGGGAGGTACCAGGATCATCTATGACCGGAAATTCCTGATGGAGTGTCGGAACTCACCTGTGACCAAAACACCCCCAAGGGATCTGCCCACCATTCCGGGGGTCACCAGCCCTTCCAGTGATGAGCCCCCCATGGAAGCCAGCCAGAGCCACCTGCGCAATAGCCCAGAAGATAAGCGGGCGGGCGGTGAAGAGTCACAGTTTGAGATGGACATTGGCGGATCTGGCAGCGGTGGAGGCACACCGGTCGCCACCATGGTGAGCAAGGGCGAGGAGG ATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGA GATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCC CTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACA TCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGT GGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTC CCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACG GCGCCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGTCAAGACCAC CTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCACCTCCCACAAC GAGGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACA AGTCCGGACTCAGATCTGGCTCCGGCAGTGCTGGTGGTAGTGCTGGTGGTAGTGCTGGTGGTAGTGCTGGTGGCAGTGCTGGTGGTAGTGCTGGTGGTAGTGCTGGTGGCTCTAGAGGCGAGATCGCCAAGTCCCTGAAGGAGATCGCCAAGTCCCTCAAAGAAATTGCTTGGTCTCTCAAAGAGATAGCAAAGTCACTAAAGGGCGGTACCGAGGGCAGAGGAAGTCTGCTAACATGCGGTGACGTCGAGGAGAATCCTGGCCCAAAGCTTGACTACAAGGACGATGACGATAAGATGGCGACTGTCGAACCGGAAACCACCCCTACTCCTAATCCCCCGACTACAGAAGAGGAGAAAACGGAATCTAATCAGGAGGTTGCTAACCCAGAACACTATATTAAACATCCCCTACAGAACAGATGGGCACTCTGGTTTTTTAAAAATGATAAAAGCAAAACTTGGCAAGCAAACCTGCGGCTGATCTCCAAGTTTGATACTGTTGAAGACTTTTGGGCTCTGTACAACCATATCCAGTTGTCTAGTAATTTAATGCCTGGCTGTGACTACTCACTTTTTAAGGATGGTATTGAGCCTATGTGGGAAGATGAGAAAAACAAACGGGGAGGACGATGGCTAATTACATTGAACAAACAGCAGAGACGAAGTGACCTCGATCGCTTTTGGCTAGAGACACTTCTGTGCCTTATTGGAGAATCTTTTGATGACTACAGTGATGATGTATGTGGCGCTGTTGTTAATGTTAGAGCTAAAGGTGATAAGATAGCAATATGGACTACTGAATGTGAAAACAGAGAAGCTGTTACACATATAGGGAGGGTATACAAGGAAAGGTTAGGACTTCCTCCAAAGATAGTGATTGGTTATCAGTCCCACGCAGACACAGCTACTAAGAGCGGCTCCACCACTAAAAATAGGTTTGTTGTTGTCGACGGCTCCGGCAGTGCTGGTGGTAGTGCTGGTGGTAGTGCTGGTGGTAGTGCTGGTGGCAGTGCTGGTGGTAGTGCTGGTGGTAGTGCTGGTGGCTCTAGAACCTTGAGAGAAATCGAGGAATTGTTGAGGAAAATCATTGAAGACAGCGTGCGCAGTGTGGCCGAGCTGGAGGACATTGAGAAGTGGCTGAAGAAAATCTAA-3’(SEQ ID NO:2)。
其中,SEQ ID NO:1和SEQ ID NO:2中标有下划线的部分为单体荧光蛋白编码基因的氨基酸序列和核苷酸序列。
在本实施例中,该mTIR传感器为具有红色荧光的mTIR传感器,其红色荧光来自其中的荧光蛋白mFP编码基因。在本实施例中,荧光蛋白mFP编码基因使用的是mCherry荧光蛋白的基因序列(SEQ ID NO:1和SEQ ID NO:2中标有下划线的部分),当然,本领域技术人员也可以使用任意的单体荧光蛋白进行替换,以获得不同的荧光效果,但该替换并不会影响该mTIR传感器的生物学检测效果。
该mTIR传感器的检测原理如图2所示。
将上述mTIR传感器的基因序列克隆至细胞表达载体后,通过病毒转导或基因转染的方式将载体送入细胞中,使其开始表达。mTIR传感器能报道细胞内mTORC1活性的动态变化,当mTORC1失活时,mTORC1在细胞内蛋白磷酸酶的作用下被mTIR传感器表达出的4EBP1去磷酸化,而带有寡聚体标签的非磷酸化4EBP1荧光融合蛋白和其结合蛋白EIF4E通过蛋白质多价相互作用,形成高分子量蛋白聚集体,在荧光显微镜下用荧光蛋白相应的激发光激发,会在一定的发射光光谱范围内可接收到明亮的荧光聚集体信号。而当细胞内mTORC1被激活时,4EBP1中的多个磷酸化位点会被mTORC1磷酸化,从而使得其不能跟EIF4E相互作用,不能形成高分子量蛋白聚集体,此时,使用荧光显微镜检测到的是呈弥散状的荧光信号。因此,可以通过聚集体的荧光信号呈弥散或聚集状的状态来判断细胞内的mTORC1的活性情况,继而发挥传感器报告基因的功能。并进一步用于筛选抑制mTORC1活性的药物或者相关mTORC1活性调控基因。
mTIR传感器表达载体构建
(1)mTIR传感器DNA序列的获得:
根据上述SEQ ID NO:1和SEQ ID NO:2所示序列构建mTIR传感器。在本实施例中,上述序列是交由商业基因合成公司直接合成得到。
当然,本领域技术人员也可以基于其他本领域常规操作选择合适的合成方式进行合成。
(2)含有上述mTIR传感器的表达载体的构建:
基于KOD-Plus-Neo高保真酶,使用PCR技术扩增上述mTIR传感器DNA序列,反应体系为:
表1
试剂 | 用量 |
10×PCR Buffer | 5μL |
2mM dNTPs | 5μL |
25mM MgSO<sub>4</sub> | 3μL |
正向引物(10μM) | 1.5μL |
反向引物(10μM) | 1.5μL |
SEQ ID NO:2 | 1μL(50~200ng) |
KOD-Plus-Neo | 1μL |
ddH<sub>2</sub>O | 补至50μL |
其中,PCR Buffer和KOD-Plus-Neo购自TOYOBO(东洋纺)。
正向引物和反向引物的序列为:
正向引物:5’-GGTTGCTAGCGCCACCATGTACCCTTATGATGTGCCAGA-3’(SEQ ID NO:3);
反向引物:5’-GATGAATTCTTAGATTTTCTTCAGCCACTTCTC-3’(SEQ ID NO:4)。
反应条件为:94℃预变性2min;98℃变性10s,55℃退火30s,68℃延伸1.5min,循环30次;72℃终延伸5min。
使用琼脂糖凝胶电泳分离目的基因片段。然后用天根普通琼脂糖凝胶DNA回收试剂盒(DP209)对扩增产物进行胶回收,具体步骤参考使用说明书。
使用FastDigest限制性内切酶(Thermo Scientific)对pmCherry-C1载体(购自addgene)与上述扩增产物进行双酶切,双酶切体系如下:
表2
将配制好的酶切体系于37℃孵育30min。酶切产物进行琼脂糖凝胶电泳鉴定,正确后进行回收备用。
使用T4 DNA连接酶(T4 DNA Ligase,购自Thermo Scientific)对酶切载体和酶切片段进行连接,连接体系如下:
表3
试剂 | 用量 |
酶切载体 | 20~100ng |
酶切片段 | 酶切片段/酶切载体的摩尔比为3:1 |
10×T4 DNA Ligase Buffer | 1μL |
T4 DNA Ligase | 1μL |
ddH<sub>2</sub>O | 补至10μL |
配好的体系于22℃孵育30min~1h,得到连接产物(质粒)。
将连接产物转染至DH5α感受态细胞中,具体步骤如下:在冰上向50μLDH5α感受态细胞中加入2μL上述连接产物,孵育30min。在42℃热激感受态细胞90s,然后迅速转移至冰上静置2min。向感受态细胞中加入1mL LB培养基,然后在200rpm、37℃恒温摇床中活化45min-1h。5000rpm离心3min,弃去部分上清。挑取菌液涂布于培养基上,在37℃细菌培养箱培养12-16h。
取培养后的菌体,提取其中的质粒,使用上述酶切体系进行酶切,得到的酶切片段进行琼脂糖凝胶电泳鉴定,长度正确后送至商业测序公司进行测序确认。
最终获得的mTIR表达载体图谱(pmCherry-C-mTIR)如图3所示。
将经测序验证的含有pmCherry-C-mTIR的冻存菌液加入到有相应抗性LB液体培养基的摇菌管中,220rpm摇菌12-16h。然后将菌液用QIAGENQIAprep Spin Miniprep Kit试剂盒按说明书提取质粒,以获得无内毒素的pmCherry-C-mTIR。
mTIR传感器的表达情况
将上述实施例中获得的无内毒素pmCherry-C-mTIR转染至活细胞中,以验证其表达效果。其中,在本实施例中,所使用的活细胞样品为HEK293细胞(人胚胎肾细胞)、Hela细胞(人***细胞)和U2OS细胞(人骨肉瘤细胞)。
通过荧光显微镜和免疫印迹法(Immunoblotting)进行检测。
结果如图4所示,可以发现,使用荧光显微镜和免疫印迹法均可以检测到活细胞中mTIR的表达,说明上述实施例中的mTIR传感器可以实现活细胞中的表达与检测。
mTIR传感器的检测效果验证
(1)mTIR传感器在活细胞中对mTORC1信号的反应与概念确证(Proof of concept,POC):
为了证实上述mTIR传感器能作为活细胞mTORC1的活性传感器,发明人使用mTORC1药物或生理抑制、以及遗传操控失活两种试验进行概念确证。
具体检测方法如下
A.mTIR传感器对mTORC1活性抑制的响应:
将含有mTIR传感器的HEK293细胞分散至96孔玻底培养皿中,每个孔按照5000个细胞/100μL培养基(含10%FBS的DMEM培养基)的密度进行投放,待细胞贴壁24小时后分别予以血清饥饿(-FBS,即用不含FBS的DMEM培养基)、50nmol雷帕霉素抑制(雷帕霉素(Rapa)是已知的mTORC1的活性抑制剂)以及必需氨基酸饥饿(-EAA,通过使用不含必需氨基酸的培养基来培养细胞)。由于血清饥饿、必需氨基酸饥饿和雷帕霉素均是已知的都能抑制mTORC1的活性的手段,因此,可以通过这些实验组有效的显示出mTIR传感器的检测准确性。以不做任何处理的含有mTIR传感器的HEK293细胞作为对照。
结果如图5所示。
可以发现,通过这些生理或抑制剂处理后,都能让mTIR传感器从弥散的荧光信号形成明亮的荧光聚集点。对照组中,含荧光聚集点的细胞比例低于10%,而血清饥饿、必需氨基酸饥饿和雷帕霉素处理10个小时以上分别能促使含荧光聚集点的细胞比例上升至50%、60%、80%左右(n=3),具有显著统计学差异(p<0.01)。该试验能够说明HEK293活细胞中形成荧光聚集点是mTIR响应mTORC1活性抑制的特征,故而证实HEK293活细胞中的mTIR传感器能作为检测mTORC1活性的传感器。
为了进一步验证上述mTIR传感器在各类细胞中的广泛适用实用性,发明人又分别在多株细胞系(HEK293细胞、Hela细胞和U2OS细胞)中对其进行了同样的测试。其中,抑制剂替换为Torin1(一种mTOR ATP结合竞争性抑制剂)。
结果如图6所示。
可以发现,含有上述mTIR传感器的HEK293细胞、Hela细胞和U2OS细胞在经mTOR的激酶特异性抑制剂Torin1处理后,在这些细胞系中mTIR传感器都能正确响应mTOR激酶活性的抑制,从而说明上述mTIR传感器能在各类细胞中作为检测mTORC1活性的传感器。
B.mTIR传感器对mTOR基因操控的响应:
为了证实mTIR响应mTORC1活性,发明人采用遗传操作手段对mTORC1复合物中的mTOR进行基因敲降,然后使用上述mTIR传感器对mTOR基因敲降的响应情况。
发明人首先基于常规技术构建了mTOR基因敲低质粒sh-mTOR,并通过慢病毒将其递送至含有上述mTIR传感器的HEK293细胞中,带shRNA表达48小时后观察HEK293活细胞中的聚集状况。
结果如图7所示。
可以发现,通过使用mTOR基因敲低质粒sh-mTOR敲低HEK293细胞中的mTOR基因表达后,其显著降低了细胞中的mTORC1活性。同时,通过检测细胞内的磷酸化p4EBP和pS6,也均能发现有表达显著降低的情况(图7A),同时,HEK293细胞中mTOR敲降也导致mTIR荧光聚集点的形成(图7B),从而能够说明上述mTIR传感器能在活细胞中响应mTOR基因下调导致的mTORC1活性抑制。
综上所述,根据上述试验结果,可以认定,无论是生理刺激、药物处理还是基因调控操作,上述mTIR传感器都能作为检测活细胞mTORC1活性的传感器,其在mTORC1失活条件下从弥散态形成聚集点是其响应mTORC1活性的显著特征,这一特征使得上述mTIR传感器可作为mTORC1活性传感器的证据。
mTIR传感器在药物筛选中的应用
上述实施例中的mTIR传感器能快速、有效地从药物库中获得活细胞mTORC1抑制剂,从而可以可用于癌症、癫痫、长寿、代谢等相关疾病候选药物的初步筛选。
以下仅提供一种范例性的实施例作为参考。
取含有上述实施例中的mTIR传感器的293T细胞,待其长至70%~90%密度时,用PBS洗涤2次,然后使用1mL 0.25%的胰酶消化2~5min。镜下观察细胞,当细胞变圆且部分脱落时,加入1mL含有10%FBS和1%P/S(青霉素/链霉素)的DMEM高糖培养基终止消化。收集细胞,1000rpm离心5min,去除上清,加入1mL培养基重悬。然后以5000个细胞/孔(100μL)铺到96孔玻底皿中。待细胞贴壁12h后,开始进行给药实验。
药物库的储存浓度为10mM,工作浓度为10μM。每孔加2μL待测药物,轻轻拍打孔板使药物均匀溶解。每次实验均需要设置阴性对照组组及阳性对照组组。在本实施例中,阴性对照组为2μL PBS,阳性对照组为250nM Torin1。
将细胞置于培养箱,培养10~12h。使用尼康显微镜60×油镜进行观察(先对阴性对照组组及阳性对照组组进行观察,确定实验的可靠性)。
结果如图8所示。
可以发现,使用上述mTIR传感器能够很好的判断出药物是否具有mTORC1抑制效果,阴性对照与阳性对照也符合理论要求,没有出现检测失败的问题。发明人使用该mTIR传感器对2000多个FDA批准药物进行初步筛选,获得了至少15个能在活细胞水平引起mTIR响应的药物,进一步使用免疫印迹检测也确认了这些药物能抑制mTORC1的活性,说明上述mTIR传感器能作为一个药物筛选工具应用于跟mTORC1相关疾病的药物筛选。
mTIR传感器在用于鉴定mTORC1信号相关调控基因的缺失、下调或活性变化中的应用
鉴于上述实施例已经验证了上述mTIR传感器对mTOR基因敲降的响应,因此,可以说明其能够应用于对mTORC1活性相关调控基因下调或缺失的遗传学鉴定。在本实施例中,发明人使用上述mTIR传感器来检测使用shRNA基因敲低Raptor和RheB基因的表达的情况下mTORC1活性的变化。其中,Raptor和RheB是两个本领域中已知的mTORC1活性必需基因,敲低其表达会造成mTORC1活性的降低。
具体试验步骤为:
用常规分子克隆技术构建已知的mTORC1上游调控基因Raptor和RheB基因敲低质粒shRaptor#1,shRaptor#2,shRheB#1,shRheB#2。通过慢病毒包装将shRNA对照(空载体shCtrl)、shRaptor#1、shRaptor#2、shRheB#1、shRheB#2递送至含有上述mTIR传感器的HEK293细胞中。待shRNA表达48小时后,随机取出部分细胞液,用尼康显微镜60×油镜观察活细胞中的聚集状况。
结果如图9所示。
可以发现,Raptor和RheB基因敲低对mTIR荧光聚集有显著的影响。
随机取出部分细胞液,使用免疫印迹法检测这些细胞中的Raptor和RheB基因表达情况和mTORC1活性水平。
结果如图10所示。
可以发现,Raptor和RheB基因敲低显著降低了mTORC1活性(p4EBP1水平下降),从而说明上述mTIR传感器的检测结果准确,其能感应调控mTORC1活性的基因下调或缺失。
综上所述,mTORC1调控基因的鉴定和确证是研究癌症、长寿、癫痫等相关疾病药物靶点的重要基础,通过遗传学或药物筛选可得到较多的mTORC1调控或相关药物靶点的候选基因,而本发明实施例中的mTIR感应器可在细胞水平上对其药物效果或抑制效果进行很好的确证,可大大减少通过免疫印迹来验证的工作量并提高其检测的实时性和准确性。
mTIR传感器与常规检测方法的对比
为了能够充分说明上述mTIR传感器相比常规检测方法的技术优势,发明人将上述mTIR传感器相比常规检测方法进行对比,结果如表4所示。
表4
其中,表4中术语解释为:
IB:免疫印迹技术(Immunoblotting);
IF:免疫荧光技术(Immunofluorescence technique);
TORCAR:mTORC1活力报告***(mTORC1 activity reporter),参考ZHOU X,CLISTER TL,LOWRY P R,et al.Dynamic Visualization of mTORC1 Activity in LivingCells[J].Cell Rep,2015,10(10):1767-1777.;
BRET:生物发光共振能量转移(Bioluminescence resonance energy transfer),参考BOUQUIER N,MOUTIN E,TINTIGNAC L A,et al.AIMTOR,a BRET biosensor for liveimaging,reveals subcellular mTOR signaling and dysfunctions[J].BMC Biol,2020,18(1):81。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
SEQUENCE LISTING
<110> 中山大学
中山大学·深圳
<120> 一种用于检测活细胞中mTORC1活性的遗传编码荧光生物传感器及其构建方法
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Claims (10)
1.一种生物传感器,其特征在于,所述生物传感器包括:
融合体基因结构A、融合体基因结构B、用于连接融合体基因结构A和融合体基因结构B的连接部;
所述融合体基因结构A是由mTORC1磷酸化底物的编码基因、标记蛋白基因和寡聚化标签基因组成的融合基因;
所述融合体基因结构B是由所述mTORC1磷酸化底物的结合蛋白编码基因和寡聚化标签基因组成的融合基因;
所述连接部为自剪切多肽的编码基因。
2.根据权利要求1所述的生物传感器,其特征在于,所述mTORC1磷酸化底物为4EBP1,所述mTORC1磷酸化底物的结合蛋白为EIF4E。
3.根据权利要求1所述的生物传感器,其特征在于,所述寡聚化标签包括六聚体标签、四聚体标签中的至少一种。
4.根据权利要求1所述的生物传感器,其特征在于,所述标记蛋白基因包括荧光蛋白;所述荧光蛋白优选为单体荧光蛋白。
5.根据权利要求1~4任一项所述的生物传感器,其特征在于,所述生物传感器的氨基酸序列如SEQ ID NO:1所示。
6.编码权利要求1~5任一项所述生物传感器的核酸分子,其特征在于,所述核酸分子的核苷酸序列如SEQ ID NO:2所示。
7.一种用于检测活细胞mTORC1活性的产品,其特征在于,所述产品包括如下(1)~(6)中的至少一种:
(1)含有SEQ ID NO:1所示核酸分子的表达盒;
(2)含有SEQ ID NO:1所示核酸分子的载体;
(3)含有(1)中所述表达盒的载体;
(4)含有SEQ ID NO:1所示核酸分子的重组微生物;
(5)含有(1)中所述表达盒的重组微生物;
(6)含有(2)中所述载体的重组微生物。
8.权利要求1~4任一项所述的生物传感器在制备活细胞mTORC1活性检测产品中的应用。
9.权利要求1~4任一项所述的生物传感器在mTORC1活性抑制剂筛选中的应用。
10.权利要求1~4任一项所述的生物传感器在制备mTORC1活性调控基因筛选平台中的应用。
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Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2012142233A1 (en) * | 2011-04-14 | 2012-10-18 | St. Jude Children's Research Hospital | Methods and compositions for detecting and modulating a novel mtor complex |
WO2015123377A1 (en) * | 2014-02-12 | 2015-08-20 | Dana-Farber Cancer Institute, Inc. | P13k-mtorc1-s6k1 signaling pathway biomarkers predictive of anti-cancer responses |
KR20180040374A (ko) * | 2016-10-12 | 2018-04-20 | 경북대학교 산학협력단 | Age-1을 유효성분으로 포함하는 뇌전증의 예방 또는 치료용 조성물 |
US20180164286A1 (en) * | 2016-12-12 | 2018-06-14 | Medical Diagnostic Laboratories, Llc | NOVEL CELL-BASED ASSAY FOR DETERMINING mTOR ACTIVITY |
CN109652453A (zh) * | 2018-12-29 | 2019-04-19 | 杭州科兴生物科技有限公司 | 一种基于pd-1/pdl-1阻断功能及生物效应的抗癌药物快速筛选方法 |
CN112444509A (zh) * | 2020-11-12 | 2021-03-05 | 中国科学技术大学 | 一种活细胞内检测clk3激酶活性的生物探针及其应用 |
-
2022
- 2022-03-28 CN CN202210310036.4A patent/CN114591987B/zh active Active
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2012142233A1 (en) * | 2011-04-14 | 2012-10-18 | St. Jude Children's Research Hospital | Methods and compositions for detecting and modulating a novel mtor complex |
WO2015123377A1 (en) * | 2014-02-12 | 2015-08-20 | Dana-Farber Cancer Institute, Inc. | P13k-mtorc1-s6k1 signaling pathway biomarkers predictive of anti-cancer responses |
KR20180040374A (ko) * | 2016-10-12 | 2018-04-20 | 경북대학교 산학협력단 | Age-1을 유효성분으로 포함하는 뇌전증의 예방 또는 치료용 조성물 |
US20180164286A1 (en) * | 2016-12-12 | 2018-06-14 | Medical Diagnostic Laboratories, Llc | NOVEL CELL-BASED ASSAY FOR DETERMINING mTOR ACTIVITY |
CN109652453A (zh) * | 2018-12-29 | 2019-04-19 | 杭州科兴生物科技有限公司 | 一种基于pd-1/pdl-1阻断功能及生物效应的抗癌药物快速筛选方法 |
CN112444509A (zh) * | 2020-11-12 | 2021-03-05 | 中国科学技术大学 | 一种活细胞内检测clk3激酶活性的生物探针及其应用 |
Non-Patent Citations (7)
Title |
---|
NATHALIE BOUQUIER ET AL.: "AIMTOR, a BRET Biosensor for Live Recording of mTOR Activity in Cell Populations and Single Cells", 《BIO PROTOC》, vol. 11, no. 8, pages 1 - 12 * |
OLETA T JOHNSON ET AL.: "A Conditionally Fluorescent Peptide Reporter of Secondary Structure Modulation", 《CHEMBIOCHEM》, vol. 20, no. 1, pages 40 - 45, XP072201820, DOI: 10.1002/cbic.201800377 * |
SOHUM MEHTA ET AL.: "Biochemical Activity Architectures Visualized-Using Genetically Encoded Fluorescent Biosensors to Map the Spatial Boundaries of Signaling Compartments", 《ACC CHEM RES》, vol. 54, no. 10, pages 2409 - 2420 * |
SOHUM MEHTA ET AL.: "Single-fluorophore Biosensors for Sensitive and Multiplexed Detection of Signaling Activities", 《NAT CELL BIOL》, vol. 20, no. 10, pages 1215 - 1225, XP036904020, DOI: 10.1038/s41556-018-0200-6 * |
XIN ZHOU ET AL.: "Dynamic Visualization of mTORC1 Activity in Living Cells", 《CELL REP》, vol. 10, no. 10, pages 1767 - 1777 * |
吕博群: "mTOR信号通路抑制剂的设计、合成与活性评价", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》, no. 8, pages 079 - 35 * |
贺影忠 等: "结节性硬化伴难治性癫痫19例mTORC1信号通路活性检测及治疗", 《临床儿科杂志》, vol. 40, no. 3, pages 196 - 201 * |
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