CN114213549B - 定位于线粒体的融合蛋白、接头及其用途 - Google Patents
定位于线粒体的融合蛋白、接头及其用途 Download PDFInfo
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- CN114213549B CN114213549B CN202111602208.7A CN202111602208A CN114213549B CN 114213549 B CN114213549 B CN 114213549B CN 202111602208 A CN202111602208 A CN 202111602208A CN 114213549 B CN114213549 B CN 114213549B
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Abstract
本发明涉及生物医学基础研究领域，特别是涉及定位于线粒体的融合蛋白、接头及其用途，所述融合蛋白包括线粒体定位多肽、目的蛋白和标记蛋白，所述线粒体定位多肽和目的蛋白之间，以及目的蛋白和标记蛋白之间均通过接头连接，所述接头为通过在柔性连接肽两端连接酶切位点获得的。本发明的接头能够增强融合蛋白线粒体靶向定位表达的高效性和有效性，融合蛋白能够用于线粒体相关的研究中，用于开发与线粒体相关疾病的疫苗或药物，并实现高效定向给药，具有广泛应用价值。
Description
技术领域
本发明涉及生物医学基础研究领域，特别是涉及定位于线粒体的融合蛋白、接头及其用途。
背景技术
在分子生物学中融合蛋白起着十分重要的作用，比如融合蛋白可以是在蛋白质的末端连上标签用于Western Blot检测或者纯化等实验。融合蛋白也可以是将研究目的蛋白与另一个标记蛋白连接，当标记蛋白为荧光蛋白时，可用于目的蛋白的示踪和图像学分析；当标记白蛋白或抗体Fc片段时，可以用来延长目的蛋白的半衰期；将两个目的蛋白连接，用来研究这两个目的蛋白的相互作用及功能。
在构建融合蛋白中，影响蛋白质的折叠和稳定性的关键因素是两蛋白间的接头(Linker)，它包括以下四个要素：第一，接头序列的长度，如果接头序列太短，可能影响两个蛋白高级结构的折叠，从而相互干扰；如果接头序列太长，会引入新的抗原，涉及免疫原性的问题；第二，接头的刚性，蛋白Linker通常可分为柔性Linker、刚性Linker和可剪切Linker三种，其中柔性Linker可以增加保持蛋白结构域之间的相互作用、增加蛋白结构域之间的距离，刚性Linker可以保持蛋白结构域之间的固定距离，可剪切Linker是保证在体内可被剪切从而使结构域分离，如P2A、T2A之类。
线粒体是细胞进行三羧酸循环和氧化磷酸化的重要场所，是细胞生命活动的最主要能量来源。组成线粒体结构和参与三羧酸循环等能量代谢和电子传递活动的蛋白质绝大多数都是由核DNA编码并在细胞质核糖体上合成后，再通过线粒体定位信号肽运送到线粒体中的指定位置。所以，核基因组正是通过编码的线粒体定位信号实现了对线粒体生命活动的控制。
目前对于线粒体定位的融合蛋白及其接头还没有可靠的选择标准，对于序列和结构之间的关系的了解也很有限。
发明内容
鉴于以上所述现有技术的缺点，本发明的目的在于提供定位于线粒体的融合蛋白、接头及其用途，用于解决现有技术中的问题。
为实现上述目的及其他相关目的，本发明提供柔性连接肽在制备定位于线粒体的融合蛋白中的用途。
本发明还提供用于制备融合蛋白的接头，所述接头为通过在所述柔性连接肽两端连接酶切位点获得的，所述融合蛋白为定位于线粒体的融合蛋白。
本发明还提供一种融合蛋白，所述融合蛋白包括线粒体定位多肽、目的蛋白和标记蛋白，所述线粒体定位多肽和目的蛋白之间，以及目的蛋白和标记蛋白之间均通过所述接头连接。
本发明还提供一种核酸构建体，所述核酸构建体包括编码所述接头的多核苷酸或包括编码所述融合蛋白的多核苷酸。
本发明还提供一种细胞系，所述细胞系为线粒体内表达有所述融合蛋白的细胞系。
本发明还提供所述融合蛋白、分离的多核苷酸、核酸构建体在以下任一项或多项中的用途：
1)定位MT-ND1蛋白；
2)制备MT-ND1蛋白定位产品；
3)制备将MT-ND1导入线粒体的产品；
4)制备MT-ND1蛋白定性或定量检测产品；
5)制备研究MT-ND1与线粒体的相互作用的产品；
6)制备在线粒体中过表达MT-ND1的产品；
7)制备线粒体定位产品；
8)制备治疗与线粒体相关疾病的药物；
9)制备与线粒体相关疾病的疫苗。
本发明还提供一种将MT-ND1蛋白定位于线粒体的方法，包括以下步骤：将所述核酸构建体转导入靶细胞内，或将所述融合蛋白加入到靶细胞的生长环境中，从而使所述融合蛋白定位于靶细胞的线粒体上。
如上所述，本发明的定位于线粒体的融合蛋白、接头及其用途，具有以下有益效果：能够将目的蛋白MT-ND1靶向定位于线粒体上。将所述线粒体定位多肽、接头、MT-ND1蛋白、EGFP制备成融合蛋白，可以显示线粒体探针共定位MT-ND1现象，可作为指示线粒体MT-ND1具体位置的重要研究工具，同时也为过表达MT-ND1目的蛋白并定位在线粒体上提供了新的载体。本发明所述线粒体定位多肽、接头、目标蛋白形成融合蛋白，通过细胞内过表达或外源加入到宿主细胞生长环境中，能够增强内源及外源蛋白线粒体靶向定位的高效性和有效性，且对细胞无毒副作用，能够用于线粒体相关的研究中过表达MT-ND1基因或者作为线粒体指示MT-ND1蛋白，用于开发与线粒体相关疾病的疫苗或药物，并实现高效定向给药，具有广泛应用价值。
附图说明
图1显示为本发明的pcDNA3.1-MTS(SCO1)-EGFP质粒信息图谱。
图2显示为本发明的插入线粒体定位信号序列比对图。
图3显示为pcDNA3.1-MTS(SCO1)-EGFP质粒的线粒体荧光定位图。
图4显示为SCO1-MT-ND1衔接部分的序列比对图，显示MT-ND1接到SCO1末端。
图5显示为质粒pcDNA3.1-MTS(SCO1)-MT-ND1-EGFP表达的MTND1融合荧光蛋白与线粒体指示荧光图。
图6显示为插入MTS(SCO1)-Linker-MT-ND1-Linker-EGFP序列比对图。
图7显示为质粒pcDNA3.1-MTS(SCO1)-Linker-MT-ND1-Linker-EGFP表达的MTND1融合荧光蛋白与线粒体指示荧光图。
图8显示为MTS(SCO1)-Linker-MT-ND1-Long linker-EGFP质粒中的Long linker序列比对图。
图9显示为质粒pcDNA3.1-MTS(SCO1)-Linker-MT-ND1-Long linker-EGFP表达的MTND1融合荧光蛋白与线粒体指示荧光图。
具体实施方式
本发明提供柔性连接肽在制备定位于线粒体的融合蛋白中的用途。
在一种实施方式中，所述用途为柔性连接肽在制备所述融合蛋白中接头的用途。
本发明中，所述柔性连接肽选自(GGGGS)n、(G)n或(EAAAK)n，其中n≥3，n为整数。优选的，n为取值范围3～10。本发明中，所述融合蛋白的接头除包括柔性连接肽外，还可以包括酶切位点等序列。
优选的，所述柔性连接肽选自(GGGGS)n，即G(Gly)甘氨酸和S(Ser)丝氨酸组合，其中n≥3，n为整数。优选的，n为3～10。更优选的，所述(GGGGS)n中n为3或6。
本发明中，所述融合蛋白是指将两个以上蛋白质或蛋白质结构域通过接头连接而成的蛋白。融合蛋白的设计中接头的设计和选择对融合蛋白的表达、稳定性及功能活性至关重要。
在一种实施方式中，所述定位于线粒体的融合蛋白包括线粒体定位多肽(Mitochondrial targeting sequence，MTS)、目的蛋白和标记蛋白。
线粒体定位多肽具有独立引导细胞核合成的蛋白质运送到线粒体靶位点，发挥正常蛋白的生理功能。所述线粒体定位多肽选自SCO1、ATP5B、NDUFS2、MnSOD、SCO2、ATP5A1。
本发明中，目的蛋白是在线粒体定位多肽的引导下定位于线粒体的蛋白。在本发明的某些实施方式中，所述目的蛋白为线粒体NADH脱氢酶亚基1(mitochondrial NADHdehydrogenase-1，MT-ND1)。
MT-ND1作为关键因子参与细胞氧化磷酸化电子传递链的第一步，MT-ND1基因表达的改变可能会引起电子传递链成分的改变，进而影响正常电子流，从而影响线粒体氧化磷酸化功能。
在一种实施方式中，所述标记蛋白为荧光蛋白。所述荧光蛋白选自绿色荧光蛋白、青色荧光蛋白、蓝色荧光蛋白、黄色荧光蛋白、橙色荧光蛋白、红色荧光蛋白或远红光荧光蛋白中的一种或多种。
在一种实施方式中，所述融合蛋白为SCO1-MT-ND1-EGFP。
本发明提供用于制备融合蛋白的接头，所述接头为通过在所述柔性连接肽两端连接酶切位点获得的连接子，所述融合蛋白为定位于线粒体的融合蛋白。
柔性连接肽两端的酶切位点为不同的酶切位点。
所述酶切位点即限制性内切酶识别序列，可以是本领域内常用的酶切位点，例如选自BamHI、EcoRI、HindIII、NdeI、XhoI等。
两端的酶切位点的设置便于更换不同的(GGGGS)n。
所述接头选自氨基酸序列如SEQ ID NO.9所示的第一接头；和/或，所述接头选自氨基酸序列如SEQ ID NO.10所示的第二接头；和/或，所述接头选自氨基酸序列如SEQ IDNO.11所示的第三接头。
在一种实施方式中，本发明还包括在SEQ ID NO.9～11所示的氨基酸序列上经过一个或几个氨基酸残基的取代和/或缺失和/或添加后得到的接头。
在一种实施方式中，所述第一接头、第二接头、第三接头的核苷酸序列依次如SEQID NO.4～5、7所示，或与SEQ ID NO.4～5、7所示的序列具有85％以上、90％以上、95％以上、97％以上、98％以上、99％以上相似性的序列。
在一种实施方式中，所述第一接头用于连接线粒体定位多肽和目的蛋白；所述第二接头或第三接头用于连接目的蛋白和标记蛋白。
本发明还提供一种融合蛋白，所述融合蛋白包括线粒体定位多肽、目的蛋白和标记蛋白，所述线粒体定位多肽和目的蛋白之间，以及目的蛋白和标记蛋白之间均通过所述接头连接。
所述线粒体定位多肽选SCO1、ATP5B、NDUFS2、MnSOD、SCO2、ATP5A1。在一种实施方式中，所述线粒体定位多肽选自SCO1，核苷酸序列如SEQ ID NO.13所示。所述线粒体定位多肽位于所述目的蛋白的N端或C端。优选为N端。
在本发明的某些实施方式中，所述目的蛋白为MT-ND1。所述MT-ND1的核苷酸序列如SEQ ID NO.1所示。
在一种实施方式中，所述标记蛋白为荧光蛋白。所述荧光蛋白选自绿色荧光蛋白、青色荧光蛋白、蓝色荧光蛋白、黄色荧光蛋白、橙色荧光蛋白、红色荧光蛋白或远红光荧光蛋白中的一种或多种。
所述线粒体定位多肽和目的蛋白之间通过第一接头、第二接头或第三接头中的任一接头连接。所述目的蛋白和标记蛋白之间通过第一接头、第二接头或第三接头中的任一接头连接。
在一种实施方式中，所述线粒体定位多肽和目的蛋白之间通过第一接头连接，目的蛋白和标记蛋白之间通过第二接头或第三接头连接。
所述融合蛋白自N端至C端的结构选自以下任一：
1)线粒体定位多肽—第一接头—目的蛋白—第二接头—标记蛋白；
2)线粒体定位多肽—第一接头—目的蛋白—第三接头—标记蛋白；
3)标记蛋白—第二接头—目的蛋白—第一接头—线粒体定位多肽；
4)标记蛋白—第三接头—目的蛋白—第一接头—线粒体定位多肽；
在一种实施方式中，所述融合蛋白为SCO1-MT-ND1-EGFP。所述SCO1-MT-ND1-EGFP融合蛋白的氨基酸序列如SEQ ID NO.13所示：MAMLVLVPGRVMRPLGGQLWRFLPRGLEFWGPAEGTARVLLRQFCARQAEGILMPMANLLLLIVPILIAMAFLMLTERKILGYMQLRKGPNVVGPYGLLQPFADAMKLFTKEPLKPATSTITLYITAPTLALTIALLLWTPLPMPNPLVNLNLGLLFILATSSLAVYSILWSGWASNSNYALIGALRAVAQTISYEVTLAIILLSTLLMSGSFNLSTLITTQEHLWLLLPSWPLAMMWFISTLAETNRTPFDLAEGESELVSGFNIEYAAGPFALFFMAEYTNIIMMNTLTTTIFLGTTYDALSPELYTTYFVTKTLLLTSLFLWIRTAYPRFRYDQLMHLLWKNFLPLTLALLMWYVSMPITISSIPPQTTDPPVATMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK(SEQ ID NO.13)
在一种实施方式中，所述融合蛋白的核苷酸序列如SEQ ID NO.6或8所示，以及与SEQ ID NO.6或8所示序列比较至少具有75％，80％，85％，90％，95％，96％，97％，98％，或99％相似性的核苷酸序列。
本发明还提供编码所述接头或所述融合蛋白的分离的多核苷酸。
在一种实施方式中，编码所述接头的多核苷酸的序列选自SEQ ID NO.4～5、7，或与SEQ ID NO.4～5、7所示的序列具有85％以上、90％以上、95％以上、97％以上、98％以上、99％以上相似性的序列。
在一种实施方式中，编码所述融合蛋白的分离的多核苷酸选自SEQ ID NO.6或8，或与SEQ ID NO.6或8所示的序列具有85％以上、90％以上、95％以上、97％以上、98％以上、99％以上相似性的序列。
本发明还提供一种核酸构建体，所述核酸构建体包括编码所述接头的多核苷酸或包括编码所述融合蛋白的多核苷酸。
术语“核酸构建体”是指可以被引入靶细胞或组织中的人工构建的核酸区段，所述核酸构建体可以为质粒，质粒包括载体骨架即空载体与表达框架。所述载体骨架可以自制或选自市场上出售的合适载体骨架。在一种实施方式中，所述载体骨架选自pcDNA3.1-MCS-EGFP，质粒图谱如图1所示。本发明中，所述表达框架即表达融合蛋白的多核苷酸。所述核酸构建体选自pcDNA3.1(+)-MTS-EGFP、pcDNA3.1-MTS(SCO1)-MT-ND1-EGFP、pcDNA3.1-MTS(SCO1)-Linker-MT-ND1-Linker-EGFP或pcDNA3.1-MTS(SCO1)-Linker-MT-ND1-Longlinker-EGFP，核苷酸序列依次如SEQ ID NO.14～17所示。
本发明还提供一种细胞系，所述细胞系为线粒体内表达有所述融合蛋白的细胞系。
所述细胞系可以通过如下任一方式获得：所述核酸构建体转染后得到；或将所述融合蛋白加入宿主细胞的生长环境中，从而使融合蛋白通过细胞膜进入宿主细胞中，并定位于线粒体上。
所述宿主细胞为真核细胞。所述真核细胞选自原生生物细胞、动物细胞、植物细胞和真菌细胞。进一步地，所述真核细胞是选自哺乳动物细胞、鸟类细胞和昆虫细胞的动物细胞。更进一步地，所述宿主细胞是CHO细胞、COS、酵母细胞、昆虫Sf9细胞等。
本发明还提供所述融合蛋白、分离的多核苷酸、核酸构建体在以下任一项或多项中的用途：
1)定位MT-ND1蛋白；
2)制备MT-ND1蛋白定位产品；
3)制备将MT-ND1导入线粒体的产品；
4)制备MT-ND1蛋白定性或定量检测产品；
5)制备研究MT-ND1与线粒体的相互作用的产品；
6)制备在线粒体中过表达MT-ND1的产品；
7)制备线粒体定位产品；
8)制备治疗与线粒体相关疾病的药物；
9)制备与线粒体相关疾病的疫苗。
本发明还提供一种将MT-ND1蛋白定位于线粒体的方法，包括以下步骤：将所述核酸构建体转导入靶细胞内，或将所述融合蛋白加入到靶细胞的生长环境中，从而使所述融合蛋白通过靶细胞的细胞膜进入所述靶细胞中，并定位于靶细胞的线粒体上。
以下通过特定的具体实例说明本发明的实施方式，本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用，本说明书中的各项细节也可以基于不同观点与应用，在没有背离本发明的精神下进行各种修饰或改变。
在进一步描述本发明具体实施方式之前，应理解，本发明的保护范围不局限于下述特定的具体实施方案；还应当理解，本发明实施例中使用的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围；在本发明说明书和权利要求书中，除非文中另外明确指出，单数形式“一个”、“一”和“这个”包括复数形式。
当实施例给出数值范围时，应理解，除非本发明另有说明，每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义，本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外，根据本技术领域的技术人员对现有技术的掌握及本发明的记载，还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。
本发明的目的在于提供一种线粒体定位的MTS-MT-ND1-EGFP融合蛋白间的接头及其应用，该线粒体定位的MTS-MT-ND1-EGFP融合蛋白间的接头可用于线粒体编码的目的蛋白MT-ND1的定位。本发明首次提出的线粒体定位的MTS-MT-ND1-EGFP融合蛋白间的接头具体序列，通过基因克隆技术和荧光蛋白融合在一起，可以实现指示线粒体编码的MT-ND1蛋白的定位，为研究目的蛋白MT-ND1与线粒体的相关功能提供新的载体。
实施例1 pcDNA3.1-MTS(SCO1)-EGFP质粒的构建以及荧光实验验证SCO1线粒体定位信号
本实施例的目的是验证定位信号肽MTS(SCO1)是否可以实现线粒体定位。以自构建质粒pcDNA3.1-MCS-EGFP为载体(图1)，选用上游Hind III和下游EcoR I两酶切位点，并且插入如SEQ ID NO.12所示的线粒体定位序列和EGFP蛋白表达翻译成融合蛋白，且保证不发生框移突变或者提前终止翻译过程。
ATGGCGATGCTGGTCCTAGTACCCGGACGAGTTATGCGGCCTCTGGGTGGCCAACTTTGGCGCTTCTTGCCTCGCGGACTCGAGTTTTGGGGCCCAGCCGAGGGGACTGCGAGAGTCTTGCTGAGGCAGTTCTGCGCGCGGCAAGCGGAGG(SEQ ID NO.12)
线粒体定位信号DNA经HindⅢ和EcoRⅠ在37℃水浴双酶切2h后，再次以1％琼脂糖凝胶电泳回收目的片段。同样pcDNA3.1(+)-MCS-EGFP空载质粒也经HindⅢ和EcoRⅠ在37℃水浴双酶切2h，经双酶切后的片段和载体经过T4 DNA连接酶在16℃下连接过夜。将连接好的片段移至DH5α感受态大肠杆菌中，冰浴5min，42℃水浴1min后立即冰浴5min，加入600μL空白LB培养基在37℃下摇菌1h，1200rpm离心3min，弃上清，保留20μL吹匀后涂布到氨苄抗性的LB培养板上，37℃过夜培养14h。挑取单菌落进行菌落PCR鉴定，选取阳性克隆菌落扩大培养，抽提质粒，测序验证，通过Seqman软件比对序列信息，与预期插入片段序列一致(图2)，核苷酸序列如SEQ ID NO.14所示：
ATGGCGATGCTGGTCCTAGTACCCGGACGAGTTATGCGGCCTCTGGGTGGCCAACTTTGGCGCTTCTTGCCTCGCGGACTCGAGTTTTGGGGCCCAGCCGAGGGGACTGCGAGAGTCTTGCTGAGGCAGTTCTGCGCGCGGCAAGCGGAGGGAATTCTGCAGTCGACGGTACCGCGGGCCCGGGATCCACCGGTCGCCACCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA。
利用已构建的pcDNA3.1(+)-MTS-EGFP融合表达质粒，Lipo3000转染Hela细胞，4-6h换液，24h后用MitoTracker-Red染色指示线粒体(碧云天，货号：C1035)，观察线粒体定位情况。具体步骤包括：在24孔板中每孔加入2×10^4个Hela细胞，培养24h后换成OPTI-MEM(Gibco，货号：31985088)培养，分别在两管50μL OPTI-MEM中单独加入pcDNA3.1(+)-MTS-EGFP融合表达质粒250ng、0.5μL Lipo3000(Invitrogen，货号：L3000150)，分别在室温放置5min，再将含有质粒的OPTI-MEM加入到含有Lipo2000的OPTI-MEM中，轻轻吹匀后37℃孵育30min，每10min混匀一下。将其分为两份均匀铺到24孔板中，培养4-6h后换成10％FBS的DMEM培养基，37℃、5％CO2培养24h。MitoTracker-Red用空白DMEM稀释后染色细胞，37℃染色30min，用PBS洗两遍后在荧光显微镜下观察，拍照记录细胞内的MTS-EGFP的定位情况。
荧光定位结果如下所示，定位信号SCO1融合绿色荧光蛋白可以和线粒体指示试剂MitoTracker-Red的红色荧光发生共定位，重叠后的荧光为黄色荧光(如图中Merged-Yellow)，显示二者发生共定位现象(图3)。绿色荧光为自发绿色荧光蛋白荧光，红色为线粒体探针荧光，发生共定位表示融合蛋白定位在线粒体上。可见，SCO1融合绿色荧光蛋白可以显示线粒体探针共定位现象，可以实现线粒体定位。
实施例2 pcDNA3.1-MTS(SCO1)-MT-ND1-EGFP质粒的构建
为了将MT-ND1构建到pcDNA3.1-MTS(SCO1)-EGFP质粒上，先将MT-ND1序列转为核编码，并补齐序列，经核编码转化后的MT-ND1序列为：TTATGCCCATGGCCAACCTCCTACTCCTCATTGTACCCATTCTAATCGCAATGGCATTCCTAATGCTTACCGAACGAAAAATTCTAGGCTATATGCAACTACGCAAAGGCCCCAACGTTGTAGGCCCCTACGGGCTACTACAACCCTTCGCTGACGCCATGAAACTCTTCACCAAAGAGCCCCTAAAACCCGCCACATCTACCATCACCCTCTACATCACCGCCCCGACCTTAGCTCTCACCATCGCTCTTCTACTATGGACCCCCCTCCCCATGCCCAACCCCCTGGTCAACCTCAACCTAGGCCTCCTATTTATTCTAGCCACCTCTAGCCTAGCCGTTTACTCAATCCTCTGGTCAGGGTGGGCATCAAACTCAAACTACGCCCTGATCGGCGCACTGCGAGCAGTAGCCCAAACAATCTCATATGAAGTCACCCTAGCCATCATTCTACTATCAACATTACTAATGAGTGGCTCCTTTAACCTCTCCACCCTTATCACAACACAAGAACACCTCTGGTTACTCCTGCCATCATGGCCCTTGGCCATGATGTGGTTTATCTCCACACTAGCAGAGACCAACCGAACCCCCTTCGACCTTGCCGAAGGGGAGTCCGAACTAGTCTCAGGCTTCAACATCGAATACGCCGCAGGCCCCTTCGCCCTATTCTTCATGGCCGAATACACAAACATTATTATGATGAACACCCTCACCACTACAATCTTCCTAGGAACAACATATGACGCACTCTCCCCTGAACTCTACACAACATATTTTGTCACCAAGACCCTACTTCTAACCTCCCTGTTCTTATGGATTCGAACAGCATACCCCCGATTCCGCTACGACCAACTCATGCACCTCCTATGGAAAAACTTCCTACCACTCACCCTAGCATTACTTATGTGGTATGTCTCCATGCCCATTACAATCTCCAGCATTCCCCCTCAAACCAC(SEQ ID NO.1),可用位点EcoR1/Bamh1设计引物分别为MT-ND1-EcoR1-F：GGAATTCTTATGCCCATGGCCAACCTC(SEQ ID NO.2)和MT-ND1-Bamh1-R：CGGGATCCGTGGTTTGAGGGGGAATGCT(SEQ ID NO.3)，扩增产物经Bamh1和EcorⅠ在37℃水浴双酶切2h后，再次以1％琼脂糖凝胶电泳回收目的片段。同样pcDNA3.1(+)-MTS(SCO1)-EGFP空载质粒也经Bamh1和EcorⅠ在37℃水浴双酶切2h，片段和载体经过T4 DNA连接酶在16℃下连接过夜。将连接好的片段移至DH5α感受态大肠杆菌中，冰浴5min，42℃水浴1min后立即冰浴5min，加入600μL空白LB培养基在37℃下摇菌1h，1200rpm离心3min，弃上清，保留20μL吹匀后涂布到氨苄抗性的LB培养板上，37℃过夜培养14h。挑取单菌落进行菌落PCR鉴定，选取阳性克隆菌落扩大培养，抽提质粒，测序验证，通过NCBI-Blast比对序列信息，与预期插入片段序列一致(图4)，核苷酸序列如SEQ ID NO.15所示：ATGGCGATGCTGGTCCTAGTACCCGGACGAGTTATGCGGCCTCTGGGTGGCCAACTTTGGCGCTTCTTGCCTCGCGGACTCGAGTTTTGGGGCCCAGCCGAGGGGACTGCGAGAGTCTTGCTGAGGCAGTTCTGCGCGCGGCAAGCGGAGGGAATTCTTATGCCCATGGCCAACCTCCTACTCCTCATTGTACCCATTCTAATCGCAATGGCATTCCTAATGCTTACCGAACGAAAAATTCTAGGCTATATGCAACTACGCAAAGGCCCCAACGTTGTAGGCCCCTACGGGCTACTACAACCCTTCGCTGACGCCATGAAACTCTTCACCAAAGAGCCCCTAAAACCCGCCACATCTACCATCACCCTCTACATCACCGCCCCGACCTTAGCTCTCACCATCGCTCTTCTACTATGGACCCCCCTCCCCATGCCCAACCCCCTGGTCAACCTCAACCTAGGCCTCCTATTTATTCTAGCCACCTCTAGCCTAGCCGTTTACTCAATCCTCTGGTCAGGGTGGGCATCAAACTCAAACTACGCCCTGATCGGCGCACTGCGAGCAGTAGCCCAAACAATCTCATATGAAGTCACCCTAGCCATCATTCTACTATCAACATTACTAATGAGTGGCTCCTTTAACCTCTCCACCCTTATCACAACACAAGAACACCTCTGGTTACTCCTGCCATCATGGCCCTTGGCCATGATGTGGTTTATCTCCACACTAGCAGAGACCAACCGAACCCCCTTCGACCTTGCCGAAGGGGAGTCCGAACTAGTCTCAGGCTTCAACATCGAATACGCCGCAGGCCCCTTCGCCCTATTCTTCATGGCCGAATACACAAACATTATTATGATGAACACCCTCACCACTACAATCTTCCTAGGAACAACATATGACGCACTCTCCCCTGAACTCTACACAACATATTTTGTCACCAAGACCCTACTTCTAACCTCCCTGTTCTTATGGATTCGAACAGCATACCCCCGATTCCGCTACGACCAACTCATGCACCTCCTATGGAAAAACTTCCTACCACTCACCCTAGCATTACTTATGTGGTATGTCTCCATGCCCATTACAATCTCCAGCATTCCCCCTCAAACCacGGATCCACCGGTCGCCACCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA。
利用已构建的pcDNA3.1-MTS(SCO1)-MT-ND1-EGFP融合表达质粒，Lipo3000转染Hela细胞，4-6h换液，24h后用MitoTracker Red染色指示线粒体(碧云天，货号：C1035)，观察线粒体定位情况。在24孔板中每孔加入2×10^4个Hela细胞，培养24h后换成OPTI-MEM(Gibco，货号：31985088)培养，分别在两管50μLOPTI-MEM中单独加入融合表达质粒250ng、0.5μL Lipo3000(Invitrogen，货号：L3000150)，各自室温放置5min，再将含有质粒的MEM加入到含有Lipo3000的MEM中，轻轻吹匀后37℃孵育30min，每10min混匀一下。将其分为两份均匀铺到24孔板中，培养4-6h后换成10％FBS的DMEM培养基，37℃、5％CO2培养24h。MitoTracker Red用空白DMEM稀释后染色细胞，37℃染色30min，用PBS洗两遍后再荧光显微镜下观察，拍照记录细胞内的EGFP的表达情况。
荧光结果如下所示，pcDNA3.1-MTS(SCO1)-MT-ND1-EGFP融合表达质粒只能在最少比例的细胞中与线粒体指示试剂MitoTracker-Red的红色荧光发生共定位，重叠后的荧光为黄色荧光(如图中Merged-Yellow)，同时显示MT-ND1表达定位不清晰(图5)。
实施例3选择插入柔性接头缓解MTS-MT-ND1-EGFP刚性结构氨基酸的空间折叠弊端
在已构建的pcDNA3.1-MTS(SCO1)-MT-ND1-EGFP融合表达质粒中，MTS(SCO1)和MT-ND1之间以及MT-ND1和EGFP之间存在很多刚性氨基酸如脯氨酸、丙氨酸等，所以我们通过插入(GGGGS)3组合的Linker增加蛋白结构域之间的柔软度，以保证不同结构域可以实现完整的空间构象，同时在Linker两端预设酶切位点组合，便于后期更换Linker。在MTS(SCO1)和MT-ND1之间加上如SEQ ID NO.4所示的接头
GGTACCGGTGGCGGAGGGTCAGGTGGCGGAGGGTCAGGTGGCGGAGGGTCAGGATCC(SEQ IDNO.4)；
其氨基酸序列为：GTGGGGSGGGGSGGGGSGS(SEQ ID NO.9)
在MT-ND1和EGFP之间加上如SEQ ID NO.5所示的接头
GATATCGGTGGCGGAGGGTCAGGTGGCGGAGGGTCAGGTGGCGGAGGGTCAGAATTC(SEQ IDNO.5)。
其氨基酸序列为：DIGGGGSGGGGSGGGGSEF(SEQ ID NO.10)
插入pcDNA3.1的序列(即指SCO1-linker-MT-ND1-linker-EGFP的序列)为
AAGCTTATGGCGATGCTGGTCCTAGTACCCGGACGAGTTATGCGGCCTCTGGGTGGCCAACTTTGGCGCTTCTTGCCTCGCGGACTCGAGTTTTGGGGCCCAGCCGAGGGGACTGCGAGAGTCTTGCTGAGGCAGTTCTGCGCGCGGCAAGCGGAGGGTACCGGTGGCGGAGGGTCAGGTGGCGGAGGGTCAGGTGGCGGAGGGTCAGGATCCATGCCCATGGCCAACCTCCTACTCCTCATTGTACCCATTCTAATCGCAATGGCATTCCTAATGCTTACCGAACGAAAAATTCTAGGCTATATGCAACTACGCAAAGGCCCCAACGTTGTAGGCCCCTACGGGCTACTACAACCCTTCGCTGACGCCATGAAACTCTTCACCAAAGAGCCCCTAAAACCCGCCACATCTACCATCACCCTCTACATCACCGCCCCGACCTTAGCTCTCACCATCGCTCTTCTACTATGGACCCCCCTCCCCATGCCCAACCCCCTGGTCAACCTCAACCTAGGCCTCCTATTTATTCTAGCCACCTCTAGCCTAGCCGTTTACTCAATCCTCTGGTCAGGGTGGGCATCAAACTCAAACTACGCCCTGATCGGCGCACTGCGAGCAGTAGCCCAAACAATCTCATATGAAGTCACCCTAGCCATCATTCTACTATCAACATTACTAATGAGTGGCTCCTTTAACCTCTCCACCCTTATCACAACACAAGAACACCTCTGGTTACTCCTGCCATCATGGCCCTTGGCCATGATGTGGTTTATCTCCACACTAGCAGAGACCAACCGAACCCCCTTCGACCTTGCCGAAGGGGAGTCCGAACTAGTCTCAGGCTTCAACATCGAATACGCCGCAGGCCCCTTCGCCCTATTCTTCATGGCCGAATACACAAACATTATTATGATGAACACCCTCACCACTACAATCTTCCTAGGAACAACATATGACGCACTCTCCCCTGAACTCTACACAACATATTTTGTCACCAAGACCCTACTTCTAACCTCCCTGTTCTTATGGATTCGAACAGCATACCCCCGATTCCGCTACGACCAACTCATGCACCTCCTATGGAAAAACTTCCTACCACTCACCCTAGCATTACTTATGTGGTATGTCTCCATGCCCATTACAATCTCCAGCATTCCCCCTCAAACCGATATCGGTGGCGGAGGGTCAGGTGGCGGAGGGTCAGGTGGCGGAGGGTCAGAATTCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAATCTAGA(SEQ ID NO.6)，由测序公司直接合成，Bamh1和EcorⅠ在37℃水浴双酶切2h后，再次以1％琼脂糖凝胶电泳回收目的片段。同样pcDNA3.1(+)-MTS(SCO1)-EGFP空载质粒也经Bamh1和EcorⅠ在37℃水浴双酶切2h，片段和载体经过T4 DNA连接酶在16℃下连接过夜。将连接好的片段移至DH5α感受态大肠杆菌中，冰浴5min，42℃水浴1min后立即冰浴5min，加入600μL空白LB培养基在37℃下摇菌1h，1200rpm离心3min，弃上清，保留20μL吹匀后涂布到氨苄抗性的LB培养板上，37℃过夜培养14h。挑取单菌落进行菌落PCR鉴定，选取阳性克隆菌落扩大培养，抽提质粒，测序验证，通过NCBI-Blast比对序列信息，与预期插入片段序列一致(图6)，核苷酸序列如SEQ IDNO.16所示：
AAGCTTATGGCGATGCTGGTCCTAGTACCCGGACGAGTTATGCGGCCTCTGGGTGGCCAACTTTGGCGCTTCTTGCCTCGCGGACTCGAGTTTTGGGGCCCAGCCGAGGGGACTGCGAGAGTCTTGCTGAGGCAGTTCTGCGCGCGGCAAGCGGAGGGTACCGGTGGCGGAGGGTCAGGTGGCGGAGGGTCAGGTGGCGGAGGGTCAGGATCCATGCCCATGGCCAACCTCCTACTCCTCATTGTACCCATTCTAATCGCAATGGCATTCCTAATGCTTACCGAACGAAAAATTCTAGGCTATATGCAACTACGCAAAGGCCCCAACGTTGTAGGCCCCTACGGGCTACTACAACCCTTCGCTGACGCCATGAAACTCTTCACCAAAGAGCCCCTAAAACCCGCCACATCTACCATCACCCTCTACATCACCGCCCCGACCTTAGCTCTCACCATCGCTCTTCTACTATGGACCCCCCTCCCCATGCCCAACCCCCTGGTCAACCTCAACCTAGGCCTCCTATTTATTCTAGCCACCTCTAGCCTAGCCGTTTACTCAATCCTCTGGTCAGGGTGGGCATCAAACTCAAACTACGCCCTGATCGGCGCACTGCGAGCAGTAGCCCAAACAATCTCATATGAAGTCACCCTAGCCATCATTCTACTATCAACATTACTAATGAGTGGCTCCTTTAACCTCTCCACCCTTATCACAACACAAGAACACCTCTGGTTACTCCTGCCATCATGGCCCTTGGCCATGATGTGGTTTATCTCCACACTAGCAGAGACCAACCGAACCCCCTTCGACCTTGCCGAAGGGGAGTCCGAACTAGTCTCAGGCTTCAACATCGAATACGCCGCAGGCCCCTTCGCCCTATTCTTCATGGCCGAATACACAAACATTATTATGATGAACACCCTCACCACTACAATCTTCCTAGGAACAACATATGACGCACTCTCCCCTGAACTCTACACAACATATTTTGTCACCAAGACCCTACTTCTAACCTCCCTGTTCTTATGGATTCGAACAGCATACCCCCGATTCCGCTACGACCAACTCATGCACCTCCTATGGAAAAACTTCCTACCACTCACCCTAGCATTACTTATGTGGTATGTCTCCATGCCCATTACAATCTCCAGCATTCCCCCTCAAACCGATATCGGTGGCGGAGGGTCAGGTGGCGGAGGGTCAGGTGGCGGAGGGTCAGAATTCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAATCTAGA
利用已构建的pcDNA3.1-MTS(SCO1)-Linker-MT-ND1-Linker-EGFP融合表达质粒，Lipo3000转染Hela细胞，4-6h换液，24h后用MitoTracker Red染色指示线粒体(碧云天，货号：C1035)，观察线粒体定位情况。在24孔板中每孔加入2×10^4个Hela细胞，培养24h后换成OPTI-MEM(Gibco，货号：31985088)培养，分别在两管50μLOPTI-MEM中单独加入融合表达质粒250ng、0.5μL Lipo3000(Invitrogen，货号：L3000150)，各自室温放置5min，再将含有质粒的MEM加入到含有Lipo3000的MEM中，轻轻吹匀后37℃孵育30min，每10min混匀一下。将其分为两份均匀铺到24孔板中，培养4-6h后换成10％FBS的DMEM培养基，37℃、5％CO2培养24h。MitoTracker Red用空白DMEM稀释后染色细胞，37℃染色30min，用PBS洗两遍后再荧光显微镜下观察，拍照记录细胞内的EGFP的表达情况。
荧光结果如下所示，pcDNA3.1-MTS(SCO1)-Linker-MT-ND1-Linker-EGFP融合表达质粒能在最多的细胞中与线粒体指示试剂MitoTracker-Red的红色荧光发生共定位，重叠后的荧光为黄色荧光(如图中Merged-Yellow)，同时显示MT-ND1表达定位较清晰，相比pcDNA3.1-MTS(SCO1)-MT-ND1-EGFP融合表达质粒的表达定位效果有一定的提升(图7)。
实施例4选择插入长链柔性接头缓解MTS-MT-ND1-EGFP刚性结构氨基酸的空间折叠弊端
为了提升各个蛋白结构域空间折叠的柔性，通过选择增加MT-ND1与EGFP之间柔性Linker的长度，更大程度提升结构域空间折叠的柔性，在MT-ND1和EGFP之间插入(GGGGS)6组合，即在MT-ND1和EGFP之间加上含有酶切位点的接头序列(Long linker)为：GATATCGGTGGCGGAGGGTCAGGTGGCGGAGGGTCAGGTGGCGGAGGGTCAGGTGGCGGAGGGTCAGGTGGCGGAGGGTCAGGTGGCGGAGGGTCAGAATTC(SEQ ID NO.7)，其氨基酸序列为：DIGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEF(SEQ ID NO.11)
同时保持MTS-MT-ND1之间的接头与实施例3中相同，插入pcDNA3.1的总的序列为
AAGCTTATGGCGATGCTGGTCCTAGTACCCGGACGAGTTATGCGGCCTCTGGGTGGCCAACTTTGGCGCTTCTTGCCTCGCGGACTCGAGTTTTGGGGCCCAGCCGAGGGGACTGCGAGAGTCTTGCTGAGGCAGTTCTGCGCGCGGCAAGCGGAGGGTACCGGTGGCGGAGGGTCAGGTGGCGGAGGGTCAGGTGGCGGAGGGTCAGGATCCATGCCCATGGCCAACCTCCTACTCCTCATTGTACCCATTCTAATCGCAATGGCATTCCTAATGCTTACCGAACGAAAAATTCTAGGCTATATGCAACTACGCAAAGGCCCCAACGTTGTAGGCCCCTACGGGCTACTACAACCCTTCGCTGACGCCATGAAACTCTTCACCAAAGAGCCCCTAAAACCCGCCACATCTACCATCACCCTCTACATCACCGCCCCGACCTTAGCTCTCACCATCGCTCTTCTACTATGGACCCCCCTCCCCATGCCCAACCCCCTGGTCAACCTCAACCTAGGCCTCCTATTTATTCTAGCCACCTCTAGCCTAGCCGTTTACTCAATCCTCTGGTCAGGGTGGGCATCAAACTCAAACTACGCCCTGATCGGCGCACTGCGAGCAGTAGCCCAAACAATCTCATATGAAGTCACCCTAGCCATCATTCTACTATCAACATTACTAATGAGTGGCTCCTTTAACCTCTCCACCCTTATCACAACACAAGAACACCTCTGGTTACTCCTGCCATCATGGCCCTTGGCCATGATGTGGTTTATCTCCACACTAGCAGAGACCAACCGAACCCCCTTCGACCTTGCCGAAGGGGAGTCCGAACTAGTCTCAGGCTTCAACATCGAATACGCCGCAGGCCCCTTCGCCCTATTCTTCATGGCCGAATACACAAACATTATTATGATGAACACCCTCACCACTACAATCTTCCTAGGAACAACATATGACGCACTCTCCCCTGAACTCTACACAACATATTTTGTCACCAAGACCCTACTTCTAACCTCCCTGTTCTTATGGATTCGAACAGCATACCCCCGATTCCGCTACGACCAACTCATGCACCTCCTATGGAAAAACTTCCTACCACTCACCCTAGCATTACTTATGTGGTATGTCTCCATGCCCATTACAATCTCCAGCATTCCCCCTCAAACCGATATCGGTGGCGGAGGGTCAGGTGGCGGAGGGTCAGGTGGCGGAGGGTCAGGTGGCGGAGGGTCAGGTGGCGGAGGGTCAGGTGGCGGAGGGTCAGAATTCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAATCTAGA(SEQ ID NO.8)。具体操作是将SEQ ID NO.7序列由测序公司直接合成，利用预设的Ecor1和Ecor5双酶切位点，在37℃水浴酶切2h后，再次以1％琼脂糖凝胶电泳回收目的片段。同样pcDNA3.1-MTS(SCO1)-Linker-MT-ND1-Linker-EGFP融合表达质粒也经Ecor1和Ecor5双酶切在37℃水浴酶切2h，片段和载体经过T4 DNA连接酶在16℃下连接过夜。将连接好的片段移至DH5α感受态大肠杆菌中，冰浴5min，42℃水浴1min后立即冰浴5min，加入600μL空白LB培养基在37℃下摇菌1h，1200rpm离心3min，弃上清，保留20μL吹匀后涂布到氨苄抗性的LB培养板上，37℃过夜培养14h。挑取单菌落进行菌落PCR鉴定，选取阳性克隆菌落扩大培养，抽提质粒，测序验证，通过NCBI-Blast比对序列信息，与预期插入片段序列一致(图8)，核苷酸序列如SEQ ID NO.17所示：
AAGCTTATGGCGATGCTGGTCCTAGTACCCGGACGAGTTATGCGGCCTCTGGGTGGCCAACTTTGGCGCTTCTTGCCTCGCGGACTCGAGTTTTGGGGCCCAGCCGAGGGGACTGCGAGAGTCTTGCTGAGGCAGTTCTGCGCGCGGCAAGCGGAGGGTACCGGTGGCGGAGGGTCAGGTGGCGGAGGGTCAGGTGGCGGAGGGTCAGGATCCATGCCCATGGCCAACCTCCTACTCCTCATTGTACCCATTCTAATCGCAATGGCATTCCTAATGCTTACCGAACGAAAAATTCTAGGCTATATGCAACTACGCAAAGGCCCCAACGTTGTAGGCCCCTACGGGCTACTACAACCCTTCGCTGACGCCATGAAACTCTTCACCAAAGAGCCCCTAAAACCCGCCACATCTACCATCACCCTCTACATCACCGCCCCGACCTTAGCTCTCACCATCGCTCTTCTACTATGGACCCCCCTCCCCATGCCCAACCCCCTGGTCAACCTCAACCTAGGCCTCCTATTTATTCTAGCCACCTCTAGCCTAGCCGTTTACTCAATCCTCTGGTCAGGGTGGGCATCAAACTCAAACTACGCCCTGATCGGCGCACTGCGAGCAGTAGCCCAAACAATCTCATATGAAGTCACCCTAGCCATCATTCTACTATCAACATTACTAATGAGTGGCTCCTTTAACCTCTCCACCCTTATCACAACACAAGAACACCTCTGGTTACTCCTGCCATCATGGCCCTTGGCCATGATGTGGTTTATCTCCACACTAGCAGAGACCAACCGAACCCCCTTCGACCTTGCCGAAGGGGAGTCCGAACTAGTCTCAGGCTTCAACATCGAATACGCCGCAGGCCCCTTCGCCCTATTCTTCATGGCCGAATACACAAACATTATTATGATGAACACCCTCACCACTACAATCTTCCTAGGAACAACATATGACGCACTCTCCCCTGAACTCTACACAACATATTTTGTCACCAAGACCCTACTTCTAACCTCCCTGTTCTTATGGATTCGAACAGCATACCCCCGATTCCGCTACGACCAACTCATGCACCTCCTATGGAAAAACTTCCTACCACTCACCCTAGCATTACTTATGTGGTATGTCTCCATGCCCATTACAATCTCCAGCATTCCCCCTCAAACCGATATCGGTGGCGGAGGGTCAGGTGGCGGAGGGTCAGGTGGCGGAGGGTCAGGTGGCGGAGGGTCAGGTGGCGGAGGGTCAGGTGGCGGAGGGTCAGAATTCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAATCTAGA。
利用已构建的pcDNA3.1-MTS(SCO1)-Linker-MT-ND1-Long linker-EGFP融合表达质粒，Lipo3000转染Hela细胞，4-6h换液，24h后用MitoTracker Red染色指示线粒体(碧云天，货号：C1035)，观察线粒体定位情况。在24孔板中每孔加入2×10^4个Hela细胞，培养24h后换成OPTI-MEM(Gibco，货号：31985088)培养，分别在两管50μLOPTI-MEM中单独加入融合表达质粒250ng、0.5μL Lipo3000(Invitrogen，货号：L3000150)，各自室温放置5min，再将含有质粒的MEM加入到含有Lipo3000的MEM中，轻轻吹匀后37℃孵育30min，每10min混匀一下。将其分为两份均匀铺到24孔板中，培养4-6h后换成10％FBS的DMEM培养基，37℃、5％CO2培养24h。MitoTracker Red用空白DMEM稀释后染色细胞，37℃染色30min，用PBS洗两遍后再荧光显微镜下观察，拍照记录细胞内的EGFP的表达情况。
荧光结果如下所示，pcDNA3.1-MTS(SCO1)-Linker-MT-ND1-Long linker-EGFP融合表达质粒能几乎在所有的细胞中与线粒体指示试剂MitoTracker-Red的红色荧光发生共定位，重叠后的荧光为黄色荧光(如图中Merged-Yellow)，同时显示MT-ND1表达定位十分清晰，相比pcDNA3.1-MTS(SCO1)-MT-ND1-EGFP融合表达质粒与pcDNA3.1-MTS(SCO1)-Linker-MT-ND1-Linker-EGFP融合表达质粒的表达定位效果总体上有明显的提升(图9)。
以上的实施例是为了说明本发明公开的实施方案，并不能理解为对本发明的限制。此外，本文所列出的各种修改以及发明中方法的变化，在不脱离本发明的范围和精神的前提下对本领域内的技术人员来说是显而易见的。虽然已结合本发明的多种具体优选实施例对本发明进行了具体的描述，但应当理解，本发明不应仅限于这些具体实施例。事实上，各种如上所述的对本领域内的技术人员来说显而易见的修改来获取发明都应包括在本发明的范围内。
序列表
<110> 上海生物芯片有限公司
<120> 定位于线粒体的融合蛋白、接头及其用途
<160> 17
<170> SIPOSequenceListing 1.0
<210> 1
<211> 958
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
ttatgcccat ggccaacctc ctactcctca ttgtacccat tctaatcgca atggcattcc 60
taatgcttac cgaacgaaaa attctaggct atatgcaact acgcaaaggc cccaacgttg 120
taggccccta cgggctacta caacccttcg ctgacgccat gaaactcttc accaaagagc 180
ccctaaaacc cgccacatct accatcaccc tctacatcac cgccccgacc ttagctctca 240
ccatcgctct tctactatgg acccccctcc ccatgcccaa ccccctggtc aacctcaacc 300
taggcctcct atttattcta gccacctcta gcctagccgt ttactcaatc ctctggtcag 360
ggtgggcatc aaactcaaac tacgccctga tcggcgcact gcgagcagta gcccaaacaa 420
tctcatatga agtcacccta gccatcattc tactatcaac attactaatg agtggctcct 480
ttaacctctc cacccttatc acaacacaag aacacctctg gttactcctg ccatcatggc 540
ccttggccat gatgtggttt atctccacac tagcagagac caaccgaacc cccttcgacc 600
ttgccgaagg ggagtccgaa ctagtctcag gcttcaacat cgaatacgcc gcaggcccct 660
tcgccctatt cttcatggcc gaatacacaa acattattat gatgaacacc ctcaccacta 720
caatcttcct aggaacaaca tatgacgcac tctcccctga actctacaca acatattttg 780
tcaccaagac cctacttcta acctccctgt tcttatggat tcgaacagca tacccccgat 840
tccgctacga ccaactcatg cacctcctat ggaaaaactt cctaccactc accctagcat 900
tacttatgtg gtatgtctcc atgcccatta caatctccag cattccccct caaaccac 958
<210> 2
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
ggaattctta tgcccatggc caacctc 27
<210> 3
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
cgggatccgt ggtttgaggg ggaatgct 28
<210> 4
<211> 57
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
ggtaccggtg gcggagggtc aggtggcgga gggtcaggtg gcggagggtc aggatcc 57
<210> 5
<211> 57
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
gatatcggtg gcggagggtc aggtggcgga gggtcaggtg gcggagggtc agaattc 57
<210> 6
<211> 1950
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
aagcttatgg cgatgctggt cctagtaccc ggacgagtta tgcggcctct gggtggccaa 60
ctttggcgct tcttgcctcg cggactcgag ttttggggcc cagccgaggg gactgcgaga 120
gtcttgctga ggcagttctg cgcgcggcaa gcggagggta ccggtggcgg agggtcaggt 180
ggcggagggt caggtggcgg agggtcagga tccatgccca tggccaacct cctactcctc 240
attgtaccca ttctaatcgc aatggcattc ctaatgctta ccgaacgaaa aattctaggc 300
tatatgcaac tacgcaaagg ccccaacgtt gtaggcccct acgggctact acaacccttc 360
gctgacgcca tgaaactctt caccaaagag cccctaaaac ccgccacatc taccatcacc 420
ctctacatca ccgccccgac cttagctctc accatcgctc ttctactatg gacccccctc 480
cccatgccca accccctggt caacctcaac ctaggcctcc tatttattct agccacctct 540
agcctagccg tttactcaat cctctggtca gggtgggcat caaactcaaa ctacgccctg 600
atcggcgcac tgcgagcagt agcccaaaca atctcatatg aagtcaccct agccatcatt 660
ctactatcaa cattactaat gagtggctcc tttaacctct ccacccttat cacaacacaa 720
gaacacctct ggttactcct gccatcatgg cccttggcca tgatgtggtt tatctccaca 780
ctagcagaga ccaaccgaac ccccttcgac cttgccgaag gggagtccga actagtctca 840
ggcttcaaca tcgaatacgc cgcaggcccc ttcgccctat tcttcatggc cgaatacaca 900
aacattatta tgatgaacac cctcaccact acaatcttcc taggaacaac atatgacgca 960
ctctcccctg aactctacac aacatatttt gtcaccaaga ccctacttct aacctccctg 1020
ttcttatgga ttcgaacagc atacccccga ttccgctacg accaactcat gcacctccta 1080
tggaaaaact tcctaccact caccctagca ttacttatgt ggtatgtctc catgcccatt 1140
acaatctcca gcattccccc tcaaaccgat atcggtggcg gagggtcagg tggcggaggg 1200
tcaggtggcg gagggtcaga attcatggtg agcaagggcg aggagctgtt caccggggtg 1260
gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc 1320
gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc 1380
aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc 1440
agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc 1500
tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag 1560
gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag 1620
gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat 1680
atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc 1740
gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc 1800
cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc 1860
aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc 1920
ggcatggacg agctgtacaa gtaatctaga 1950
<210> 7
<211> 102
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
gatatcggtg gcggagggtc aggtggcgga gggtcaggtg gcggagggtc aggtggcgga 60
gggtcaggtg gcggagggtc aggtggcgga gggtcagaat tc 102
<210> 8
<211> 1995
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
aagcttatgg cgatgctggt cctagtaccc ggacgagtta tgcggcctct gggtggccaa 60
ctttggcgct tcttgcctcg cggactcgag ttttggggcc cagccgaggg gactgcgaga 120
gtcttgctga ggcagttctg cgcgcggcaa gcggagggta ccggtggcgg agggtcaggt 180
ggcggagggt caggtggcgg agggtcagga tccatgccca tggccaacct cctactcctc 240
attgtaccca ttctaatcgc aatggcattc ctaatgctta ccgaacgaaa aattctaggc 300
tatatgcaac tacgcaaagg ccccaacgtt gtaggcccct acgggctact acaacccttc 360
gctgacgcca tgaaactctt caccaaagag cccctaaaac ccgccacatc taccatcacc 420
ctctacatca ccgccccgac cttagctctc accatcgctc ttctactatg gacccccctc 480
cccatgccca accccctggt caacctcaac ctaggcctcc tatttattct agccacctct 540
agcctagccg tttactcaat cctctggtca gggtgggcat caaactcaaa ctacgccctg 600
atcggcgcac tgcgagcagt agcccaaaca atctcatatg aagtcaccct agccatcatt 660
ctactatcaa cattactaat gagtggctcc tttaacctct ccacccttat cacaacacaa 720
gaacacctct ggttactcct gccatcatgg cccttggcca tgatgtggtt tatctccaca 780
ctagcagaga ccaaccgaac ccccttcgac cttgccgaag gggagtccga actagtctca 840
ggcttcaaca tcgaatacgc cgcaggcccc ttcgccctat tcttcatggc cgaatacaca 900
aacattatta tgatgaacac cctcaccact acaatcttcc taggaacaac atatgacgca 960
ctctcccctg aactctacac aacatatttt gtcaccaaga ccctacttct aacctccctg 1020
ttcttatgga ttcgaacagc atacccccga ttccgctacg accaactcat gcacctccta 1080
tggaaaaact tcctaccact caccctagca ttacttatgt ggtatgtctc catgcccatt 1140
acaatctcca gcattccccc tcaaaccgat atcggtggcg gagggtcagg tggcggaggg 1200
tcaggtggcg gagggtcagg tggcggaggg tcaggtggcg gagggtcagg tggcggaggg 1260
tcagaattca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc 1320
gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat 1380
gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc 1440
tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac 1500
cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc 1560
accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc 1620
gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc 1680
ctggggcaca agctggagta caactacaac agccacaacg tctatatcat ggccgacaag 1740
cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg 1800
cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc 1860
gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat 1920
cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg 1980
tacaagtaat ctaga 1995
<210> 9
<211> 19
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Gly Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
1 5 10 15
Ser Gly Ser
<210> 10
<211> 19
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Asp Ile Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
1 5 10 15
Ser Glu Phe
<210> 11
<211> 34
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Asp Ile Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
1 5 10 15
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
20 25 30
Glu Phe
<210> 12
<211> 151
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
atggcgatgc tggtcctagt acccggacga gttatgcggc ctctgggtgg ccaactttgg 60
cgcttcttgc ctcgcggact cgagttttgg ggcccagccg aggggactgc gagagtcttg 120
ctgaggcagt tctgcgcgcg gcaagcggag g 151
<210> 13
<211> 617
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
Met Ala Met Leu Val Leu Val Pro Gly Arg Val Met Arg Pro Leu Gly
1 5 10 15
Gly Gln Leu Trp Arg Phe Leu Pro Arg Gly Leu Glu Phe Trp Gly Pro
20 25 30
Ala Glu Gly Thr Ala Arg Val Leu Leu Arg Gln Phe Cys Ala Arg Gln
35 40 45
Ala Glu Gly Ile Leu Met Pro Met Ala Asn Leu Leu Leu Leu Ile Val
50 55 60
Pro Ile Leu Ile Ala Met Ala Phe Leu Met Leu Thr Glu Arg Lys Ile
65 70 75 80
Leu Gly Tyr Met Gln Leu Arg Lys Gly Pro Asn Val Val Gly Pro Tyr
85 90 95
Gly Leu Leu Gln Pro Phe Ala Asp Ala Met Lys Leu Phe Thr Lys Glu
100 105 110
Pro Leu Lys Pro Ala Thr Ser Thr Ile Thr Leu Tyr Ile Thr Ala Pro
115 120 125
Thr Leu Ala Leu Thr Ile Ala Leu Leu Leu Trp Thr Pro Leu Pro Met
130 135 140
Pro Asn Pro Leu Val Asn Leu Asn Leu Gly Leu Leu Phe Ile Leu Ala
145 150 155 160
Thr Ser Ser Leu Ala Val Tyr Ser Ile Leu Trp Ser Gly Trp Ala Ser
165 170 175
Asn Ser Asn Tyr Ala Leu Ile Gly Ala Leu Arg Ala Val Ala Gln Thr
180 185 190
Ile Ser Tyr Glu Val Thr Leu Ala Ile Ile Leu Leu Ser Thr Leu Leu
195 200 205
Met Ser Gly Ser Phe Asn Leu Ser Thr Leu Ile Thr Thr Gln Glu His
210 215 220
Leu Trp Leu Leu Leu Pro Ser Trp Pro Leu Ala Met Met Trp Phe Ile
225 230 235 240
Ser Thr Leu Ala Glu Thr Asn Arg Thr Pro Phe Asp Leu Ala Glu Gly
245 250 255
Glu Ser Glu Leu Val Ser Gly Phe Asn Ile Glu Tyr Ala Ala Gly Pro
260 265 270
Phe Ala Leu Phe Phe Met Ala Glu Tyr Thr Asn Ile Ile Met Met Asn
275 280 285
Thr Leu Thr Thr Thr Ile Phe Leu Gly Thr Thr Tyr Asp Ala Leu Ser
290 295 300
Pro Glu Leu Tyr Thr Thr Tyr Phe Val Thr Lys Thr Leu Leu Leu Thr
305 310 315 320
Ser Leu Phe Leu Trp Ile Arg Thr Ala Tyr Pro Arg Phe Arg Tyr Asp
325 330 335
Gln Leu Met His Leu Leu Trp Lys Asn Phe Leu Pro Leu Thr Leu Ala
340 345 350
Leu Leu Met Trp Tyr Val Ser Met Pro Ile Thr Ile Ser Ser Ile Pro
355 360 365
Pro Gln Thr Thr Asp Pro Pro Val Ala Thr Met Val Ser Lys Gly Glu
370 375 380
Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp
385 390 395 400
Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala
405 410 415
Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu
420 425 430
Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gln
435 440 445
Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys
450 455 460
Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys
465 470 475 480
Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp
485 490 495
Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp
500 505 510
Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn
515 520 525
Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe
530 535 540
Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His
545 550 555 560
Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp
565 570 575
Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu
580 585 590
Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile
595 600 605
Thr Leu Gly Met Asp Glu Leu Tyr Lys
610 615
<210> 14
<211> 921
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
atggcgatgc tggtcctagt acccggacga gttatgcggc ctctgggtgg ccaactttgg 60
cgcttcttgc ctcgcggact cgagttttgg ggcccagccg aggggactgc gagagtcttg 120
ctgaggcagt tctgcgcgcg gcaagcggag ggaattctgc agtcgacggt accgcgggcc 180
cgggatccac cggtcgccac catggtgagc aagggcgagg agctgttcac cggggtggtg 240
cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag 300
ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag 360
ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc 420
cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac 480
gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg 540
aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag 600
gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc 660
atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag 720
gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc 780
gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac 840
gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc 900
atggacgagc tgtacaagta a 921
<210> 15
<211> 1854
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
atggcgatgc tggtcctagt acccggacga gttatgcggc ctctgggtgg ccaactttgg 60
cgcttcttgc ctcgcggact cgagttttgg ggcccagccg aggggactgc gagagtcttg 120
ctgaggcagt tctgcgcgcg gcaagcggag ggaattctta tgcccatggc caacctccta 180
ctcctcattg tacccattct aatcgcaatg gcattcctaa tgcttaccga acgaaaaatt 240
ctaggctata tgcaactacg caaaggcccc aacgttgtag gcccctacgg gctactacaa 300
cccttcgctg acgccatgaa actcttcacc aaagagcccc taaaacccgc cacatctacc 360
atcaccctct acatcaccgc cccgacctta gctctcacca tcgctcttct actatggacc 420
cccctcccca tgcccaaccc cctggtcaac ctcaacctag gcctcctatt tattctagcc 480
acctctagcc tagccgttta ctcaatcctc tggtcagggt gggcatcaaa ctcaaactac 540
gccctgatcg gcgcactgcg agcagtagcc caaacaatct catatgaagt caccctagcc 600
atcattctac tatcaacatt actaatgagt ggctccttta acctctccac ccttatcaca 660
acacaagaac acctctggtt actcctgcca tcatggccct tggccatgat gtggtttatc 720
tccacactag cagagaccaa ccgaaccccc ttcgaccttg ccgaagggga gtccgaacta 780
gtctcaggct tcaacatcga atacgccgca ggccccttcg ccctattctt catggccgaa 840
tacacaaaca ttattatgat gaacaccctc accactacaa tcttcctagg aacaacatat 900
gacgcactct cccctgaact ctacacaaca tattttgtca ccaagaccct acttctaacc 960
tccctgttct tatggattcg aacagcatac ccccgattcc gctacgacca actcatgcac 1020
ctcctatgga aaaacttcct accactcacc ctagcattac ttatgtggta tgtctccatg 1080
cccattacaa tctccagcat tccccctcaa accacggatc caccggtcgc caccatggtg 1140
agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac 1200
gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag 1260
ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg 1320
accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac 1380
gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag 1440
gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac 1500
cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg 1560
gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc 1620
aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac 1680
taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg 1740
agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg 1800
gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtaa 1854
<210> 16
<211> 1950
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
aagcttatgg cgatgctggt cctagtaccc ggacgagtta tgcggcctct gggtggccaa 60
ctttggcgct tcttgcctcg cggactcgag ttttggggcc cagccgaggg gactgcgaga 120
gtcttgctga ggcagttctg cgcgcggcaa gcggagggta ccggtggcgg agggtcaggt 180
ggcggagggt caggtggcgg agggtcagga tccatgccca tggccaacct cctactcctc 240
attgtaccca ttctaatcgc aatggcattc ctaatgctta ccgaacgaaa aattctaggc 300
tatatgcaac tacgcaaagg ccccaacgtt gtaggcccct acgggctact acaacccttc 360
gctgacgcca tgaaactctt caccaaagag cccctaaaac ccgccacatc taccatcacc 420
ctctacatca ccgccccgac cttagctctc accatcgctc ttctactatg gacccccctc 480
cccatgccca accccctggt caacctcaac ctaggcctcc tatttattct agccacctct 540
agcctagccg tttactcaat cctctggtca gggtgggcat caaactcaaa ctacgccctg 600
atcggcgcac tgcgagcagt agcccaaaca atctcatatg aagtcaccct agccatcatt 660
ctactatcaa cattactaat gagtggctcc tttaacctct ccacccttat cacaacacaa 720
gaacacctct ggttactcct gccatcatgg cccttggcca tgatgtggtt tatctccaca 780
ctagcagaga ccaaccgaac ccccttcgac cttgccgaag gggagtccga actagtctca 840
ggcttcaaca tcgaatacgc cgcaggcccc ttcgccctat tcttcatggc cgaatacaca 900
aacattatta tgatgaacac cctcaccact acaatcttcc taggaacaac atatgacgca 960
ctctcccctg aactctacac aacatatttt gtcaccaaga ccctacttct aacctccctg 1020
ttcttatgga ttcgaacagc atacccccga ttccgctacg accaactcat gcacctccta 1080
tggaaaaact tcctaccact caccctagca ttacttatgt ggtatgtctc catgcccatt 1140
acaatctcca gcattccccc tcaaaccgat atcggtggcg gagggtcagg tggcggaggg 1200
tcaggtggcg gagggtcaga attcatggtg agcaagggcg aggagctgtt caccggggtg 1260
gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc 1320
gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc 1380
aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc 1440
agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc 1500
tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag 1560
gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag 1620
gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat 1680
atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc 1740
gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc 1800
cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc 1860
aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc 1920
ggcatggacg agctgtacaa gtaatctaga 1950
<210> 17
<211> 1995
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
aagcttatgg cgatgctggt cctagtaccc ggacgagtta tgcggcctct gggtggccaa 60
ctttggcgct tcttgcctcg cggactcgag ttttggggcc cagccgaggg gactgcgaga 120
gtcttgctga ggcagttctg cgcgcggcaa gcggagggta ccggtggcgg agggtcaggt 180
ggcggagggt caggtggcgg agggtcagga tccatgccca tggccaacct cctactcctc 240
attgtaccca ttctaatcgc aatggcattc ctaatgctta ccgaacgaaa aattctaggc 300
tatatgcaac tacgcaaagg ccccaacgtt gtaggcccct acgggctact acaacccttc 360
gctgacgcca tgaaactctt caccaaagag cccctaaaac ccgccacatc taccatcacc 420
ctctacatca ccgccccgac cttagctctc accatcgctc ttctactatg gacccccctc 480
cccatgccca accccctggt caacctcaac ctaggcctcc tatttattct agccacctct 540
agcctagccg tttactcaat cctctggtca gggtgggcat caaactcaaa ctacgccctg 600
atcggcgcac tgcgagcagt agcccaaaca atctcatatg aagtcaccct agccatcatt 660
ctactatcaa cattactaat gagtggctcc tttaacctct ccacccttat cacaacacaa 720
gaacacctct ggttactcct gccatcatgg cccttggcca tgatgtggtt tatctccaca 780
ctagcagaga ccaaccgaac ccccttcgac cttgccgaag gggagtccga actagtctca 840
ggcttcaaca tcgaatacgc cgcaggcccc ttcgccctat tcttcatggc cgaatacaca 900
aacattatta tgatgaacac cctcaccact acaatcttcc taggaacaac atatgacgca 960
ctctcccctg aactctacac aacatatttt gtcaccaaga ccctacttct aacctccctg 1020
ttcttatgga ttcgaacagc atacccccga ttccgctacg accaactcat gcacctccta 1080
tggaaaaact tcctaccact caccctagca ttacttatgt ggtatgtctc catgcccatt 1140
acaatctcca gcattccccc tcaaaccgat atcggtggcg gagggtcagg tggcggaggg 1200
tcaggtggcg gagggtcagg tggcggaggg tcaggtggcg gagggtcagg tggcggaggg 1260
tcagaattca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc 1320
gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat 1380
gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc 1440
tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac 1500
cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc 1560
accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc 1620
gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc 1680
ctggggcaca agctggagta caactacaac agccacaacg tctatatcat ggccgacaag 1740
cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg 1800
cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc 1860
gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat 1920
cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg 1980
tacaagtaat ctaga 1995
Claims (7)
1.柔性连接肽在制备定位于线粒体的融合蛋白中接头的用途，所述定位于线粒体的融合蛋白包括线粒体定位多肽、目的蛋白和标记蛋白，所述融合蛋白自N端至C端的结构为：线粒体定位多肽—第一接头—目的蛋白—第二接头或第三接头—标记蛋白，所述线粒体定位多肽和目的蛋白之间通过氨基酸序列如SEQ ID NO.9所示的第一接头连接，目的蛋白和标记蛋白之间通过氨基酸序列如SEQ ID NO.10所示的第二接头或氨基酸序列如SEQ IDNO.11所示的第三接头连接，所述融合蛋白为SCO1-MT-ND1-EGFP；所述SCO1-MT-ND1-EGFP融合蛋白的核苷酸序列如SEQ ID NO.16或17所示。
2.一种融合蛋白，其特征在于，所述融合蛋白包括线粒体定位多肽、目的蛋白和标记蛋白，所述线粒体定位多肽和目的蛋白之间，以及目的蛋白和标记蛋白之间均通过接头连接，所述融合蛋白自N端至C端的结构为：线粒体定位多肽—第一接头—目的蛋白—第二接头或第三接头—标记蛋白，所述线粒体定位多肽和目的蛋白之间通过氨基酸序列如SEQ IDNO.9所示的第一接头连接，目的蛋白和标记蛋白之间通过氨基酸序列如SEQ ID NO.10所示的第二接头或氨基酸序列如SEQ ID NO.11所示的第三接头连接，所述融合蛋白为SCO1-MT-ND1-EGFP；所述SCO1-MT-ND1-EGFP融合蛋白的核苷酸序列如SEQ ID NO.16或17所示。
3.编码权利要求2所述的融合蛋白的分离的多核苷酸。
4.根据权利要求3所述的多核苷酸，其特征在于，编码所述融合蛋白的分离的多核苷酸选自SEQ ID NO.6或8。
5.一种核酸构建体，其特征在于，所述核酸构建体包括编码权利要求2所述的融合蛋白的多核苷酸。
6.一种细胞系，其特征在于，所述细胞系为线粒体内表达有权利要求2所述的融合蛋白的细胞系，所述细胞系不是植物细胞系。
7.权利要求2所述的融合蛋白、权利要求3或4所述的分离的多核苷酸、权利要求5所述的核酸构建体在以下任一项或多项中的用途：
1)制备MT-ND1蛋白定位产品；
2)制备将MT-ND1导入线粒体的产品；
3)制备MT-ND1蛋白定性或定量检测产品；
4)制备研究MT-ND1与线粒体的相互作用的产品；
5)制备在线粒体中过表达MT-ND1的产品；
6)制备线粒体定位产品。
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