CN112851765B - 蛋白或肽与核酸共价连接的方法 - Google Patents
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Abstract
本发明提供了一种蛋白或肽与核酸共价连接的方法,涉及生物交联的技术领域。上述蛋白或肽与核酸共价连接的方法,包括以下步骤:(1)目标蛋白或肽与Trwc酶形成融合蛋白;(2)融合蛋白中Trwc酶催化目标蛋白或肽与目标核酸共价连接。本发明利用Trwc酶能够识别、剪切和连接特定的目标核酸序列且能与目标核酸共价连接的性质,实现了稳定、高效、定点、定向共价连接目标蛋白或肽与目标核酸的目的,反应条件温和,反应步骤简单,耗时短,无需引入任何化学试剂,只需加入金属离子,适合产业化推广。
Description
技术领域
本发明涉及生物交联的技术领域,具体涉及一种蛋白与核酸共价连接的方法。
背景技术
蛋白核酸复合物由于具备了蛋白质功能多样性以及核酸的可编程性,使其在生物技术上研究十分广泛。目前蛋白核酸复合物在核酸纳米结构上构建的多酶体系、固相载体如生物芯片上的蛋白固定、生物分子递送、高分辨成像技术上已有大量工作报导。
目前蛋白核酸连接方法主要有以下几种:生物素与链霉亲和素的高亲附作用、镍离子介导的次氮基三乙酸(NTA)与六聚组氨酸的结合、特异性抗原抗体相互作用、异源双官能交联剂介导、适配体-蛋白介导的组装等。但上述各种方法都不可避免的需要对蛋白或核酸进行特定基因、功能进行修饰,其过程比较繁琐,易对DNA结构或蛋白的固有性质造成损失,以及修饰连接时出现非特异性结合、随机性大、连接效率低等问题。
实际运用中需依据不同目的选择不同的修饰方法,然而这并不能完全满足实际要求,这也是限制蛋白核酸复合物发展的一个瓶颈,因此目前蛋白核酸连接技术有待发展,急需要一种稳定、高效、反应条件温和、易于使用的共价定点连接技术。
发明内容
有鉴于此,本发明致力于提供一种蛋白或肽与核酸共价连接的方法,克服了现有技术中几种常用的蛋白核酸连接方法存在的操作过程繁琐、易损伤蛋白或核酸的性质、随机性大、连接效率低等问题,实现了稳定、高效、定点、定向共价连接目标蛋白或肽与目标核酸的目的。
为了达到本发明的目的,采用了下述的技术方案:
本发明第一方面提供了一种蛋白或肽与核酸共价连接的方法,包括以下步骤:
(1)目标蛋白或肽与Trwc酶形成融合蛋白;
(2)融合蛋白中Trwc酶催化目标蛋白或肽与目标核酸共价连接。
本发明的发明人发现了现有技术中几种常用的蛋白核酸连接方法存在着操作过程繁琐、易损伤蛋白或核酸的性质、随机性大、连接效率低等问题。因此,经过不断的努力研究发现了可以利用Trwc酶作为连接载体催化目标蛋白或肽与目标核酸共价连接,先将Trwc酶与目标蛋白制备成融合蛋白,融合蛋白中的Trwc酶座位连接载体去结合目标核酸,以此达到了目标蛋白和目标核酸通过Trwc酶共价连接的目的。
Trwc为DNA链转移酶(Trwc蛋白酶),因为最初是在质粒弛缓复合物中发现的,也称为弛缓酶,Trwc酶发现于质粒R388。Trwc DNA链转移酶活性的决定因素位于其N端结构域(1-293),而C端结构域包含5’→3’DNA解旋酶活性。在体外,全长Trwc及其N端结构域都能以类似于I型拓扑异构酶的方式裂解含nic位点的单链寡核酸和完整的链转移反应。
示例性地,Trwc酶包括其相关突变体或者部分结构域,所述突变体或部分结构域仍然具有Trwc酶的功能能够识别、剪切和连接特定序列的DNA。
示例性地,所述的目标蛋白可为多聚蛋白、寡聚蛋白、单体蛋白等。按其来源可为植物蛋白、动物蛋白或人工合成蛋白等。所述目标肽可为有生物活性多肽或人工合成多肽等。示例性地,所述目标肽可为有生物活性多肽或人工合成多肽等。例如,细胞因子模拟肽、抗菌性活性肽、用于心血管疾病的多肽、其它药用小肽以及诊断用多肽等。
本发明中,对目标蛋白或肽不作具体限定,可以根据实际需要进行选择。
本发明利用Trwc酶在催化DNA链的断裂和结合方面的性质,将Trwc酶先与目标蛋白或肽组装成融合蛋白,再利用融合蛋白中的Trwc酶催化共价连接目标核酸,实现了稳定、高效、定点、定向共价连接目标蛋白或肽与目标核酸。
在本发明一种具体实施方式中,所述Trwc酶的氨基酸序列如SEQ ID NO.1所示:MLSHMVLTRQDIPRAASYYEDPADDYYAKDPDASEWQGKGAEELGLSGEVDSKRFRELLAGNIGEGHRIMRSATRQDSKERIGLDLTFSAPKSVSLQALVAGDAEIIKAHDRAVARTLEQAEARAQARQKIQGKTRIETTPNLVIGKFRHETSRERDPQLHTHAVILNMTKRSDGQWRALKNDEIVKATRYLGAVYNAELAHELQKLGYQLRYGKDGNFDLAHIDRQQIEGFSKRTEQIAEWYAARGLDPNSVSLEQKQAAKVLSRAKKTSVDREALRAEWQATAKELGIDFS或其同源序列。本发明中的Trwc蛋白酶有4处碱基位点进行突变,突变位点为G13P、G22P、G31P、G141P(序列中横线加粗的氨基酸突变位点),突变后的Trwc蛋白酶性质更加稳定。
示例性地,所述同源序列与原序列的同源性约80%或以上、81%或以上、82%或以上、83%或以上、84%或以上、85%或以上、86%或以上、87%或以上、88%或以上、89%或以上、90%或以上、91%或以上、92%或以上、93%或以上、94%或以上、95%或以上、96%或以上、97%或以上、98%或以上、99%或以上、99.1%或以上、99.2%或以上、99.3%或以上、99.4%或以上、99.5%或以上、99.6%或以上、99.7%或以上、99.8%或以上、或99.9%或以上仍具有Trwc蛋白酶活性的氨基酸序列。
在本发明一种具体实施方式中,所述Trwc酶的核苷酸序列如SEQ ID NO:2所示或其简并序列。
示例性地,所述简并序列与原序列的同源性约80%或以上、81%或以上、82%或以上、83%或以上、84%或以上、85%或以上、86%或以上、87%或以上、88%或以上、89%或以上、90%或以上、91%或以上、92%或以上、93%或以上、94%或以上、95%或以上、96%或以上、97%或以上、98%或以上、99%或以上、99.1%或以上、99.2%或以上、99.3%或以上、99.4%或以上、99.5%或以上、99.6%或以上、99.7%或以上、99.8%或以上、或99.9%或以上仍具有编码Trwc蛋白酶功能的核苷酸序列。
在本发明一种具体实施方式中,所述Trwc酶识别的核酸序列为5’-n-TGCGTATTGTCT-n-3’(SEQ ID NO:3),其中n代表零个、一个或多个碱基。
示例性地,所述n为0-30个碱基,例如n可以为0、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30个碱基。
在本发明一种具体实施方式中,所述Trwc酶识别的核酸序列为ATTGACTTACGCGCACCGAAAGGTGCGTATTGTCTATAGCCCAGTTTA(SEQ ID NO:4),画横线部分为Trwc酶的识别位点。
在本发明一种具体实施方式中,所述Trwc酶识别的核酸序列为ATTGACTTACGCGCACCGAAAGGTGCGTATTGTCTATAGCCCAGTTTAAGGATAGG(SEQ ID NO:5),画横线部分为Trwc酶的识别位点。
进一步,在本发明提供的技术方案的基础上,所述目标蛋白或肽与Trwc酶直接连接或通过柔性连接肽连接形成融合蛋白。
所述柔性连接肽的序列可依据目标肽或蛋白进行选择。
示例性地,柔性连接肽的序列,例如可为GGGSGGSG、GGGGS、GGGG、GSGGSG、(GGGGS)2、(GGGGS)3、(GGGGS)4、(GGGGS)5、(GGGGS)6、GGGGSGGG、GSGGSGGG、GSGGSGGGSGGSGGG、GGGGSGGGSGG等。
在本发明的一个具体实施方式中,所述目标蛋白或肽与DNA拓扑异构酶通过柔性连接肽连接,优选柔性连接肽的序列为GGGGS。
本发明第二方面提供了一种蛋白核酸复合物,包括目标蛋白或肽、Trwc酶与目标核酸,所述目标蛋白或肽与目标核酸通过Trwc酶的催化作用而连接;其中,所述目标蛋白或肽与Trwc酶形成融合蛋白。
进一步,所述目标蛋白或肽与Trwc酶直接连接或通过柔性连接肽连接;更优选通过柔性连接肽连接。
进一步,所述Trwc酶的氨基酸序列如SEQ ID NO.1所示或其同源序列。
在本发明的一个具体实施方式中,所述目标蛋白为链球菌G蛋白、荧光蛋白ECFP、荧光蛋白Venus、丙酮酸氧化酶、磷酸乙酰转移酶中的一种。
蛋白核酸复合物不会影响或增强目标蛋白的功能。示例性地,荧光蛋白与目标核酸形成复合物后,经过检测发现其显示的荧光强度不变或者荧光强度增强。某蛋白酶与目标核酸形成复合物后,蛋白酶的活性不受影响或酶活力增强。
蛋白核酸复合物可以应用于蛋白类药物的靶向运输。示例性地,蛋白类药物作为生物大分子不易进入肿瘤细胞,并且在体内循环过程中较易被生物降解。DNA结构可被用作蛋白类生物大分子的运输载体,例如将DNA结构和蛋白类药物制备成蛋白核酸药物复合物,具有结构可设计,尺寸可控,位点精确定位,生物相容性好,无明显细胞毒性,易于功能化修饰等优势,用以实现对蛋白类药物的靶向运输和可控释放,具有重大的理论和实际意义。
本发明第三方面提供了蛋白核酸复合物的制备方法,包括以下步骤:
通过基因重组将目标蛋白与Trwc酶融合形成融合蛋白,并将所述融合蛋白与目标核酸混合,反应后获得蛋白核酸复合物。
在本发明的一个具体实施方式中,所述制备方法还包括在融合蛋白与目标核酸相混合的混合物中加入金属离子,例如镁离子、钙离子、锰离子等,优选镁离子。
在本发明的一个具体实施方式中,所述蛋白核酸复合物的制备方法包括:(1)融合蛋白的克隆:通过分子克隆将目标蛋白基因与Trwc酶基因融合形成融合蛋白的基因;
(2)融合蛋白的表达、纯化:将融合蛋白的基因克隆入表达载体,并将构建的表达载体转化到宿主细胞表达株中进行诱导表达,即获得融合蛋白;
(3)将融合蛋白与目标核酸混合,加入镁离子,37度反应30min,即可获得核酸共价连接的蛋白。
在本发明一种具体实施方式中,SPG蛋白-目标核酸复合物的制备方法包括以下步骤:
(1)融合蛋白的克隆:通过分子克隆将SPG蛋白基因与Trwc酶基因融合形成融合蛋白的基因;
(2)融合蛋白的表达、纯化:将融合蛋白的基因克隆入表达载体,并将构建的表达载体转化到宿主细胞表达株中进行诱导表达,即获得融合蛋白;
(3)将融合蛋白与目标核酸(SEQ ID NO.4)混合,加入终浓度为1mM的镁离子,37度反应30min,即可获得核酸共价连接的蛋白。
在本发明一种具体实施方式中,ECFP蛋白-目标核酸复合物的制备方法包括以下步骤:
(1)融合蛋白的克隆:通过分子克隆将ECFP蛋白基因与Trwc酶基因融合形成融合蛋白的基因;
(2)融合蛋白的表达、纯化:将融合蛋白的基因克隆入表达载体,并将构建的表达载体转化到宿主细胞表达株中进行诱导表达,即获得融合蛋白;
(3)将融合蛋白与目标核酸(SEQ ID NO.5)混合,加入终浓度为1mM的镁离子,37度反应30min,即可获得核酸共价连接的蛋白。
本发明第四方面提供了一种融合蛋白,所述融合蛋白包括目标蛋白或肽和Trwc酶。
示例性地,融合蛋白选自链球菌G蛋白-Trwc融合蛋白、荧光蛋白ECFP-Trwc融合蛋白、荧光蛋白Venus-Trwc融合蛋白、丙酮酸氧化酶-Trwc融合蛋白、磷酸乙酰转移酶-Trwc融合蛋白。
将目标蛋白与trwc酶融合的方案,可替代为使用其他方式连接,如:化学修饰或非共价结合等。
进一步,所述目标蛋白或肽与所述DNA拓扑异构酶直接连接或通过柔性连接肽连接;更优选通过柔性连接肽连接。
融合蛋白作为制备蛋白核酸复合物的中间产物,仅需融合一个小蛋白(Trwc酶仅有约293个氨基酸组成),对目标蛋白几乎无影响,融合蛋白不仅会保留目标蛋白与Trwc酶两者的功能,而且可能会增强目标蛋白的功能。
本发明第五方面提供了Trwc酶在目标蛋白或肽与目标核酸连接中的用途。
本发明采用上述技术方案具有以下有益效果:
(1)本发明提供了一种蛋白或肽与核酸共价连接的方法,利用Trwc酶能够识别、剪切和连接特定的目标核酸序列且能与目标核酸共价连接的性质,实现了稳定、高效、定点、定向共价连接目标蛋白或肽与目标核酸的目的,克服了目前现有技术中众多蛋白核酸连接出现的问题。
(2)本发明提供的蛋白或肽与核酸共价连接的方法不会影响目标蛋白或肽与目标核酸的结构或性能,可以应用于增强目标蛋白的功能、蛋白类药物的靶向运输等方面。
(3)本发明提供了蛋白核酸复合物及其制备方法,通过Trwc酶催化连接的方式获得,反应条件温和,反应步骤简单,耗时短,无需引入任何化学试剂,只需加入金属离子,适合产业化推广。
附图说明
图1所示为所示为本发明Trwc酶催化共价连接目标核酸与目标蛋白的原理示意图。其中,POI表示目标蛋白。
图2所示为融合蛋白SPG-Trwc和SPG-Trwc-核酸复合物的电泳结果图。其中,泳道1为融合蛋白SPG-Trwc对照;泳道2为SPG-Trwc-核酸复合物。
图3所示为融合蛋白ECFP-Trwc和ECFP-Trwc-核酸复合物的电泳结果图。其中,泳道1为融合蛋白ECFP-Trwc对照;泳道2为ECFP-Trwc-核酸复合物。
图4所示为实施例5中验证Trwc蛋白酶突变前后稳定性差异结果图。
具体实施方式
除非另有定义,本发明中所使用的所有科学和技术术语具有与本发明涉及技术领域的技术人员通常理解的相同的含义。
术语“Trwc酶”包括其相关突变体或者部分结构域,所述突变体或部分结构域仍然具有Trwc酶的功能,能够催化目标蛋白或肽与目标核酸进行连接。
术语“目标蛋白”可为任意的蛋白,例如目标蛋白可以为链球菌G蛋白、荧光蛋白ECFP、荧光蛋白Venus、丙酮酸氧化酶、磷酸乙酰转移酶中的一种,也可根据需要进行自由选择。
术语“目标核酸”可为任意的核酸序列,只需包含Trwc酶识别的核酸序列5’-TGCGTATTGTCT-3’,对于核酸序列的长度不作具体限定。
在本文中所披露的范围的端点和任何值都不限于该精确的范围或值,这些范围或值应当理解为包含接近这些范围或值的值。对于数值范围来说,各个范围的端点值之间、各个范围的端点值和单独的点值之间,以及单独的点值之间可以彼此组合而得到一个或多个新的数值范围,这些数值范围应被视为在本文中具体公开。
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
下面结合具体实施例详细描述本发明,这些实施例用于理解而不是限制本发明。
本发明中Trwc酶催化共价连接目标核酸与目标蛋白的原理如图1所示。
实施例1SPG与Trwc酶的融合、表达
本实施例中以SPG,(链球菌G蛋白,Streptococcus Protein G)作为目标蛋白,将其与Trwc酶进行融合与表达。
(1)融合蛋白的克隆
人工合成SPG与Trwc酶的核苷酸序列,SPG的核苷酸序列如SEQ ID NO:6所示,Trwc酶的核苷酸序列如SEQ ID NO:2所示。
本实施例中SPG与Trwc酶通过柔性连接肽GGGGS连接而形成融合蛋白,简称为SPG-Trwc,其中,融合蛋白SPG-Trwc的核苷酸序列如SEQ ID NO:7所示。
(2)融合蛋白的表达、纯化
将融合蛋白SPG-Trwc的基因克隆入表达载体PET32a(蛋白可在多种表达载体和表达宿主中良好表达,这里仅描述在大肠杆菌中的表达并利用镍亲和层析柱纯化)。将构建的表达载体转化到大肠杆菌表达株BL21(DE3)中,挑取阳性克隆。将挑取的阳性克隆转接到LB培养基,37℃、振荡培养至对数生长期(OD值约为0.5)。向培养物中加入工作终浓度为1mM的IPTG,25℃、振荡培养诱导蛋白表达8小时。Ni亲和层析纯化目标蛋白,即获得纯化的融合蛋白SPG-Trwc,其氨基酸序列如SEQ ID NO:8所示。
实施例2SPG与核酸形成的复合物的制备
将实施例1中纯化所得融合蛋白SPG-Trwc与识别核苷酸序列A:ATTGACTTACGCGCACCGAAAGGTGCGTATTGTCTATAGCCCAGTTTA(SEQ ID NO:4)混合,加入终浓度为1mM的镁离子,于37℃反应30min,即可获得与蛋白核酸复合物SPG-Trwc-核酸。
将所得蛋白核酸复合物SPG-Trwc-核酸进行SDS-PAGE电泳确认,其结果如图2所示。图2中从左向右的通道分别表示:泳道1为融合蛋白对照;泳道2为SPG-Trwc-核酸复合物。从图2中可以看出,通道2中相较于对照存在着明显的滞后条带,说明本实施例中的目标蛋白与目标核酸已共价连接形成蛋白核酸复合物SPG-Trwc-核酸。
实施例3ECFP与Trwc酶的融合、表达
本实施例中以荧光蛋白ECFP作为目标蛋白,将其与Trwc蛋白进行融合与表达。
(1)融合蛋白的克隆:人工合成ECFP与Trwc蛋白的核苷酸序列,ECFP的核苷酸序列如SEQ ID NO:9所示,Trwc酶的核苷酸序列如SEQ ID NO:2所示。
本实施例中ECFP与Trwc酶通过柔性连接肽GGGGS连接而形成融合蛋白,简称为ECFP-Trwc,其中,融合蛋白ECFP-Trwc的核苷酸序列如SEQ ID NO:10所示。
(2)融合蛋白的表达、纯化:将融合蛋白ECFP-Trwc的基因克隆入表达载体PET32a。将构建的表达载体转化到大肠杆菌表达株BL21(DE3)中,挑取阳性克隆。将挑取的阳性克隆转接到LB培养基,37℃、振荡培养至对数生长期(OD值约为0.5)。向培养物中加入工作终浓度为1mM的IPTG,25℃、振荡培养诱导蛋白表达8小时。Ni亲和层析纯化目标蛋白,即获得纯化的融合蛋白ECFP-Trwc,其氨基酸序列如SEQ ID NO:11所示。
实施例4ECFP与核酸形成的复合物的制备
将实施例3纯化所得融合蛋白ECFP-Trwc与识别核苷酸序列B:ATTGACTTACGCGCACCGAAAGGTGCGTATTGTCTATAGCCCAGTTTAAGGATAGG(SEQ ID NO:5)混合,加入终浓度为1mM的镁离子,于37℃反应30min,即可获得与蛋白核酸复合物ECFP-Trwc-核酸。
将所得蛋白核酸复合物ECFP-Trwc-核酸进行SDS-PAGE电泳确认,其结果如图3所示。图3中从左向右的通道分别表示:泳道1为融合蛋白对照;泳道2为ECFP-Trwc-核酸复合物。从图3中可以看出,通道2中相较于对照存在着明显的滞后条带,说明本实施例中的目标蛋白与目标核酸已共价连接形成蛋白核酸复合物ECFP-Trwc-核酸。
实施例5
验证突变前Trwc蛋白酶、与突变后Trwc蛋白酶(突变位点为G13P、G22P、G31P、G141P)的蛋白稳定性差异,将前述突变前后的两种Trwc蛋白酶配制成澄清溶液于4度放置一周后观察,观察结果如图4所示。
从图4可以看出,左侧离心管管内突变后的蛋白溶液依然保持澄清的状态,而右侧离心管管内突变前的蛋白蛋白溶液有明显沉淀现象,证明了突变后的Trwc蛋白酶性质更加稳定。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换等,均应包含在本发明的保护范围之内。
SEQUENCE LISTING
<110> 中国科学院武汉病毒研究所
<120> 蛋白或肽与核酸共价连接的方法
<160> 11
<170> PatentIn version 3.5
<210> 1
<211> 293
<212> PRT
<213> Unknown
<220>
<223> Trwc酶的氨基酸序列
<400> 1
Met Leu Ser His Met Val Leu Thr Arg Gln Asp Ile Pro Arg Ala Ala
1 5 10 15
Ser Tyr Tyr Glu Asp Pro Ala Asp Asp Tyr Tyr Ala Lys Asp Pro Asp
20 25 30
Ala Ser Glu Trp Gln Gly Lys Gly Ala Glu Glu Leu Gly Leu Ser Gly
35 40 45
Glu Val Asp Ser Lys Arg Phe Arg Glu Leu Leu Ala Gly Asn Ile Gly
50 55 60
Glu Gly His Arg Ile Met Arg Ser Ala Thr Arg Gln Asp Ser Lys Glu
65 70 75 80
Arg Ile Gly Leu Asp Leu Thr Phe Ser Ala Pro Lys Ser Val Ser Leu
85 90 95
Gln Ala Leu Val Ala Gly Asp Ala Glu Ile Ile Lys Ala His Asp Arg
100 105 110
Ala Val Ala Arg Thr Leu Glu Gln Ala Glu Ala Arg Ala Gln Ala Arg
115 120 125
Gln Lys Ile Gln Gly Lys Thr Arg Ile Glu Thr Thr Pro Asn Leu Val
130 135 140
Ile Gly Lys Phe Arg His Glu Thr Ser Arg Glu Arg Asp Pro Gln Leu
145 150 155 160
His Thr His Ala Val Ile Leu Asn Met Thr Lys Arg Ser Asp Gly Gln
165 170 175
Trp Arg Ala Leu Lys Asn Asp Glu Ile Val Lys Ala Thr Arg Tyr Leu
180 185 190
Gly Ala Val Tyr Asn Ala Glu Leu Ala His Glu Leu Gln Lys Leu Gly
195 200 205
Tyr Gln Leu Arg Tyr Gly Lys Asp Gly Asn Phe Asp Leu Ala His Ile
210 215 220
Asp Arg Gln Gln Ile Glu Gly Phe Ser Lys Arg Thr Glu Gln Ile Ala
225 230 235 240
Glu Trp Tyr Ala Ala Arg Gly Leu Asp Pro Asn Ser Val Ser Leu Glu
245 250 255
Gln Lys Gln Ala Ala Lys Val Leu Ser Arg Ala Lys Lys Thr Ser Val
260 265 270
Asp Arg Glu Ala Leu Arg Ala Glu Trp Gln Ala Thr Ala Lys Glu Leu
275 280 285
Gly Ile Asp Phe Ser
290
<210> 2
<211> 879
<212> DNA
<213> Unknown
<220>
<223> Trwc酶的核苷酸序列
<400> 2
atgctgagcc atatggtgct gacccgccag gatattccac gtgcggcgag ctattatgaa 60
gatcccgcgg atgattatta tgcgaaagat cccgatgcga gcgaatggca aggtaaaggc 120
gcggaagaat taggtctgag cggcgaagtt gatagcaaac gctttcgcga actgctggcg 180
ggcaacattg gtgaaggcca tcgcattatg cgttcagcga cccgccagga tagcaaagaa 240
cgcattggcc tggatctgac ctttagcgcg ccgaaaagcg ttagcctgca agcgttagtg 300
gcaggcgatg cggaaattat taaagcgcat gatcgcgcgg ttgcgcgcac cttagaacaa 360
gcggaagcgc gtgcacaagc gcgccaaaaa attcagggca aaacccgcat tgaaaccacc 420
ccaaacctgg tgattggcaa atttcgccat gaaaccagcc gtgaacgcga tccgcagtta 480
catacccatg cggtgattct gaacatgacc aaacgcagcg atggtcaatg gcgcgcgctg 540
aaaaacgatg aaattgtgaa agcgacccgc tatctgggcg cggtgtataa tgcggaactg 600
gcgcatgaac tgcagaaact gggctatcag ctgcgctatg gcaaagatgg caactttgat 660
ctggcgcata ttgatcgcca gcagattgaa ggctttagca aacgcaccga acagattgcg 720
gaatggtatg cggcacgcgg cttagatcct aatagcgtga gcctggaaca aaaacaggcg 780
gcgaaagtgt taagccgcgc gaaaaaaacc agcgtggatc gtgaagcgtt acgtgcggaa 840
tggcaggcga ctgcgaaaga actgggcatt gactttagc 879
<210> 3
<211> 14
<212> DNA
<213> Unknown
<220>
<223> Trwc酶识别的核酸序列
<220>
<221> misc_feature
<222> (14)..(14)
<223> n is a, c, g, or t
<400> 3
ntgcgtattg tctn 14
<210> 4
<211> 48
<212> DNA
<213> Unknown
<220>
<223> Trwc酶识别的核酸序列
<400> 4
attgacttac gcgcaccgaa aggtgcgtat tgtctatagc ccagttta 48
<210> 5
<211> 56
<212> DNA
<213> Unknown
<220>
<223> Trwc酶识别的核酸序列
<400> 5
attgacttac gcgcaccgaa aggtgcgtat tgtctatagc ccagtttaag gatagg 56
<210> 6
<211> 171
<212> DNA
<213> Unknown
<220>
<223> SPG的核苷酸序列
<400> 6
atgcagtaca agcttatcct gaacggtaaa accctgaaag gtgaaaccac caccgaagct 60
gttgacgctg ctaccgcgga aaaagttttc aaacagtacg ctaacgacaa cggtgttgac 120
ggtgaatgga cctacgacga cgctaccaaa accttcacgg taaccgagga t 171
<210> 7
<211> 1080
<212> DNA
<213> Unknown
<220>
<223> 融合蛋白SPG-Trwc的核苷酸序列
<400> 7
atgcagtaca agcttatcct gaacggtaaa accctgaaag gtgaaaccac caccgaagct 60
gttgacgctg ctaccgcgga aaaagttttc aaacagtacg ctaacgacaa cggtgttgac 120
ggtgaatgga cctacgacga cgctaccaaa accttcacgg taaccgagga tggtggaggt 180
ggatcgctga gccatatggt gctgacccgc caggatattc cacgtgcggc gagctattat 240
gaagatcccg cggatgatta ttatgcgaaa gatcccgatg cgagcgaatg gcaaggtaaa 300
ggcgcggaag aattaggtct gagcggcgaa gttgatagca aacgctttcg cgaactgctg 360
gcgggcaaca ttggtgaagg ccatcgcatt atgcgttcag cgacccgcca ggatagcaaa 420
gaacgcattg gcctggatct gacctttagc gcgccgaaaa gcgttagcct gcaagcgtta 480
gtggcaggcg atgcggaaat tattaaagcg catgatcgcg cggttgcgcg caccttagaa 540
caagcggaag cgcgtgcaca agcgcgccaa aaaattcagg gcaaaacccg cattgaaacc 600
accccaaacc tggtgattgg caaatttcgc catgaaacca gccgtgaacg cgatccgcag 660
ttacataccc atgcggtgat tctgaacatg accaaacgca gcgatggtca atggcgcgcg 720
ctgaaaaacg atgaaattgt gaaagcgacc cgctatctgg gcgcggtgta taatgcggaa 780
ctggcgcatg aactgcagaa actgggctat cagctgcgct atggcaaaga tggcaacttt 840
gatctggcgc atattgatcg ccagcagatt gaaggcttta gcaaacgcac cgaacagatt 900
gcggaatggt atgcggcacg cggcttagat cctaatagcg tgagcctgga acaaaaacag 960
gcggcgaaag tgttaagccg cgcgaaaaaa accagcgtgg atcgtgaagc gttacgtgcg 1020
gaatggcagg cgactgcgaa agaactgggc attgacttta gccaccacca ccaccaccac 1080
<210> 8
<211> 360
<212> PRT
<213> Unknown
<220>
<223> 融合蛋白SPG-Trwc的氨基酸序列
<400> 8
Met Gln Tyr Lys Leu Ile Leu Asn Gly Lys Thr Leu Lys Gly Glu Thr
1 5 10 15
Thr Thr Glu Ala Val Asp Ala Ala Thr Ala Glu Lys Val Phe Lys Gln
20 25 30
Tyr Ala Asn Asp Asn Gly Val Asp Gly Glu Trp Thr Tyr Asp Asp Ala
35 40 45
Thr Lys Thr Phe Thr Val Thr Glu Asp Gly Gly Gly Gly Ser Leu Ser
50 55 60
His Met Val Leu Thr Arg Gln Asp Ile Pro Arg Ala Ala Ser Tyr Tyr
65 70 75 80
Glu Asp Pro Ala Asp Asp Tyr Tyr Ala Lys Asp Pro Asp Ala Ser Glu
85 90 95
Trp Gln Gly Lys Gly Ala Glu Glu Leu Gly Leu Ser Gly Glu Val Asp
100 105 110
Ser Lys Arg Phe Arg Glu Leu Leu Ala Gly Asn Ile Gly Glu Gly His
115 120 125
Arg Ile Met Arg Ser Ala Thr Arg Gln Asp Ser Lys Glu Arg Ile Gly
130 135 140
Leu Asp Leu Thr Phe Ser Ala Pro Lys Ser Val Ser Leu Gln Ala Leu
145 150 155 160
Val Ala Gly Asp Ala Glu Ile Ile Lys Ala His Asp Arg Ala Val Ala
165 170 175
Arg Thr Leu Glu Gln Ala Glu Ala Arg Ala Gln Ala Arg Gln Lys Ile
180 185 190
Gln Gly Lys Thr Arg Ile Glu Thr Thr Pro Asn Leu Val Ile Gly Lys
195 200 205
Phe Arg His Glu Thr Ser Arg Glu Arg Asp Pro Gln Leu His Thr His
210 215 220
Ala Val Ile Leu Asn Met Thr Lys Arg Ser Asp Gly Gln Trp Arg Ala
225 230 235 240
Leu Lys Asn Asp Glu Ile Val Lys Ala Thr Arg Tyr Leu Gly Ala Val
245 250 255
Tyr Asn Ala Glu Leu Ala His Glu Leu Gln Lys Leu Gly Tyr Gln Leu
260 265 270
Arg Tyr Gly Lys Asp Gly Asn Phe Asp Leu Ala His Ile Asp Arg Gln
275 280 285
Gln Ile Glu Gly Phe Ser Lys Arg Thr Glu Gln Ile Ala Glu Trp Tyr
290 295 300
Ala Ala Arg Gly Leu Asp Pro Asn Ser Val Ser Leu Glu Gln Lys Gln
305 310 315 320
Ala Ala Lys Val Leu Ser Arg Ala Lys Lys Thr Ser Val Asp Arg Glu
325 330 335
Ala Leu Arg Ala Glu Trp Gln Ala Thr Ala Lys Glu Leu Gly Ile Asp
340 345 350
Phe Ser His His His His His His
355 360
<210> 9
<211> 720
<212> DNA
<213> Unknown
<220>
<223> ECFP的核苷酸序列
<400> 9
atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60
ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120
ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180
ctcgtgacca ccctgacctg gggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240
cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300
ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360
gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420
aagctggagt acaactacat cagccacaac gtctatatca cggccgacaa gcagaagaac 480
ggcatcaagg cgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540
gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600
tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660
ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 720
<210> 10
<211> 1668
<212> DNA
<213> Unknown
<220>
<223> 融合蛋白ECFP-Trwc的核苷酸序列
<400> 10
atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60
ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120
ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180
ctcgtgacca ccctgacctg gggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240
cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300
ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360
gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420
aagctggagt acaactacat cagccacaac gtctatatca cggccgacaa gcagaagaac 480
ggcatcaagg cgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540
gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600
tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660
ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagggt 720
ggaggtggat cgggtggagg tggatcggga tccgaaaacc tttacttcca aggcctgagc 780
catatggtgc tgacccgcca ggatattcca cgtgcggcga gctattatga agatcccgcg 840
gatgattatt atgcgaaaga tcccgatgcg agcgaatggc aaggtaaagg cgcggaagaa 900
ttaggtctga gcggcgaagt tgatagcaaa cgctttcgcg aactgctggc gggcaacatt 960
ggtgaaggcc atcgcattat gcgttcagcg acccgccagg atagcaaaga acgcattggc 1020
ctggatctga cctttagcgc gccgaaaagc gttagcctgc aagcgttagt ggcaggcgat 1080
gcggaaatta ttaaagcgca tgatcgcgcg gttgcgcgca ccttagaaca agcggaagcg 1140
cgtgcacaag cgcgccaaaa aattcagggc aaaacccgca ttgaaaccac cccaaacctg 1200
gtgattggca aatttcgcca tgaaaccagc cgtgaacgcg atccgcagtt acatacccat 1260
gcggtgattc tgaacatgac caaacgcagc gatggtcaat ggcgcgcgct gaaaaacgat 1320
gaaattgtga aagcgacccg ctatctgggc gcggtgtata atgcggaact ggcgcatgaa 1380
ctgcagaaac tgggctatca gctgcgctat ggcaaagatg gcaactttga tctggcgcat 1440
attgatcgcc agcagattga aggctttagc aaacgcaccg aacagattgc ggaatggtat 1500
gcggcacgcg gcttagatcc taatagcgtg agcctggaac aaaaacaggc ggcgaaagtg 1560
ttaagccgcg cgaaaaaaac cagcgtggat cgtgaagcgt tacgtgcgga atggcaggcg 1620
actgcgaaag aactgggcat tgactttagc caccaccacc accaccac 1668
<210> 11
<211> 556
<212> PRT
<213> Unknown
<220>
<223> 融合蛋白ECFP-Trwc的氨基酸序列
<400> 11
Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
1 5 10 15
Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly
20 25 30
Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile
35 40 45
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
50 55 60
Leu Thr Trp Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys
65 70 75 80
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu
85 90 95
Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu
100 105 110
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
115 120 125
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
130 135 140
Asn Tyr Ile Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn
145 150 155 160
Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser
165 170 175
Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly
180 185 190
Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu
195 200 205
Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe
210 215 220
Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Gly
225 230 235 240
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Ser Glu Asn Leu Tyr Phe
245 250 255
Gln Gly Leu Ser His Met Val Leu Thr Arg Gln Asp Ile Pro Arg Ala
260 265 270
Ala Ser Tyr Tyr Glu Asp Pro Ala Asp Asp Tyr Tyr Ala Lys Asp Pro
275 280 285
Asp Ala Ser Glu Trp Gln Gly Lys Gly Ala Glu Glu Leu Gly Leu Ser
290 295 300
Gly Glu Val Asp Ser Lys Arg Phe Arg Glu Leu Leu Ala Gly Asn Ile
305 310 315 320
Gly Glu Gly His Arg Ile Met Arg Ser Ala Thr Arg Gln Asp Ser Lys
325 330 335
Glu Arg Ile Gly Leu Asp Leu Thr Phe Ser Ala Pro Lys Ser Val Ser
340 345 350
Leu Gln Ala Leu Val Ala Gly Asp Ala Glu Ile Ile Lys Ala His Asp
355 360 365
Arg Ala Val Ala Arg Thr Leu Glu Gln Ala Glu Ala Arg Ala Gln Ala
370 375 380
Arg Gln Lys Ile Gln Gly Lys Thr Arg Ile Glu Thr Thr Pro Asn Leu
385 390 395 400
Val Ile Gly Lys Phe Arg His Glu Thr Ser Arg Glu Arg Asp Pro Gln
405 410 415
Leu His Thr His Ala Val Ile Leu Asn Met Thr Lys Arg Ser Asp Gly
420 425 430
Gln Trp Arg Ala Leu Lys Asn Asp Glu Ile Val Lys Ala Thr Arg Tyr
435 440 445
Leu Gly Ala Val Tyr Asn Ala Glu Leu Ala His Glu Leu Gln Lys Leu
450 455 460
Gly Tyr Gln Leu Arg Tyr Gly Lys Asp Gly Asn Phe Asp Leu Ala His
465 470 475 480
Ile Asp Arg Gln Gln Ile Glu Gly Phe Ser Lys Arg Thr Glu Gln Ile
485 490 495
Ala Glu Trp Tyr Ala Ala Arg Gly Leu Asp Pro Asn Ser Val Ser Leu
500 505 510
Glu Gln Lys Gln Ala Ala Lys Val Leu Ser Arg Ala Lys Lys Thr Ser
515 520 525
Val Asp Arg Glu Ala Leu Arg Ala Glu Trp Gln Ala Thr Ala Lys Glu
530 535 540
Leu Gly Ile Asp Phe Ser His His His His His His
545 550 555
Claims (11)
1.蛋白或肽与核酸共价连接的方法,其特征在于,包括以下步骤:
(1)目标蛋白或肽与Trwc酶形成融合蛋白;
(2)融合蛋白中Trwc酶催化目标蛋白或肽与目标核酸共价连接;
所述Trwc酶的氨基酸序列如SEQ ID NO.1所示。
2.根据权利要求1所述的方法,其特征在于,所述Trwc酶识别的核酸序列为5’-n-TGCGTATTGTCT-n-3’,其中n代表零个、一个或多个碱基。
3.根据权利要求2所述的方法,其特征在于,所述n为0-30个碱基。
4.根据权利要求1-3中任一项所述的方法,其特征在于,所述目标蛋白或肽与Trwc酶直接连接或通过柔性连接肽连接形成融合蛋白。
5.一种蛋白核酸复合物,其特征在于,包括目标蛋白或肽、Trwc酶与目标核酸,所述目标蛋白或肽与目标核酸通过Trwc酶的催化连接;其中,所述目标蛋白或肽与Trwc酶形成融合蛋白;
所述Trwc酶的氨基酸序列如SEQ ID NO.1所示。
6.权利要求5所述复合物的制备方法,其特征在于,包括以下步骤:
通过基因重组将目标蛋白与Trwc酶融合形成融合蛋白,并将所述融合蛋白与目标核酸混合,反应后获得蛋白核酸复合物。
7.一种融合蛋白,其特征在于,所述融合蛋白包括目标蛋白或肽和Trwc酶;所述Trwc酶的氨基酸序列如SEQ ID NO.1所示。
8.根据权利要求7所述的融合蛋白,其特征在于,所述目标蛋白或肽与Trwc酶直接连接或通过柔性连接肽连接。
9.Trwc酶在目标蛋白或肽与目标核酸连接中的用途,其特征在于,所述Trwc酶的氨基酸序列如SEQ ID NO.1所示。
10.一种Trwc酶,其特征在于,所述Trwc酶的氨基酸序列如SEQ ID NO.1所示。
11.编码权利要求10所述Trwc酶的基因。
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