CN112759626B - 一种核定位信号肽及其序列和应用 - Google Patents
一种核定位信号肽及其序列和应用 Download PDFInfo
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Abstract
本发明公开了一种核定位信号肽及其序列和应用，所述信号肽的序列为PGPIRCRHRSKVS，编码所述信号肽的基因，得到信号肽的基因序列。所述的信号肽基因序列为5’‑CCGGGTCCTATCCGCTGCAGGCATCGATCGAAGGTTTCC‑3’。采用多肽作为核定位信号肽将目的蛋白定位于细胞核中，所述多肽序列为PGPIRCRHRSKVS。采用多肽作为核定位信号肽将目的蛋白定位于细胞核核仁中，所述多肽序列为PGPIRCRHRSKVS的2~5个串联。本发明是一种未被发现的入核信号序列，并且串联之后可以特异性定位于核仁区域。
Description
技术领域
本发明涉及生物钟转录抑制因子CHRONO蛋白核定位NLS序列，以及该NLS序列在表达载体领域的应用技术领域，尤其涉及一种核定位信号肽及其序列和应用。
背景技术
将外源基因人工导入细胞的转基因技术是一种重要的技术，不仅仅是一种分析各种生物现象的基本技术，而且因为其在诸如基因治疗和有益动物生产方面的应用。
生物钟系统的核心主要是由BMAL1/CLOCK构成的转录激活复合物与转录抑制因子PER和CRY构成的负反馈调控环路。在2014年，研究发现这个负反馈环路中还存在另外一个全新的核心生物钟元件——CHRONO。虽然研究发现CHRONO是一个转录抑制因子，但是其在负反馈环路中执行功能的分子机制、对于生物钟系统的影响，以及CHRONO对于生物钟下游其它生理活动的影响仍不清楚。为了全面认识生物钟的调控系统，我们对于CHRONO展开了探索研究。CHRONO作为一个脊椎动物特有的基因，目前并没有同源基因或者蛋白的研究。CHRONO作为转录因子，需要进入细胞核内执行功能，而对于CHRONO的入核机制却没有任何信息。
发明内容
本发明目的就是为了弥补已有技术的缺陷，提供一种核定位信号肽及其序列和应用，可将基因导入细胞核中。
本发明是通过以下技术方案实现的：
CHRONO作为转录因子，进入细胞核对于CHRONO来说是至关重要的。为了研究其入核的机制，通过对一系列截短体或者突变体与cpVenus融合蛋白的在不同细胞中的定位统计发现，CHRONO入核是依靠其N端的Pro Gly Pro Ile Arg Cys Arg His Arg Ser Lys ValSer序列，该核定位信号与经典的单簇信号K(K/R)X(K/R)相似，然而之前对CHRONO或者直接对该序列进行经软件预测后并未判定为核定位序列，说明CHRONO上发现的Pro Gly ProIle Arg Cys Arg His Arg Ser Lys Val Ser序列是一个新型的核定位序列。
将NLS信号与cpVenus蛋白融合表达，cpVenus获得细胞核定位的能力。
一种核定位信号肽，所述信号肽的序列为Pro Gly Pro Ile Arg Cys Arg HisArg Ser Lys Val Ser，编码所述信号肽的基因，得到信号肽的基因序列。
所述的信号肽基因序列为5’-CCGGGTCCTATCCGCTGCAGGCATCGATCGAAGGTTTCC-3’。
一种重组表达载体，载体包括所述的信号肽基因。
所述信号肽基因包括多个拷贝，即信号肽基因是通过多个信号肽基因序列并列组成的。
所述的多个拷贝是无间断的连续拷贝。
一种核定位信号肽的应用，采用多肽作为核定位信号肽将目的蛋白定位于细胞核中，所述多肽序列为Pro Gly Pro Ile Arg Cys Arg His Arg Ser Lys Val Ser。
一种核定位信号肽的应用，采用多肽作为核定位信号肽将目的蛋白定位于细胞核核仁中，所述多肽序列为Pro Gly Pro Ile Arg Cys Arg His Arg Ser Lys Val Ser的2～5个串联。
本发明的优点是：本发明得到了未被发现的入核信号序列，并且串联之后可以特异性定位于核仁区域，采用多肽作为核定位信号肽将目的蛋白定位于细胞核中，采用多肽作为核定位信号肽将目的蛋白定位于细胞核核仁中，应用广泛。
附图说明
图1为构建的系列小肽在细胞内的分布与统计结果图。A为系列融合蛋白在细胞内的定位图，B为系列融合蛋白在细胞内分布的统计结果，C为不同融合蛋白在核内定位的差异分析结果。
图2为DAPI染核，行荧光共聚焦分析结果图。
具体实施方式
一种核定位信号肽，所述信号肽的序列为Pro Gly Pro Ile Arg Cys Arg Hi sArg Ser Lys Val Ser，编码所述信号肽的基因，得到信号肽的基因序列。
所述的信号肽基因序列为5’-CCGGGTCCTATCCGCTGCAGGCATCGATCG AAGGTTTCC-3’。
一种重组表达载体，载体包括所述的信号肽基因。
所述信号肽基因包括多个拷贝。
所述的多个拷贝是无间断的连续拷贝。
一种核定位信号肽的应用，采用多肽作为核定位信号肽将目的蛋白定位于细胞核中，所述多肽序列为Pro Gly Pro Ile Arg Cys Arg His Arg Ser Lys Val Ser。
一种核定位信号肽的应用，采用多肽作为核定位信号肽将目的蛋白定位于细胞核核仁中，所述多肽序列为Pro Gly Pro Ile Arg Cys Arg His Arg Ser Lys Val Ser的2～5个串联。
具体实施例如下：
质粒与细胞：pcDNA3.1(+)/Hygro-cpVenus与pEGFP-N1质粒为实验室保存质粒。HEK-293T、NIH 3T3、Hela和U2-OS细胞购于中科院上海细胞所。大肠杆菌DH5α菌种购自全式金公司。
各种抗体：抗Nucleoin抗体购自CST公司；Cy3标记山羊抗兔IgG二抗购自生工生物工程股份有限公司。
各种酶类与试剂：各种T4连接酶和限制性内切酶购于Thermo Scientific的FastDigest系列产品；DNA Polymerase购于Vazyme公司；去内毒素质粒提取试剂盒购于康为世纪公司；DMEM培养基购于GE公司；胎牛血清购于PAN-Biotech公司；Lipo8000转染试剂购于Bioyotime公司；DAPI购自Sigma公司。其它普通试剂购自国产试剂公司。
实施例1将确定CHRONO中的NLS序列：Pro Gly Pro Ile Arg Cys Arg His ArgSer Lys Val Ser。
1材料与方法
1.1质粒
pEGFP-N1为市售产品；pcDNA3.1(+)/Hygro-CHRONO-cpVenus、pcDNA3.1(+)/Hygro-01/02/03/04/05/06/07/08/09-cpVenus的真核表达载体为实验室自己制备，将质粒转化大肠杆菌DH5α，提取质粒DNA，用DNA/RNA定量仪测定DNA浓度，置-20℃保存备用。
1.2 01～09小肽融合cpVenus蛋白的定位分析
1.2.1CHRONO蛋白序列的分析
利用Predict Protein等软件分析CHRONO蛋白序列并未发现已知的NLS序列。通过人工分析发现CHRONO的N端有较多的正点氨基酸(精氨酸、赖氨酸)富集，表现出类似单簇NLS的特征。
1.2.2pcDNA3.1(+)/Hygro-CHRONO-cpVenus、pcDNA3.1(+)/Hygro-01/02/03/04/0
5/06/07/08/09-cpVenus真核表达载体的构建
利用人源的cDNA进行CHRONO的扩增，利用pcDNA3.1(+)/Hygro-cpVenus为模板扩增cpVenus，利用融合PCR获得CHRONO-cpVenus的融合片段后利用HindⅢ和XhoⅠ插入pcDNA3.1(+)/Hygro的载体中。构建pcDNA3.1(+)/Hygro-01-cpVenus载体后；之后在其基础上构建pcDNA3.1(+)/Hygro-02-cpVenus载体；利用点突变在pcDNA3.1(+)/Hygro-02-cpVenus载体上构建pcDNA3.1(+)/Hygro-03-cpVenus载体；在pcDNA3.1(+)/Hygro-02-cpVenus载体上构建pcDNA3.1(+)/Hygro-04-cpVenus载体；利用点突变在pcDNA3.1(+)/Hygro-04-cpVenus载体上构建pcDNA3.1(+)/Hygro-05-cpVenus载体；利用点突变在pcDNA3.1(+)/Hygro-05-cpVenus载体上构建pcDNA3.1(+)/Hygro-06-cpVenus载体；利用点突变在pcDNA3.1(+)/Hygro-06-cpVenus载体上构建pcDNA3.1(+)/Hygro-07-cpVenus载体；利用点突变在pcDNA3.1(+)/Hygro-06-cpVenus载体上构建pcDNA3.1(+)/Hygro-08-cpVenus载体；在pcDNA3.1(+)/Hygro-04-cpVenus载体上构建pcDNA3.1(+)/Hygro-09-cpVenus载体。
引物：
pcDNA3.1(+)/Hygro-CHRONO-cpVenus-F1：GAGAAGCTTATGGATTCTCCATCTAGCG
pcDNA3.1(+)/Hygro-CHRONO-cpVenus-R1：GCCGCCGTCGGATCCACCACAACCTTGGGTGTTTAGAC
pcDNA3.1(+)/Hygro-CHRONO-cpVenus-F2：CAAGGTTGTGGTGGATCCGACGGCGGCGTGCAGCTCGCC
pcDNA3.1(+)/Hygro-CHRONO-cpVenus-R2：CTACTCGAGTTACTCGATGTTGTGGCGG
pcDNA3.1(+)/Hygro-01-cpVenus-F：GAGAAGCTTATGATCCGCTGCAGGCATCGATCGGGTGGATCCGACGGCGGCGTGC
pcDNA3.1(+)/Hygro-01-cpVenus-R：同pcDNA3.1(+)/Hygro-CHRONO-cpVenus-R2
pcDNA3.1(+)/Hygro-02-cpVenus-F：GAGAAGCTTATGATCCGCTGCAGGCATCGATCGAAGGTTGGTGGATCCGACGGCGGCGTGC
pcDNA3.1(+)/Hygro-02-cpVenus-R：同pcDNA3.1(+)/Hygro-CHRONO-cpVenus-R2
pcDNA3.1(+)/Hygro-03-cpVenus-F：CATCGATCGAAGGTTTCCGGTGGATCCGACGGCG
pcDNA3.1(+)/Hygro-03-cpVenus-R：GCCGTCGGATCCACCGGAAACCTTCGATCGATGC
pcDNA3.1(+)/Hygro-04-cpVenus-F：GAGAAGCTTATGCCGGGTCCTATCCGCTGCAGGCATCGATCGAAGGTTGG
pcDNA3.1(+)/Hygro-04-cpVenus-R：同pcDNA3.1(+)/Hygro-CHRONO-cpVenus-R2
pcDNA3.1(+)/Hygro-05-cpVenus-F：同pcDNA3.1(+)/Hygro-03-cpVenus-F
pcDNA3.1(+)/Hygro-05-cpVenus-R：同pcDNA3.1(+)/Hygro-03-cpVenus-R
pcDNA3.1(+)/Hygro-06-cpVenus-F：CGATCGAAGGTTTCCGGTGGTGGATCCGACGGCG
pcDNA3.1(+)/Hygro-06-cpVenus-R：GCCGTCGGATCCACCACCGGAAACCTTCGATCGA
pcDNA3.1(+)/Hygro-07-cpVenus-F：GATCGAAGGTTTCCGGTAACGGTGGATCCGACGGCG
pcDNA3.1(+)/Hygro-07-cpVenus-R：CCGCCGTCGGATCCACCGTTACCGGAAACCTTCGATpcDNA3.1(+)/Hygro-08-cpVenus-F：GCATCGATCGAAGGTTTCCGGTAACCAGGGTGGATCCGACGGCGGCGTG
pcDNA3.1(+)/Hygro-08-cpVenus-R：GCACGCCGCCGTCGGATCCACCCTGGTTACCGGAAACCTTCGATCGATG
pcDNA3.1(+)/Hygro-09-cpVenus-F：GAGAAGCTTATGCCCAGCCCGGGTCCTATCCGCTGCAGGCATCGATCG
pcDNA3.1(+)/Hygro-09-cpVenus-R：同pcDNA3.1(+)/Hygro-CHRONO-cpVenus-R2
1.2.3pcDNA3.1(+)/Hygro-01/02/03/04/05/06/07/08/09-cpVenus定位分析
将构建的载体转染U2-OS细胞，转染24小时后，4％的多聚甲醛固定细胞，DAPI染核，进行荧光共聚焦分析，结果如图1所示，05信号肽可以定位于细胞核中。
1.2.4pcDNA3.1(+)/Hygro-triple05-cpVenus载体构建
利用BamHⅠ酶切pcDNA3.1(+)/Hygro-05-cpVenus载体后，连接重复的05片段，测序后确定为三个重复的05序列。
引物：
05-F:GATCCCCGGGTCCTATCCGCTGCAGGCATCGATCGAAGGTTTCCGGTG
05-R:GATCCACCGGAAACCTTCGATCGATGCCTGCAGCGGATAGGACCCGGG
1.2.5pcDNA3.1(+)/Hygro-triple05-cpVenus的定位分析
将构建的载体转染U2-OS细胞，转染24小时后，4％的多聚甲醛固定细胞，之后用PBS漂洗三次，每次5分钟；用冰预冷的100％甲醇溶液将细胞覆盖，-20℃下甲醇中孵育10分钟，用PBS漂洗5分钟；在1×PBS/5％正常血清/0.3％TritonX-100的封闭液中封闭标本1小时；吸干封闭液，加入稀释后的一抗；4℃下孵育过夜，用PBS漂洗三次，每次5分钟；加入稀释后的荧光物质标记的二抗，室温下避光孵育1小时，用PBS漂洗三次，每次5分钟；DAPI染核，行荧光共聚焦分析，结果如图2所示，与EGFP和CHRONO相比，05信号肽可以在细胞核与核仁内均有分布，而triple05信号肽定位在细胞核的核仁之中。
序列表
<110> 安徽大学
<120> 一种核定位信号肽及其序列和应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 39
<212> DNA
<213> 人(Homo sapiens)
<400> 1
ccgggtccta tccgctgcag gcatcgatcg aaggtttcc 39
<210> 2
<211> 13
<212> PRT
<213> 人(Homo sapiens)
<400> 2
Pro Gly Pro Ile Arg Cys Arg His Arg Ser Lys Val Ser
1 5 10
Claims (7)
1.一种核定位信号肽，其特征在于：所述信号肽的序列为Pro Gly Pro Ile Arg CysArg His Arg Ser Lys Val Ser。
2.根据权利要求1所述的一种核定位信号肽，其特征在于：所述的信号肽基因序列为5’-CCGGGTCCTATCCGCTGCAGGCATCGATCGAAGGTTTCC-3’。
3.一种重组表达载体，其特征在于：载体包括如权利要求2所述的信号肽基因。
4.根据权利要求3所述的一种重组表达载体，其特征在于：所述信号肽基因包括多个拷贝。
5.根据权利要求4所述的一种重组表达载体，其特征在于：所述的多个拷贝是无间断的连续拷贝。
6.一种核定位信号肽的应用，其特征在于：采用多肽作为核定位信号肽将目的蛋白定位于细胞核中，所述多肽序列为Pro Gly Pro Ile Arg Cys Arg His Arg Ser Lys ValSer。
7.一种核定位信号肽的应用，其特征在于：采用多肽作为核定位信号肽将目的蛋白定位于细胞核核仁中，所述多肽序列为Pro Gly Pro Ile Arg Cys Arg His Arg Ser LysVal Ser的2~5个串联。
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| US6057125A (en) * | 1997-06-30 | 2000-05-02 | The Board Of Trustees Of Northwestern University | Clock gene and gene product |
| US6476188B1 (en) * | 1998-06-19 | 2002-11-05 | The Rockefeller University | Clock gene and methods of use thereof |
| CN111411122A (zh) * | 2020-04-01 | 2020-07-14 | 华南农业大学 | 稻瘟病菌基因MoHXT2在调控植物糖转运功能中的应用 |
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| "Identification of the Repressive Domain of the Negative Circadian Clock Component CHRONO";Yu Yang等;《Int. J. Mol. Sci.》;20200402;第21卷;第1-17页 * |
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