CN114057893A - 一种编码线粒体定位的豆蔻酰化多肽及其制备方法与应用 - Google Patents
一种编码线粒体定位的豆蔻酰化多肽及其制备方法与应用 Download PDFInfo
- Publication number
- CN114057893A CN114057893A CN202111307669.1A CN202111307669A CN114057893A CN 114057893 A CN114057893 A CN 114057893A CN 202111307669 A CN202111307669 A CN 202111307669A CN 114057893 A CN114057893 A CN 114057893A
- Authority
- CN
- China
- Prior art keywords
- polypeptide
- amino acid
- acid sequence
- seq
- myristoylation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70571—Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/033—Fusion polypeptide containing a localisation/targetting motif containing a motif for targeting to the internal surface of the plasma membrane, e.g. containing a myristoylation motif
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/07—Fusion polypeptide containing a localisation/targetting motif containing a mitochondrial localisation signal
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Plant Pathology (AREA)
- Microbiology (AREA)
- Optics & Photonics (AREA)
- Cell Biology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Neurology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明提供了一种编码线粒体定位豆蔻酰化的多肽及其制备方法与应用,属于分子遗传学技术领域。本发明所述多肽序列具有如M‑N式表示的氨基酸序列,其中,M为SEQ ID No.1所示的氨基酸序列以及N为SEQ ID No.2所示的氨基酸序列。本发明包括序列如SEQ ID No.3所述的多肽、多肽组合物、核酸、多肽偶联物在表达豆蔻酰化线粒体定位蛋白中的应用。本发明所述的序列的氨基末端带有豆蔻酰化保守序列,能够被豆蔻酰化,进而***在线粒体细胞膜中。
Description
技术领域
本发明涉及分子遗传学技术领域,尤其是指一种编码线粒体定位的豆蔻酰化多肽及其制备方法与应用。
背景技术
本发明属于分子遗传学领域。线粒体功能研究依赖于线粒体共定位的标记基因和相关荧光染料。但是,如何将脂肪酸修饰的目的蛋白人为导入线粒体,是目前研究的难点。
观察和研究线粒体功能,依赖于线粒体定位的标记基因和荧光染料。同时,将目的基因和线粒体导肽融合,能够将目的基因编码的目的蛋白导入线粒体。这些方法使得研究线粒体功能变得简单和直接。
脂肪酸修饰的目的蛋白(即豆蔻酰化)导入线粒体,是目前相关技术的难点。现有的线粒体导肽,不能将脂肪酸修饰的外源蛋白导入线粒体。
发明内容
为解决上述技术问题,本发明提供了一种编码线粒体定位的豆蔻酰化的多肽及其制备方法。
一种编码线粒体定位的豆蔻酰化的多肽,所述多肽序列具有如M-N式表示的氨基酸序列,且所述氨基酸序列的氨基末端进行了豆蔻酰化,其中, M为SEQ ID No.1所示的氨基酸序列以及N为SEQ ID No.2所示的氨基酸序列,或具有与所述M-N式表示的氨基酸序列具有至少70%同源性的氨基酸序列。
进一步的,所述多肽的氨基酸序列如SEQ ID No.3所示。
一种多肽组合物,所述多肽组合物包括所述氨基酸序列如SEQ ID No.3 所示的多肽。
一种核酸,所述核酸含有氨基酸序列如SEQ ID No.3所示的多肽的编码序列。
一种多肽偶联物,所述多肽偶联物包括多肽部分,其中多肽部分含有所述的多肽。
进一步的,所述多肽偶联物还包括偶联部分,所述偶联部分与所述多肽部分连接。
进一步的,所述偶联部分选自血蓝蛋白、卵清蛋白及牛血清白蛋白中的至少一种。
所述的多肽、所述的多肽组合物、所述的核酸、所述多肽偶联物在制备用于靶向定位亚细胞器荧光成像的荧光探针中的应用。
所述的多肽、所述的多肽组合物、所述的核酸、所述多肽偶联物在表达线粒体定位蛋白中的应用。
进一步的,所述亚细胞器包括线粒体。
本发明的上述技术方案相比现有技术具有以下优点:
本发明所述的序列的氨基末端带有豆蔻酰化保守序列,能够被豆蔻酰化,进而***在线粒体细胞膜中。同时本发明序列羧基末端的序列,来源于 NMDA受体GluN1亚基的C0区域,C0区域能够结合Calmodulin,进而促进和线粒体的结合。可见,本发明中得到的序列可以靶向定位线粒体。
附图说明
为了使本发明的内容更容易被清楚的理解,下面根据本发明的具体实施例并结合附图,对本发明作进一步详细的说明,其中。
图1是本发明实施例2中豆蔻酰化线粒体定位多肽序列的定位表达荧光图;
图2是本发明实施例3豆蔻酰化线粒体定位的GFP融合质粒 (myr-mito-GFP)与商业化的线粒体定位质粒(Mito-RFP)的荧光图。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明,以使本领域的技术人员可以更好地理解本发明并能予以实施,但所举实施例不作为对本发明的限定。
实施例1
豆蔻酰化线粒体定位多肽序列筛选和确认:
(1)豆蔻酰化保守序列来源于蛋白MARCKS前端序列:MGAQFSK。
其对应的核苷酸为:ATGGGTGCCCAGTTCTCCAAG。
(2)定制带有豆蔻酰化保守序列的GluN1 C0区域的PCR引物,其序列如下:
GluN1:5’-3’(带有豆蔻酰化保守序列):TCGGCTAGCACCATG GGTGCCCAGTTCTCCAAGATCGCCTACAAGCGACACAAGGATGCC; 3’-5’:AGCTGTCGACCTGCAGGTTCTTCCTCCACACGTTCAC。
(3)GluN1质粒作为模板,进行常规PCR。(PCR试剂盒购于宝生物技术(TaKaRa)有限公司)体系。PCR体系(25μL)如下(单位:μL):
10×Buffer:2.5,
dNTP:2,
Taq:0.5;
primer:2;
vector:300ng。
(4)进行PCR,其中PCR的条件如下:
(4.1)一个循环,95℃30秒;
(4.2)30个循环,95℃30秒,58℃30秒,72℃2分钟;
(4.3)一个循环,72℃10分钟。
(5)进行琼脂糖凝胶电泳,核酸胶回收后使用NEB快切酶Nhe I和 Sal I酶切20分钟。核酸胶回收酶切后的产物。
(6)NEB快切酶Nhe I和Sal I酶切pEGFP-N3载体1-3小时,经过核酸胶回收酶切后的载体。
(7)利用宝生物技术(TaKaRa)有限公司连接酶Solution I,在16度条件下,连接1-2小时(具体方法见产品说明)。
(8)连接产物加入感受态细胞DH5α(全式金生物)中,先在冰上孵育20分钟,接着42度热激1分钟,最后在冰上放置2-5分钟,涂板后挑菌测序正确。
实施例2
豆蔻酰化线粒体定位多肽序列(myr-mito)的表达:
首先将HEK293细胞接种于24孔板中,接着在24小时后,利用lipo2000 转染试剂以每孔300ng的质粒量将豆蔻酰化线粒体定位序列(myr-mito)的 GFP融合质粒转染进HEK293细胞,转染24小时后荧光显微镜观察其定位 (比例尺:20微米)。可以看出,本发明中带有豆蔻酰化线粒体定位序列能够在HEK293细胞中表达,并且呈现出线粒体定位。
实施例3
豆蔻酰化线粒体定位多肽序列(myr-mito)的定位:
首先将HEK293细胞接种于24孔板中,接着在24小时后,利用lipo2000 转染试剂以每孔300ng的质粒量将豆蔻酰化线粒体定位序列(myr-mito)的 GFP融合质粒和商业化的线粒体定位质粒(Mito-RFP)或内质网定位质粒(ER-RFP)转染进HEK293细胞,转染24小时后荧光显微镜观察其定位(比例尺:20微米)。可以看出,本发明中带有豆蔻酰化线粒体定位序列能够地定位在线粒体,而不是内质网。
SEQ ID No.1:Met GlyAla Gln Phe Ser Lys;
SEQ ID No.2:Ile Ala Tyr Lys Arg His Lys Asp Ala Arg Arg Lys Gln MetGln LeuAla PheAlaAlaValAsnVal TrpArg Lys Asn Leu Gln;
SEQ ID No.3:Met Gly Ala Gln Phe Ser Lys Ile Ala Tyr Lys Arg His LysAsp AlaArgArg Lys Gln Met Gln LeuAla Phe AlaAla Val Asn Val Trp Arg Lys AsnLeu Gln。
显然,上述实施例仅仅是为清楚地说明所作的举例,并非对实施方式的限定。对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其它不同形式变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
SEQUENCE LISTING
<110> 苏州大学
<120> 一种编码线粒体定位的豆蔻酰化的多肽及其制备方法与应用
<130> 3
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 7
<212> PRT
<213> (人工序列)
<400> 1
Met Gly Ala Gln Phe Ser Lys
1 5
<210> 2
<211> 29
<212> PRT
<213> (人工序列)
<400> 2
Ile Ala Tyr Lys Arg His Lys Asp Ala Arg Arg Lys Gln Met Gln Leu
1 5 10 15
Ala Phe Ala Ala Val Asn Val Trp Arg Lys Asn Leu Gln
20 25
<210> 3
<211> 36
<212> PRT
<213> (人工序列)
<400> 3
Met Gly Ala Gln Phe Ser Lys Ile Ala Tyr Lys Arg His Lys Asp Ala
1 5 10 15
Arg Arg Lys Gln Met Gln Leu Ala Phe Ala Ala Val Asn Val Trp Arg
20 25 30
Lys Asn Leu Gln
35
Claims (10)
1.一种编码线粒体定位豆蔻酰化的多肽,其特征在于:所述多肽序列具有如M-N式表示的氨基酸序列,且所述氨基酸序列的氨基末端进行了豆蔻酰化,其中,M为SEQ ID No.1所示的氨基酸序列以及N为SEQ ID No.2所示的氨基酸序列,或具有与所述M-N式表示的氨基酸序列具有至少70%同源性的氨基酸序列。
2.根据权利要求1所述的编码线粒体定位的豆蔻酰化多肽,其特征在于:所述多肽的氨基酸序列如SEQ ID No.3所示。
3.一种多肽组合物,其特征在于:所述多肽组合物包括权利要求2中所述氨基酸序列如SEQ ID No.3所示的多肽。
4.一种核酸,其特征在于:所述核酸含有氨基酸序列如SEQ ID No.3所示的多肽的编码序列。
5.一种多肽偶联物,其特征在于:所述多肽偶联物包括多肽部分,其中多肽部分含有权利要求1-2任一项所述的多肽。
6.根据权利要求5所述的多肽偶联物,其特征在于:所述多肽偶联物还包括偶联部分,所述偶联部分与所述多肽部分连接。
7.根据权利要求6所述的多肽偶联物,其特征在于:所述偶联部分选自血蓝蛋白、卵清蛋白及牛血清白蛋白中的至少一种。
8.权利要求1-2任一项所述的多肽、权利要求3所述的多肽组合物、权利要求4所述的核酸、权利要求5-7任一项所述多肽偶联物在制备用于靶向定位亚细胞器荧光成像的荧光探针中的应用。
9.权利要求1-2任一项所述的多肽、权利要求3所述的多肽组合物、权利要求4所述的核酸、权利要求5-7任一项所述多肽偶联物在表达线粒体定位蛋白中的应用。
10.根据权利要求8所述的应用,其特征在于:所述亚细胞器包括线粒体。
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN2021104549855 | 2021-04-26 | ||
CN202110454985 | 2021-04-26 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN114057893A true CN114057893A (zh) | 2022-02-18 |
CN114057893B CN114057893B (zh) | 2022-12-30 |
Family
ID=80274762
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202111307669.1A Active CN114057893B (zh) | 2021-04-26 | 2021-11-05 | 一种编码线粒体定位的豆蔻酰化多肽及其制备方法与应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN114057893B (zh) |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1998040477A1 (en) * | 1997-03-14 | 1998-09-17 | The Regents Of The University Of California | Fluorescent protein sensors for detection of analytes |
CN107446027A (zh) * | 2017-09-05 | 2017-12-08 | 苏州大学 | 具有核定位能力的透皮短肽及其应用 |
US20180126003A1 (en) * | 2016-05-04 | 2018-05-10 | Curevac Ag | New targets for rna therapeutics |
CN109575116A (zh) * | 2018-11-09 | 2019-04-05 | 广东海洋大学 | 一种线粒体定位导肽及其发现方法与应用 |
WO2020101740A1 (en) * | 2018-11-16 | 2020-05-22 | Codiak Biosciences, Inc. | Engineered extracellular vesicles and uses thereof |
-
2021
- 2021-11-05 CN CN202111307669.1A patent/CN114057893B/zh active Active
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1998040477A1 (en) * | 1997-03-14 | 1998-09-17 | The Regents Of The University Of California | Fluorescent protein sensors for detection of analytes |
US20180126003A1 (en) * | 2016-05-04 | 2018-05-10 | Curevac Ag | New targets for rna therapeutics |
CN107446027A (zh) * | 2017-09-05 | 2017-12-08 | 苏州大学 | 具有核定位能力的透皮短肽及其应用 |
CN109575116A (zh) * | 2018-11-09 | 2019-04-05 | 广东海洋大学 | 一种线粒体定位导肽及其发现方法与应用 |
WO2020101740A1 (en) * | 2018-11-16 | 2020-05-22 | Codiak Biosciences, Inc. | Engineered extracellular vesicles and uses thereof |
Also Published As
Publication number | Publication date |
---|---|
CN114057893B (zh) | 2022-12-30 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP2023156355A (ja) | 細胞または生物のゲノムへのDNA配列の標的化組み込みのためのCas9レトロウイルスインテグラーゼおよびCas9レコンビナーゼ系 | |
CA3024184A1 (en) | Variants of a dna polymerase of the polx family | |
KR101915740B1 (ko) | 목적 단백질의 발현 효율을 증진시키기 위한 신규한 펩타이드 및 이를 포함하는 융합 단백질 | |
CN108165593A (zh) | 胶原蛋白7及相关方法 | |
KR20190002363A (ko) | 콜라겐을 생산하기 위한 효모 균주 및 방법 | |
CN109153990A (zh) | 用于基因编辑的组合物和方法 | |
US20200308239A1 (en) | Genetically encoded fluorescent-iron ferritin nanoparticle probes for detecting an intracellular target by fluorescent and electron microscopy | |
CN113249408B (zh) | 一种靶向激活体液免疫和细胞免疫的核酸疫苗载体构建及应用 | |
CN113307886A (zh) | 一种编码线粒体定位的豆蔻酰化多肽及其制备方法与应用 | |
KR102014826B1 (ko) | 목적 단백질의 발현 효율을 증진시키기 위한 신규한 펩타이드 및 이를 포함하는 융합 단백질 | |
CN114057893B (zh) | 一种编码线粒体定位的豆蔻酰化多肽及其制备方法与应用 | |
Anderson et al. | Construction and expression of a synthetic wheat storage protein gene | |
US7994148B2 (en) | Transmembrane delivery peptide and bio-material comprising the same | |
CN114057861A (zh) | 一种靶向UBE2C的bio-PROTAC人工蛋白 | |
CN113661247A (zh) | 细胞穿透转座酶 | |
CN105198999A (zh) | 一种融合蛋白、其制备方法及其应用 | |
KR102617593B1 (ko) | 바이러스 뉴클레오캡시드를 이용한 목적 단백질 발현 플랫폼 | |
CN114213549B (zh) | 定位于线粒体的融合蛋白、接头及其用途 | |
CN109777807A (zh) | 一种在线粒体中定位表达外源蛋白的方法 | |
Pichon et al. | A method for the large-scale cloning of nuclear proteins and nuclear targeting sequences on a functional basis | |
CN114075554B (zh) | NCre/CCre***及Cre-GFP融合蛋白的构建方法与应用 | |
WO2004016748A2 (en) | Biotinylation of proteins | |
RU2380373C2 (ru) | ПЕПТИД DED И ЕГО ПРИМЕНЕНИЕ ДЛЯ ИДЕНТИФИКАЦИИ И/ИЛИ ОЧИСТКИ РЕКОМБИНАНТНЫХ БЕЛКОВ, ВЕКТОР pDED | |
CN109336957B (zh) | 用于检测人参根尖干细胞的特异性基因PgWOX5及其检测方法与应用 | |
CN115960247A (zh) | 基于t7rna聚合酶的融合多肽及其制备方法、用途 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |