WO2022246934A1 - Myristoylated polypetide encoding mitochondrial localization, and preparation method therefor and use thereof - Google Patents
Myristoylated polypetide encoding mitochondrial localization, and preparation method therefor and use thereof Download PDFInfo
- Publication number
- WO2022246934A1 WO2022246934A1 PCT/CN2021/101154 CN2021101154W WO2022246934A1 WO 2022246934 A1 WO2022246934 A1 WO 2022246934A1 CN 2021101154 W CN2021101154 W CN 2021101154W WO 2022246934 A1 WO2022246934 A1 WO 2022246934A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- polypeptide
- amino acid
- acid sequence
- myristoylated
- seq
- Prior art date
Links
- 230000025608 mitochondrion localization Effects 0.000 title claims abstract description 11
- 238000002360 preparation method Methods 0.000 title abstract description 5
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 56
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 55
- 229920001184 polypeptide Polymers 0.000 claims abstract description 53
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 20
- 230000002438 mitochondrial effect Effects 0.000 claims abstract description 12
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 12
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 12
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 12
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 11
- 239000000203 mixture Substances 0.000 claims abstract description 9
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 7
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims abstract description 4
- 210000003470 mitochondria Anatomy 0.000 claims description 10
- 230000007498 myristoylation Effects 0.000 claims description 10
- 230000008878 coupling Effects 0.000 claims description 5
- 238000010168 coupling process Methods 0.000 claims description 5
- 238000005859 coupling reaction Methods 0.000 claims description 5
- 239000007850 fluorescent dye Substances 0.000 claims description 4
- 210000003463 organelle Anatomy 0.000 claims description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 2
- 108091026890 Coding region Proteins 0.000 claims description 2
- 108010058846 Ovalbumin Proteins 0.000 claims description 2
- 229940098773 bovine serum albumin Drugs 0.000 claims description 2
- 238000000799 fluorescence microscopy Methods 0.000 claims description 2
- 108060003552 hemocyanin Proteins 0.000 claims description 2
- 229940092253 ovalbumin Drugs 0.000 claims description 2
- 210000000170 cell membrane Anatomy 0.000 abstract description 2
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 abstract 1
- 239000013612 plasmid Substances 0.000 description 9
- 210000004027 cell Anatomy 0.000 description 6
- 102100022645 Glutamate receptor ionotropic, NMDA 1 Human genes 0.000 description 3
- 108091036078 conserved sequence Proteins 0.000 description 3
- 230000004927 fusion Effects 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 230000004807 localization Effects 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 230000004898 mitochondrial function Effects 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 101710121995 Glutamate receptor ionotropic, NMDA 1 Proteins 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 239000012096 transfection reagent Substances 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 102000000584 Calmodulin Human genes 0.000 description 1
- 108010041952 Calmodulin Proteins 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 102000015695 Myristoylated Alanine-Rich C Kinase Substrate Human genes 0.000 description 1
- 108010063737 Myristoylated Alanine-Rich C Kinase Substrate Proteins 0.000 description 1
- 238000009004 PCR Kit Methods 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 1
- 230000030583 endoplasmic reticulum localization Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002073 fluorescence micrograph Methods 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
-
- 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
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
-
- 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
- C07K14/76—Albumins
- C07K14/77—Ovalbumin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5076—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving cell organelles, e.g. Golgi complex, endoplasmic reticulum
- G01N33/5079—Mitochondria
-
- 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
Definitions
- the invention relates to the technical field of molecular genetics, in particular to a myristoylated polypeptide encoding mitochondrial location and its preparation method and application.
- the present invention belongs to the field of molecular genetics. Mitochondrial function studies rely on mitochondrial co-localized marker genes and associated fluorescent dyes. However, how to artificially introduce fatty acid-modified target proteins into mitochondria is a difficult point in current research.
- Observing and studying mitochondrial function relies on mitochondrial localized marker genes and fluorescent dyes. At the same time, by fusing the target gene with the mitochondrial guide peptide, the target protein encoded by the target gene can be introduced into the mitochondria. These methods make studying mitochondrial function simple and straightforward.
- fatty acid-modified target protein i.e. myristoylation
- the present invention provides a polypeptide encoding mitochondria-localized myristoylation and a preparation method thereof.
- a polypeptide encoding mitochondrial localized myristoylation has an amino acid sequence represented by the formula M-N, and the amino terminus of the amino acid sequence is myristoylated, wherein M is shown in SEQ ID No.1 and N is the amino acid sequence shown in SEQ ID No.2, or has an amino acid sequence with at least 70% homology with the amino acid sequence represented by the M-N formula.
- amino acid sequence of the polypeptide is shown in SEQ ID No.3.
- polypeptide composition comprising said amino acid sequence as shown in SEQ ID No.3 polypeptide.
- nucleic acid contains the coding sequence of the polypeptide with amino acid sequence as shown in SEQ ID No.3.
- polypeptide conjugate includes a polypeptide part, wherein the polypeptide part contains the polypeptide.
- polypeptide conjugate further includes a coupling part, and the coupling part is connected to the polypeptide part.
- the coupling moiety is at least one selected from hemocyanin, ovalbumin and bovine serum albumin.
- polypeptide for targeting and localizing subcellular organelle fluorescence imaging.
- subcellular organelles include mitochondria.
- the amino terminal of the sequence described in the present invention has a myristoylation conservative sequence, which can be myristoylated and then inserted into the mitochondrial cell membrane.
- the carboxy-terminal sequence of the sequence of the present invention is derived from the C0 region of the NMDA receptor GluN1 subunit, and the C0 region can bind Calmodulin, thereby promoting the combination with mitochondria. It can be seen that the sequence obtained in the present invention can target mitochondria.
- Fig. 1 is the localized expression fluorescence map of the myristoylated mitochondrial localized polypeptide sequence in Example 2 of the present invention
- Fig. 2 is a fluorescence image of the myristoylated mitochondrial localization GFP fusion plasmid (myr-mito-GFP) and the commercial mitochondrial localization plasmid (Mito-RFP) in Example 3 of the present invention.
- the myristoylation conserved sequence is derived from the front sequence of protein MARCKS: MGAQFSK.
- the corresponding nucleotide is: ATGGGTGCCCAGTTCTCCAAG.
- GluN1 5'-3' (with myristoylation conserved sequence): TCGGCTAGCACCATG GGTGCCCAGTTCTCCAAGATCGCCTACAAGCGACACAAGGATGCC; 3'-5': AGCTGTCGACCTGCAGGTTCTTCCTCCACACGTTCAC.
- the GluN1 plasmid was used as a template for conventional PCR.
- the PCR kit was purchased from TaKaRa Co., Ltd.) system.
- the PCR system (25 ⁇ L) is as follows (unit: ⁇ L):
- NEB fast cutting enzymes Nhe I and Sal I digest the pEGFP-N3 vector for 1-3 hours, and recover the digested vector through nucleic acid gel.
- the ligation product was added to the competent cell DH5 ⁇ (full type gold bio), first incubated on ice for 20 minutes, then heat-shocked at 42 degrees for 1 minute, and finally placed on ice for 2-5 minutes, and the bacteria were picked and sequenced after plating. correct.
- myristoylated mitochondria-localized polypeptide sequence (myr-mito):
- HEK293 cells were seeded in 24-well plates, and then 24 hours later, the GFP fusion plasmid of myristoylated mitochondrial localization sequence (myr-mito) was transfected into HEK293 cells with lipo2000 transfection reagent at a plasmid quantity of 300 ng per well , 24 hours after transfection, the localization was observed by fluorescence microscope (scale bar: 20 ⁇ m). It can be seen that the mitochondrial localization sequence with myristoylation in the present invention can be expressed in HEK293 cells, and exhibits mitochondrial localization.
- myr-mito myristoylated mitochondrial localization sequence
- HEK293 cells were seeded in 24-well plates, and then 24 hours later, the GFP fusion plasmid of myristoylated mitochondrial localization sequence (myr-mito) and commercial mitochondria were transfected with lipo2000 transfection reagent at a plasmid quantity of 300 ng per well.
- the localization plasmid (Mito-RFP) or endoplasmic reticulum localization plasmid (ER-RFP) was transfected into HEK293 cells, and its localization was observed by fluorescence microscope 24 hours after transfection (scale bar: 20 ⁇ m). It can be seen that the mitochondrial localization sequence with myristoylation in the present invention can be localized to the mitochondria instead of the endoplasmic reticulum.
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Biomedical Technology (AREA)
- Toxicology (AREA)
- Hematology (AREA)
- Zoology (AREA)
- Urology & Nephrology (AREA)
- Microbiology (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Provided are a myristoylated polypeptide encoding mitochondrial localization, and a preparation method therefor and the use thereof, belonging to the technical field of molecular genetics. The polypeptide sequence has an amino acid sequence as represented by formula M-N, wherein M is an amino acid sequence as shown in SEQ ID NO: 1 and N is an amino acid sequence as shown in SEQ ID NO: 2. Further provided is the use of a polypeptide comprising a sequence as shown in SEQ ID NO: 3, a polypeptide composition, a nucleic acid and a polypeptide conjugate in the expression of a myristoylated mitochondrial localization protein. The amino terminus of the sequence contains a myristoylated conservative sequence, which can be myristoylated and then inserted into a mitochondrial cell membrane.
Description
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。The invention relates to the technical field of molecular genetics, in particular to a myristoylated polypeptide encoding mitochondrial location and its preparation method and application.
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。The present invention belongs to the field of molecular genetics. Mitochondrial function studies rely on mitochondrial co-localized marker genes and associated fluorescent dyes. However, how to artificially introduce fatty acid-modified target proteins into mitochondria is a difficult point in current research.
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。Observing and studying mitochondrial function relies on mitochondrial localized marker genes and fluorescent dyes. At the same time, by fusing the target gene with the mitochondrial guide peptide, the target protein encoded by the target gene can be introduced into the mitochondria. These methods make studying mitochondrial function simple and straightforward.
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。The introduction of fatty acid-modified target protein (i.e. myristoylation) into mitochondria is a difficult point in current related technologies. Existing mitochondrial guiding peptides cannot introduce fatty acid-modified foreign proteins into mitochondria.
发明内容Contents of the invention
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。In order to solve the above technical problems, the present invention provides a polypeptide encoding mitochondria-localized myristoylation and a preparation method thereof.
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。A polypeptide encoding mitochondrial localized myristoylation, the polypeptide sequence has an amino acid sequence represented by the formula M-N, and the amino terminus of the amino acid sequence is myristoylated, wherein M is shown in SEQ ID No.1 and N is the amino acid sequence shown in SEQ ID No.2, or has an amino acid sequence with at least 70% homology with the amino acid sequence represented by the M-N formula.
进一步的，所述多肽的氨基酸序列如SEQ ID No.3所示。Further, the amino acid sequence of the polypeptide is shown in SEQ ID No.3.
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。A polypeptide composition, said polypeptide composition comprising said amino acid sequence as shown in SEQ ID No.3 polypeptide.
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。A nucleic acid, said nucleic acid contains the coding sequence of the polypeptide with amino acid sequence as shown in SEQ ID No.3.
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。A polypeptide conjugate, the polypeptide conjugate includes a polypeptide part, wherein the polypeptide part contains the polypeptide.
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。Further, the polypeptide conjugate further includes a coupling part, and the coupling part is connected to the polypeptide part.
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。Further, the coupling moiety is at least one selected from hemocyanin, ovalbumin and bovine serum albumin.
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。Application of the polypeptide, the polypeptide composition, the nucleic acid, and the polypeptide conjugate in the preparation of fluorescent probes for targeting and localizing subcellular organelle fluorescence imaging.
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。The application of the polypeptide, the polypeptide composition, the nucleic acid, and the polypeptide conjugate in expressing a mitochondria-localized protein.
进一步的，所述亚细胞器包括线粒体。Further, the subcellular organelles include mitochondria.
本发明的上述技术方案相比现有技术具有以下优点：The above technical solution of the present invention has the following advantages compared with the prior art:
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，进而插入在线粒体细胞膜中。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。The amino terminal of the sequence described in the present invention has a myristoylation conservative sequence, which can be myristoylated and then inserted into the mitochondrial cell membrane. At the same time, the carboxy-terminal sequence of the sequence of the present invention is derived from the C0 region of the NMDA receptor GluN1 subunit, and the C0 region can bind Calmodulin, thereby promoting the combination with mitochondria. It can be seen that the sequence obtained in the present invention can target mitochondria.
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。In order to make the content of the present invention more clearly understood, the present invention will be further described in detail below according to the specific embodiments of the present invention and in conjunction with the accompanying drawings.
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；Fig. 1 is the localized expression fluorescence map of the myristoylated mitochondrial localized polypeptide sequence in Example 2 of the present invention;
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。Fig. 2 is a fluorescence image of the myristoylated mitochondrial localization GFP fusion plasmid (myr-mito-GFP) and the commercial mitochondrial localization plasmid (Mito-RFP) in Example 3 of the present invention.
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。The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and implement it, but the examples given are not intended to limit the present invention.
实施例1Example 1
豆蔻酰化线粒体定位多肽序列筛选和确认：Sequence screening and confirmation of myristoylated mitochondria-localized peptides:
(1)豆蔻酰化保守序列来源于蛋白MARCKS前端序列：MGAQFSK。(1) The myristoylation conserved sequence is derived from the front sequence of protein MARCKS: MGAQFSK.
其对应的核苷酸为：ATGGGTGCCCAGTTCTCCAAG。The corresponding nucleotide is: ATGGGTGCCCAGTTCTCCAAG.
(2)定制带有豆蔻酰化保守序列的GluN1C0区域的PCR引物，其序列如下：(2) Customize PCR primers for the GluN1C0 region with myristoylation conserved sequence, the sequence of which is as follows:
GluN1:5’-3’(带有豆蔻酰化保守序列)：TCGGCTAGCACCATG GGTGCCCAGTTCTCCAAGATCGCCTACAAGCGACACAAGGATGCC；3’-5’：AGCTGTCGACCTGCAGGTTCTTCCTCCACACGTTCAC。GluN1: 5'-3' (with myristoylation conserved sequence): 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)：(3) The GluN1 plasmid was used as a template for conventional PCR. (The PCR kit was purchased from TaKaRa Co., Ltd.) system. The PCR system (25 μL) is as follows (unit: μL):
10×Buffer:2.5,10×Buffer: 2.5,
dNTP:2,dNTP: 2,
Taq:0.5；Taq: 0.5;
primer:2；primer: 2;
vector:300ng。vector: 300ng.
(4)进行PCR，其中PCR的条件如下：(4) Carry out PCR, wherein the conditions of PCR are as follows:
(4.1)一个循环，95℃30秒；(4.1) One cycle, 95°C for 30 seconds;
(4.2)30个循环，95℃30秒，58℃30秒，72℃2分钟；(4.2) 30 cycles, 95°C for 30 seconds, 58°C for 30 seconds, 72°C for 2 minutes;
(4.3)一个循环，72℃10分钟。(4.3) One cycle, 72°C for 10 minutes.
(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。(5) Perform agarose gel electrophoresis, and use NEB fast-cutting enzymes Nhe I and Sal I to digest for 20 minutes after the nucleic acid gel is recovered. Nucleic acid gel to recover the digested product.
(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。(6) NEB fast cutting enzymes Nhe I and Sal I digest the pEGFP-N3 vector for 1-3 hours, and recover the digested vector through nucleic acid gel.
(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)。(7) Use Ligase Solution I of TaKaRa Co., Ltd., and connect for 1-2 hours at 16 degrees (see product description for specific methods).
(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。(8) The ligation product was added to the competent cell DH5α (full type gold bio), first incubated on ice for 20 minutes, then heat-shocked at 42 degrees for 1 minute, and finally placed on ice for 2-5 minutes, and the bacteria were picked and sequenced after plating. correct.
实施例2Example 2
豆蔻酰化线粒体定位多肽序列(myr-mito)的表达：Expression of myristoylated mitochondria-localized polypeptide sequence (myr-mito):
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。First, HEK293 cells were seeded in 24-well plates, and then 24 hours later, the GFP fusion plasmid of myristoylated mitochondrial localization sequence (myr-mito) was transfected into HEK293 cells with lipo2000 transfection reagent at a plasmid quantity of 300 ng per well , 24 hours after transfection, the localization was observed by fluorescence microscope (scale bar: 20 μm). It can be seen that the mitochondrial localization sequence with myristoylation in the present invention can be expressed in HEK293 cells, and exhibits mitochondrial localization.
实施例3Example 3
豆蔻酰化线粒体定位多肽序列(myr-mito)的定位：Location of myristoylated mitochondrial targeting polypeptide sequence (myr-mito):
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。First, HEK293 cells were seeded in 24-well plates, and then 24 hours later, the GFP fusion plasmid of myristoylated mitochondrial localization sequence (myr-mito) and commercial mitochondria were transfected with lipo2000 transfection reagent at a plasmid quantity of 300 ng per well. The localization plasmid (Mito-RFP) or endoplasmic reticulum localization plasmid (ER-RFP) was transfected into HEK293 cells, and its localization was observed by fluorescence microscope 24 hours after transfection (scale bar: 20 μm). It can be seen that the mitochondrial localization sequence with myristoylation in the present invention can be localized to the mitochondria instead of the endoplasmic reticulum.
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。Apparently, the above-mentioned embodiments are only examples for clear description, and are not intended to limit the implementation. For those of ordinary skill in the art, on the basis of the above description, other changes or changes in various forms can also be made. It is not necessary and impossible to exhaustively list all the implementation manners here. However, the obvious changes or changes derived therefrom are still within the scope of protection of the present invention.
Claims (10)
- 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。A polypeptide encoding mitochondrial localized myristoylation, characterized in that: the polypeptide sequence has an amino acid sequence represented by the formula M-N, and the amino terminal of the amino acid sequence is myristoylated, wherein M is SEQ ID No. The amino acid sequence shown in 1 and N is the amino acid sequence shown in SEQ ID No. 2, or an amino acid sequence having at least 70% homology with the amino acid sequence represented by the M-N formula.
- 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。The myristoylated polypeptide encoding mitochondrial localization according to claim 1, characterized in that: the amino acid sequence of the polypeptide is as shown in SEQ ID No.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。A polypeptide composition, characterized in that: said polypeptide composition comprises a polypeptide having the amino acid sequence described in claim 2 as shown in SEQ ID No.3.
- 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。A nucleic acid, characterized in that: the nucleic acid contains the coding sequence of the polypeptide whose amino acid sequence is shown in SEQ ID No.3.
- 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。A polypeptide conjugate, characterized in that: the polypeptide conjugate comprises a polypeptide part, wherein the polypeptide part contains the polypeptide according to any one of claims 1-2.
- 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。The polypeptide conjugate according to claim 5, characterized in that: the polypeptide conjugate further comprises a coupling part connected to the polypeptide part.
- 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。The polypeptide conjugate according to claim 6, characterized in that: the coupling moiety is selected from at least one of hemocyanin, ovalbumin and bovine serum albumin.
- 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。The polypeptide according to any one of claims 1-2, the polypeptide composition according to claim 3, the nucleic acid according to claim 4, and the polypeptide conjugate according to any one of claims 5-7 are prepared for target Application of Fluorescent Probes to Localize Subcellular Organelles for Fluorescence Imaging.
- 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。The polypeptide according to any one of claims 1-2, the polypeptide composition according to claim 3, the nucleic acid according to claim 4, and the polypeptide conjugate according to any one of claims 5-7 express mitochondrial localized proteins in the application.
- 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。The use according to claim 8, characterized in that: the subcellular organelles include mitochondria.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110594511.0A CN113307886A (en) | 2021-05-28 | 2021-05-28 | Myristoylation polypeptide for encoding mitochondrial localization and preparation method and application thereof |
| CN202110594511.0 | 2021-05-28 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2022246934A1 true WO2022246934A1 (en) | 2022-12-01 |
Family
ID=77376472
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/CN2021/101154 WO2022246934A1 (en) | 2021-05-28 | 2021-06-21 | Myristoylated polypetide encoding mitochondrial localization, and preparation method therefor and use thereof |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN113307886A (en) |
| WO (1) | WO2022246934A1 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114134129B (en) * | 2021-11-19 | 2022-11-29 | 上海生物芯片有限公司 | Mitochondrial localization polypeptide, localization system and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109996539A (en) * | 2016-09-26 | 2019-07-09 | 青岛普美圣医药科技有限公司 | N-methyl-D-aspartate receptor allosteric modulators and its application method |
| WO2020191361A2 (en) * | 2019-03-21 | 2020-09-24 | Codiak Biosciences, Inc. | Extracellular vesicles for vaccine delivery |
| CN112899307A (en) * | 2021-01-28 | 2021-06-04 | 苏州大学 | Use of GluN1 and/or GluN2 subunit carboxy-terminal myristoylated NMDA receptors |
-
2021
- 2021-05-28 CN CN202110594511.0A patent/CN113307886A/en active Pending
- 2021-06-21 WO PCT/CN2021/101154 patent/WO2022246934A1/en active Application Filing
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109996539A (en) * | 2016-09-26 | 2019-07-09 | 青岛普美圣医药科技有限公司 | N-methyl-D-aspartate receptor allosteric modulators and its application method |
| WO2020191361A2 (en) * | 2019-03-21 | 2020-09-24 | Codiak Biosciences, Inc. | Extracellular vesicles for vaccine delivery |
| CN112899307A (en) * | 2021-01-28 | 2021-06-04 | 苏州大学 | Use of GluN1 and/or GluN2 subunit carboxy-terminal myristoylated NMDA receptors |
Non-Patent Citations (3)
| Title |
|---|
| AICH ABHISHEK, SHAHA CHANDRIMA: "Novel Role of Calmodulin in Regulating Protein Transport to Mitochondria in a Unicellular Eukaryote", MOLECULAR AND CELLULAR BIOLOGY, AMERICAN SOCIETY FOR PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS, US, vol. 33, no. 22, 15 November 2013 (2013-11-15), US , pages 4579 - 4593, XP093007338, ISSN: 0270-7306, DOI: 10.1128/MCB.00829-13 * |
| DATABASE PROTEIN 22 March 2015 (2015-03-22), ANONYMOUS : "Glutamate [NMDA] receptor subunit zeta-1 [Myotis brandtii]", XP093007337, retrieved from NCBI Database accession no. EPQ17422.1 * |
| XU ZHENGPING, LI BOLIANG: "Myristoylation of Protein", PROGRESS IN BIOCHEMISTRY AND BIOPHYSICS, vol. 25, no. 1, 31 December 1998 (1998-12-31), XP093007245 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113307886A (en) | 2021-08-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20220315952A1 (en) | Cas 9 retroviral integrase and cas 9 recombinase systems for targeted incorporation of a dna sequence into a genome of a cell or organism | |
| CN108165593A (en) | Collagen 7 and correlation technique | |
| US8993742B2 (en) | Tubulo-vesicular structure localization signals | |
| WO2022246934A1 (en) | Myristoylated polypetide encoding mitochondrial localization, and preparation method therefor and use thereof | |
| US20200308239A1 (en) | Genetically encoded fluorescent-iron ferritin nanoparticle probes for detecting an intracellular target by fluorescent and electron microscopy | |
| WO2018082231A1 (en) | Method for establishing cd36 mutant gene stable eukaryotic expression cell line causing cd36 deletion | |
| WO2023071934A1 (en) | Novel crispr enzyme and system, and application | |
| Kataoka et al. | Identification of the factors that interact with NCBP, an 80 kDa nuclear cap binding protein | |
| CN113249408B (en) | Construction and application of nucleic acid vaccine vector for targeting activation of humoral immunity and cellular immunity | |
| US8609373B2 (en) | Fusion protein mixture for inducing human pluripotent stem cell and preparation method there of | |
| CN112708605A (en) | Proteome obtained by splitting Cas9 protein and application thereof | |
| CN110551193B (en) | Novel tag protein for protein enrichment expression and intracellular localization and application thereof | |
| CN114057893B (en) | Myristoylation polypeptide for encoding mitochondrial localization and preparation method and application thereof | |
| CN114057861B (en) | bio-PROTAC artificial protein targeting UBE2C | |
| EP2275567A1 (en) | Nucleic acid expression construct and its use as a cell proliferation marker | |
| US7994148B2 (en) | Transmembrane delivery peptide and bio-material comprising the same | |
| Hara et al. | Prion domain interaction responsible for species discrimination in yeast [PSI+] transmission | |
| CN113396221A (en) | Beta-galactosidase alpha peptide as non-antibiotic selection marker and application thereof | |
| CN112899307A (en) | Use of GluN1 and/or GluN2 subunit carboxy-terminal myristoylated NMDA receptors | |
| Biermanns et al. | The peroxisomal membrane targeting elements of human peroxin 2 (PEX2) | |
| JP2022520283A (en) | Cell-permeable transposase | |
| JP4243680B2 (en) | Foreign insert selection marker | |
| Pichon et al. | A method for the large-scale cloning of nuclear proteins and nuclear targeting sequences on a functional basis | |
| JP4024525B2 (en) | Fusion protein consisting of two different selectable markers | |
| CN114213549B (en) | Fusion proteins, linkers, and uses thereof that localize to mitochondria |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 21942504 Country of ref document: EP Kind code of ref document: A1 |
|
| NENP | Non-entry into the national phase |
Ref country code: DE |
|
| 122 | Ep: pct application non-entry in european phase |
Ref document number: 21942504 Country of ref document: EP Kind code of ref document: A1 |