CN110790821B

CN110790821B - Cell nucleus penetrating peptide and application thereof

Info

Publication number: CN110790821B
Application number: CN201911134307.XA
Authority: CN
Inventors: 韦宇平; 张曼; 张鑫; 段明嫦; 禹皓天
Original assignee: Nanyang Normal University
Current assignee: Nanyang Normal University
Priority date: 2019-11-19
Filing date: 2019-11-19
Publication date: 2022-09-02
Anticipated expiration: 2039-11-19
Also published as: CN110790821A

Abstract

The invention belongs to the technical field of biology, and particularly relates to a cell nucleus penetrating peptide and application thereof. The amino acid sequence of the invention is as follows: Lys-Lys-Arg-Pro-Pro-Arg-Lys-Lys. The cell nucleus penetrating peptide can enter nucleolus of cell nucleus through nuclear membrane, and has the advantages of good safety, simple preparation method and convenient quality control.

Description

Cell nucleus penetrating peptide and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a cell nucleus penetrating peptide and application thereof.

Background

The nucleus is a closed, membranous organelle in eukaryotic cells, which contains most of the genetic material in the cell. The structure is mainly nuclear cytoplasm and nuclear membrane, the former is mainly DNA and protein, the latter is a double-layer membrane separating cytoplasm from cytoplasm. Most molecules cannot penetrate nuclear membrane directly, so how to mediate the delivery of exogenous substances to the nucleus has been one of the hot research hotspots in cell biology.

Cell-penetrating peptides (CPPs), or penetrating peptides, are short peptides that efficiently carry macromolecular substances into cells. The partial cell-penetrating peptide can penetrate through cell membranes and nuclear membranes, so that efficient substance delivery is realized, and meanwhile, targeting property and nontoxicity are achieved through artificial sequence design. Therefore, the cell nucleus penetrating peptide is one of the most ideal carriers in the cell nucleus delivery process.

Disclosure of Invention

The present invention provides a novel cell nucleus penetrating peptide which can penetrate the nuclear membrane and enter the nucleolus.

The technical scheme adopted by the invention is as follows: a cell nucleus penetrating peptide having the amino acid sequence: Lys-Lys-Arg-Pro-Pro-Arg-Lys-Lys.

The invention also provides a compound, and the specific technical scheme is as follows: the complex comprises a cell nucleus penetrating peptide as described above and a foreign substance. For example, the nuclear penetrating peptide in the complex may be linked to the foreign substance by covalent or non-covalent bonds for the purpose of allowing the foreign substance to be transported into the nucleus.

Preferably, the foreign substance is a cargo molecule and/or a marker molecule.

Preferably, the cargo molecule is a nucleotide and/or a drug molecule precursor; the marker molecules are fluorescein and/or biotin.

The invention also provides the application of the cell nucleus penetrating peptide, and the specific technical scheme is as follows: use of the cell nucleus penetrating peptide for mediating entry of foreign substances into the cell nucleus.

Preferably, the use of said nuclear penetrating peptide for mediating entry of foreign material into the nuclear nucleoli of a cell.

The beneficial effects of the invention are: the nuclear penetrating peptides of the present invention can penetrate the nuclear membrane into the nuclear nucleoli of the nucleus.

The cell nucleus penetrating peptide only contains 8 amino acids, has no cytotoxicity and immunogenicity, and has good safety.

The cell nucleus penetrating peptide is synthesized by adopting a polypeptide solid phase synthesis method with mature technology, is convenient for quality control, and is cheap and easy to obtain.

After the nuclear penetrating peptide is subjected to fluorescence labeling by FITC, the nuclear penetrating peptide is cultured with adherent cells together, and the measured fluorescence quantity in the cell nucleus can reach 76 times of that of a group cultured with FITC and the adherent cells together.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.

FIG. 1 is a fluorescence microscopic view of example 3 of the present invention, FIG. 1(a) is a fluorescence microscopic view of FITC group, and FIG. 1(b) is a fluorescence microscopic view of FITC-labeled nuclear transit peptide group.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1 Synthesis of Nuclear penetrating peptides of the invention

1) Activating resin: 1000mg of Fmoc-Trp wang Resin was weighed and soaked in 30mL of DMF for 30min to fully swell.

2) Deprotection: and (3) performing suction filtration to remove 30mL of DMF (dimethyl formamide) soaked in the resin, adding 30mL of DMF solution containing 20% of piperidine by mass, blowing nitrogen for 25min, performing suction filtration to remove the DMF solution, alternately washing the resin three times by using 10mL of isopropanol and DMF, and detecting the resin by using an indantrione method, wherein the resin is black or purple.

3) Condensation reaction: connecting the next amino acid, weighing 1.4mmol/g resin of Fmoc-amino acid, taking TBTU/HOBt/DIEA 30mL DMF as reaction liquid, and blowing nitrogen at room temperature for 2 h. After the reaction, the resin was washed three times with 10mL of isopropanol and DMF alternately. And detecting the amino group.

4) Repeating the processes of the steps 2) to 3): the polypeptide is extended from the C-terminus to the N-terminus in the order of the polypeptide. The polypeptide sequence is as follows: Lys-Lys-Arg-Pro-Pro-Arg-Lys-Lys.

5) Polypeptide cleavage: the polypeptide-resin complex was blown dry with nitrogen and mixed to 30mL of cleavage reagent at the ratio of TFA/phenol/ultrapure water/thioanisole/EDT/TIS (80/5/5/5/3/2, vol.). Placing the peptide resin in a round-bottom flask, adding cutting fluid, magnetically stirring for 2h, removing the resin by using a sand core filter, directly dripping the filtrate into frozen ether, centrifugally precipitating at 5000r/min, and freeze-drying to constant weight to obtain the crude peptide.

6) The crude peptide was purified by HPLC under a gradient of 10% to 100% acetonitrile for 30min to give a product purity of 97%. Obtaining the purified polypeptide, wherein the sequence of the polypeptide is as follows: Lys-Lys-Arg-Pro-Pro-Arg-Lys-Lys.

EXAMPLE 2 cytotoxicity test

1) Taking 48-well plate, adding 5 × 10 of the solution into each well ⁴ Hela cell culture solution for cervical cancer at 37 ℃ in a 5% carbon dioxide incubator for 24 hours until the cells adhere to the wall.

2) A medium containing 1mM of cell nucleus-penetrating peptide (Lys-Lys-Arg-Pro-Pro-Arg-Lys-Lys) was prepared, and the medium without cell nucleus-penetrating peptide was used as a negative Control well (Control) and incubated at 37 ℃ for 48 hours with 5% carbon dioxide. The formula of the culture medium is as follows: DMEM medium containing 10% fetal bovine serum.

3) Adherent cells were added with 20. mu.l MTT per well, incubated for an additional 4h and then the medium was discarded, 150. mu.l DMSO per well and shaken for 10 min.

4) The 490nm wavelength was selected and the cell viability was calculated by light absorbance on a microplate reader immunoassay.

TABLE 1 toxicity test of the Nuclear penetrating peptides of the invention

As can be seen from Table 1, the cell nucleus penetrating peptide of the present invention has no significant effect on the survival rate of cells when cultured at a high concentration (1mM) for a long period of time (48 hours), and is safe and non-toxic.

Example 3 Membrane-penetrating Nuclear fluorescence detection assay of Nuclear penetrating peptides of the invention

1) Media containing a certain concentration of FITC and FITC fluorescently-labeled nuclear penetrating peptide (Lys-Lys-Arg-Pro-Pro-Arg-Lys-Lys) were added to adherent cells, the media were carefully aspirated after 0.5min incubation, and the adherent cells were washed three times with heparin-PBS. The formula of the culture medium is as follows: DMEM medium containing 10%.

2) The photographs were taken under a fluorescence microscope (see the attached figure 1 of the specification, fig. 1(a) is a fluorescence microscope observation image of a FITC group, and fig. 1(b) is a fluorescence microscope observation image of FITC fluorescence-labeled cell nucleus-penetrating peptide).

As can be seen from FIG. 1, the cell nucleus-penetrating peptide of the present invention can efficiently enter nucleoli (shown as a bright spot).

Example 4 fluorescence quantification assay of the Nuclear penetrating peptides of the invention into the nucleus

1) Media containing a certain concentration of FITC, FITC fluorescently-labeled nuclear penetrating peptide (Lys-Lys-Arg-Pro-Arg-Lys-Lys), FITC fluorescently-labeled reference peptide 1 (Glu-Lys-Arg-Pro-Arg-Lys-Glu) and FITC fluorescently-labeled reference peptide 2(Lys-Lys-Arg-Asp-Asp-Arg-Lys-Lys) were added to adherent cells, and after incubation for 30min to 4h, the media were carefully aspirated, and the adherent cells were washed three times with PBS. The formula of the culture medium is as follows: DMEM medium containing 10%.

2) Adding pancreatin to react for 5min until the cells float, and adding fresh culture medium to stop the reaction.

3) The amount of fluorescence that 10000 cells had was measured in a flow cytometer. FITC was used as a control and reference (100%).

TABLE 2 measurement of the amount of fluorescence of the nuclear penetrating peptide into the nucleus

As can be seen from Table 2, the total intracellular fluorescence of the FITC fluorescence labeled nuclear penetrating peptide group is increased by geometric orders of magnitude compared with the FITC group, the nuclear penetrating peptide can efficiently penetrate through a nuclear membrane, the FITC is successfully brought into the nucleus, and meanwhile, experiments also show that the nuclear penetrating peptide can be disabled due to the replacement of amino acid residues, so that the sequence of the nuclear penetrating peptide has remarkable uniqueness.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and should not be taken as limiting the scope of the present invention, which is intended to cover any modifications, equivalents, improvements, etc. within the spirit and scope of the present invention.

Sequence listing

<110> south Yang college of learning

<120> a cell nucleus penetrating peptide and use thereof

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Lys Lys Arg Pro Pro Arg Lys Lys

1 5

<210> 2

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Glu Lys Arg Pro Pro Arg Lys Glu

1 5

<210> 3

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Lys Lys Arg Asp Asp Arg Lys Lys

1 5

Claims

1. A cell nucleus penetrating peptide having an amino acid sequence of: Lys-Lys-Arg-Pro-Pro-Arg-Lys-Lys.

2. A complex comprising the cell nucleus-penetrating peptide of claim 1 and a foreign substance.

3. The complex of claim 2, wherein the foreign substance is a cargo molecule and/or a marker molecule.

4. The complex of claim 3, wherein the cargo molecule is a nucleotide and/or a drug molecule precursor; the marker molecules are fluorescein and/or biotin.

5. The use of the nuclear penetrating peptide of claim 1, wherein said nuclear penetrating peptide is used to mediate entry of foreign material into the nucleus of a cell.

6. Use of a nuclear penetrating peptide according to claim 5 for mediating the entry of foreign material into the nuclear core.