A membrane-penetrating peptide specific to human cervical cancer cells and its application

Abstract

This invention discloses a specific membrane-penetrating peptide for human cervical cancer cells and its application. The amino acid sequence of the specific membrane-penetrating peptide is shown in SEQ ID No.1: Lys-Lys-Ile-Ile-Pro-Trp-Trp-Arg-Arg. This specific membrane-penetrating peptide is safe and non-toxic, has low immunogenicity, and is obtained by solid-phase synthesis. It is inexpensive, readily available, and easy to control in terms of quality. It can selectively and efficiently penetrate the membrane of human cervical cancer cells.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to a membrane-penetrating peptide specific to human cervical cancer cells and its application. Background Technology

[0002] Human cervical cancer cells (HeLa) are the first aneuploid epithelioid cell line obtained through continuous culture from human tissue. They were established by Gey et al. in 1951 from cervical cancer tissue of a 31-year-old Black woman. Compared to normal human cells, human cervical cancer cells exhibit significant differences in membrane electrical properties and composition.

[0003] Cell-penetrating peptides are a class of short peptides capable of carrying macromolecules into cells, and their cell-penetrating ability does not depend on classical endocytosis. Cell-penetrating peptides have wide applications in drug delivery; however, naturally occurring cell-penetrating peptides typically lack specific cell-penetrating capabilities, making the development of cell-specific cell-penetrating peptides a hot research topic. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a transmembrane peptide with low toxicity and side effects, high efficiency, and specificity in penetrating human cervical cancer cells, as well as its applications.

[0005] The specific technical solution is as follows:

[0006] In a first aspect, the present invention provides a human cervical cancer cell-specific transmembrane peptide, the amino acid sequence of which is shown in SEQ ID No. 1: Lys-Lys-Ile-Ile-Pro-Trp-Trp-Arg-Arg.

[0007] The specific membrane-penetrating peptide described herein can selectively and efficiently penetrate the membrane of human cervical cancer cells.

[0008] In a second aspect, the present invention provides the application of specific membrane-penetrating peptides in cases where specific entry into human cervical cancer cells is required for non-disease diagnosis and treatment purposes.

[0009] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0010] This invention provides a human cervical cancer cell-specific transmembrane peptide that is safe, non-toxic, and has low immunogenicity. It is synthesized via solid-phase synthesis, making it inexpensive, readily available, and easy to control in terms of quality. This transmembrane peptide is non-cytotoxic and can selectively and efficiently penetrate the membrane of human cervical cancer cells while remaining unaffected by normal human cell membranes. Detailed Implementation

[0011] The following is a detailed description of specific embodiments of the present invention, which is only used to provide further explanation of the present invention, but does not constitute a limitation of the present invention.

[0012] Example 1: Synthesis of membrane-penetrating peptides

[0013] (1) Activating the resin: Weigh 2000mg of Fmoc-Arg wang Resin, add 20mL of DMF and soak for 30min to allow it to swell fully.

[0014] (2) Deprotection: Remove the DMF soaking the resin by pressure filtration, add 10 mL of DMF solution containing 20% ​​piperidine, boil under nitrogen for 15 min, then remove by pressure filtration to remove the Fmoc group of amino group, wash the resin three times alternately with 10 mL of isopropanol and 10 mL of DMF, and then test the resin by ninhydrin method to see if it turns black or purple.

[0015] (3) Condensation reaction: To connect the next amino acid, weigh 1.4 mmol / g of Fmoc-amino acid from the resin, and use 20 mL of DMF containing 910 mg TBTU, 0.45 g HOBt, and 0.52 mL DIEA as the reaction solution. Boil under nitrogen at room temperature for 3 h. After the reaction, wash the resin three times alternately with 10 mL of isopropanol and 10 mL of DMF. Detect the amino group.

[0016] (4) Repeat steps (2)-(3): Extend the polypeptide from the C-terminus to the N-terminus in sequence. Repeat the deprotection, washing, and condensation process until the remaining amino acids are linked, thus completing the polypeptide linkage.

[0017] (5) Peptide cleavage: The peptide-resin complex was dried with nitrogen. A 20 mL mixed cleavage reagent was prepared by mixing TFA / phenol / ultrapure water / thioanisole / EDT / TIS in a volume ratio of 80 / 5 / 5 / 5 / 3 / 2. The peptide resin was placed in a round-bottom flask, the cleavage solution was added, and the mixture was magnetically stirred for 3 h. The resin was removed through a 200-mesh sintered glass filter. The filtrate was directly added dropwise to frozen ether, centrifuged at 5000 r / min to precipitate, and freeze-dried to constant weight to obtain the crude peptide.

[0018] (6) The crude peptide was purified by HPLC to a purity greater than 95%.

[0019] The amino acid sequence synthesized according to the above method is Lys-Lys-Ile-Ile-Pro-Trp-Trp-Arg-Arg.

[0020] Example 2: HeLa cytotoxicity experiment

[0021] (1) Cell seeding: Take a 96-well plate and add 5×10⁶ cells to each well. 3 HeLa cell culture medium was incubated at 37°C in a 5% CO2 incubator for 24 hours to allow the cells to adhere to the culture vessel.

[0022] (2) Cell culture: Prepare a culture medium containing 1 mM of membrane-penetrating peptide, and use a culture medium without membrane-penetrating peptide as a negative control well (Control). Culture at 37°C and 5% carbon dioxide for 24 h.

[0023] (3) Color development: Add 20 μl MTT to each well of adherent cells, continue incubation for 4 h, then discard the culture medium, add 150 μl DMSO to each well, and shake for 10 min.

[0024] (4) Colorimetric analysis: Select a wavelength of 490nm and calculate the cell viability by measuring the light absorption value on the ELISA reader.

[0025] The membrane-penetrating peptide provided by this invention did not cause significant damage to HeLa cells under high concentration (1mM) and long duration (24h) conditions, and is safe and non-toxic.

[0026] Example 3: Normal Cytotoxicity Experiment

[0027] (1) Cell seeding: Take a 96-well plate and add 7×10⁶ cells to each well. 3 Incubate epithelial cells in a culture medium at 37°C with a 5% carbon dioxide incubator for 24 hours to allow the cells to adhere to the culture vessel.

[0028] (2) Cell culture: Prepare a culture medium containing 0.5 mM of membrane-penetrating peptide, and use a culture medium without membrane-penetrating peptide as a negative control well. Culture at 37°C and 5% carbon dioxide for 24 h.

[0029] (3) Color development: Add 20 μl MTT to each well of adherent cells, continue incubation for 4 h, then discard the culture medium, add 150 μl DMSO to each well, and shake for 10 min.

[0030] (4) Colorimetric analysis: Select a wavelength of 490nm and calculate the cell viability by measuring the light absorption value on the ELISA reader.

[0031] The membrane-penetrating peptide provided by this invention does not cause significant damage to normal cells under high concentration (1mM) and long duration (24h) conditions, and is safe and non-toxic.

[0032] Example 4: Cell Transmembrane Experiment

[0033] (1) HeLa cells were incubated with a medium containing 1 mM transmembrane peptide for 30 min. The medium was carefully aspirated and the adherent cells were washed three times with PBS. Human epithelial cells under the same conditions were used as a control.

[0034] 2) Add fresh cell culture medium and culture for 24 hours.

[0035] 3) Add 150 μl DMSO to each well of HeLa cells, shake for 10 min, centrifuge at 10000 rpm to collect the supernatant and obtain the intracellular extract.

[0036] 4) HPLC determination of the membrane-penetrating peptide concentration in the intracellular extract. Specific test results are shown in Table 1:

[0037] Table 1. Results of cell penetration test

[0038]

[0039] As shown in Table 1, the membrane-penetrating peptide of the present invention can selectively and efficiently penetrate the HeLa cell membrane and enter the cell, while hardly penetrating the human epithelial cell membrane. The intracellular membrane-penetrating peptide concentration in HeLa cells is more than 15 times that of the control group.

[0040] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A human cervical cancer cell-specific cell-penetrating peptide, characterized in that, The amino acid sequence of the specific cell-penetrating peptide is shown as SEQ ID No. 1, and the specific cell-penetrating peptide can selectively and efficiently penetrate the membrane of human cervical cancer cells.

2. The use of the human cervical cancer cell-specific cell-penetrating peptide of claim 1 for specific entry into human cervical cancer cells for non-disease diagnosis and treatment purposes.

Images