Synthetic peptide sp2 and application thereof

Abstract

A synthetic peptide sp2 has the amino acid sequence shown in SEQ ID No. 1. sp2 has obvious anti-tumor growth activity on xenograft tumours such as subcutaneous human pancreatic cancer, human cervical cancer, and human ovarian cancer in nude mice, with a dose-effect and time-effect relation; and has the effect of delaying metastasis and prolonging the survival time of nude mouse having in situ transplantation of human pancreatic cancer and lung adenocarcinoma. After the animals were given intravenous injection of sp2 at a dose of 2000 mg / kgBW, no toxicity reaction and death were observed. The applications of sp2 also include: (1) preventing and / or treating of tumor; (2) inhibiting proliferation and / or growth and / or invasion of tumor cells; (3) enhancing the anti-tumor immune response; (4) inducing tumor cell differentiation; (5) preparing anti-tumor drugs; (6) inhibiting tumor telomerase activity; and (7) regulating the tumor cell cycle.

Description

INCORPORATION OF SEQUENCE LISTING

[0001] This application contains a sequence listing submitted in Computer Readable Form (CRF). The CFR file containing the sequence listing entitled “PA128-0088_ST25.txt”, which was created on Nov. 17, 2020, and is 512 bytes in size. The information in the sequence listing is incorporated herein by reference in its entirety.TECHNICAL FIELD

[0002] The present disclosure belongs to the technical field of biomedicine, and relates to a synthetic peptide sp2 and an application thereof, especially an application in preparing new non-cytotoxic anti-tumor drugs.BACKGROUND

[0003] Cancer patients urgently need safe and effective anti-tumor drugs. Pancreatic cancer is extremely malignant and has an insidious onset. At first diagnosis, metastasis to peripancreatic organs or lymph nodes, liver metastasis, and distant metastasis have occurred. It is called the king of cancer. The most widely used in traditional chemotherapy for pancreatic cancer are cytotoxic anti-tumo...

Examples

example 1

[0058]Amino acid composition, solid-phase chemical synthesis, chromatographic purification and mass spectrometry identification

[0059]1) Analysis of the amino acid composition of sp2: 10.2 mg sample was weighed and was dissolved with 7 mL of 6N HCl, and hydrolyzed at 110° C. under nitrogen protection for 22 hours. The reaction solution was transferred to a 10 mL volumetric flask after cooling, and was made to volume. 0.2 mL of the solution was taken and blown dry with nitrogen at 55° C.; 1 mL of distilled water was added and dried, and repeated three times. The dried product was dissolved thoroughly with 1.2 mL of deionized water (pH was adjusted with 0.02 mol / L HCl) and mixed well. It was filtered with 0.45 μM filter membrane, and injected 20 μL for computer testing (Hitachi L-8900 amino acid analyzer).

[0060]2) Solid-phase chemical synthesis, purity detection and molecular weight confirmation of sp2:

[0061]The solid-phase chemical synthesis of sp2 used the polypeptide Fmoc solid-phas...

example 2

Cell Proliferation Inhibition Test

[0064]Cells were digested and counted to prepare a cell suspension with a concentration of 1×105 cells / mL. 100 μL of the cell suspension was added to each well of a 96-well plate (1×104 cells per well); the 96-well plate was placed in a 37° C., 5% CO2 incubator for 24 h; 100 μL of the corresponding drug-containing medium was added to each well, and a negative control group, a menstruum control group, and a positive control group were set up at the same time with 5 replicate wells in each group; the 96-well plate was placed in a 37° C., 5% CO2 incubator for 72 h; 10 μL of CCK-8 solution was added to each well, and the culture plate was incubated in the incubator for 4 h; the OD value at 450 nm was measured with a microplate reader to calculate the inhibitory rate and IC50 value of sp2 on BxPC-3 tumor cell lines. The evaluation standard ploted different concentrations of the same sample versus the tumor cell inhibition rate to obtain a dose-effect cur...

example 3

[0065]In vivo efficacy evaluation of sp2 on human pancreatic cancer in situ, human cervical cancer, human ovarian cancer, human osteosarcoma, and human poorly differentiated pregastric cancer subcutaneously in nude mice[0066]I. Cell lines: human cervical cancer cell line SiHa, human ovarian cancer cell line A2780, human pancreatic cancer in situ BxPC-3, human poorly differentiated pregastric cancer BGC-823, and human osteosarcoma cell line MG-63 were cultured in the RPMI-1640 medium containing 10% fetal bovine serum.[0067]II. Modeling method: subcutaneous injection of cell line, tumor formation in the armpit[0068]III. Experimental steps:[0069](i)Cell Preparation Stage

[0070]1. The MG-63 cell lines in a good state of recovery was inoculated into a T75 cell culture flask and cultured at 37° C., 5% CO2.

[0071]2. The medium was changed every 2-3 days, and when the cell healing degree reached about 80%, a 1:3 passage was performed, and about 20 bottles were needed.

[0072]3. After the number...

INCORPORATION OF SEQUENCE LISTING

[0001] This application contains a sequence listing submitted in Computer Readable Form (CRF). The CFR file containing the sequence listing entitled “PA128-0088_ST25.txt”, which was created on Nov. 17, 2020, and is 512 bytes in size. The information in the sequence listing is incorporated herein by reference in its entirety.TECHNICAL FIELD

[0002] The present disclosure belongs to the technical field of biomedicine, and relates to a synthetic peptide sp2 and an application thereof, especially an application in preparing new non-cytotoxic anti-tumor drugs.BACKGROUND

[0003] Cancer patients urgently need safe and effective anti-tumor drugs. Pancreatic cancer is extremely malignant and has an insidious onset. At first diagnosis, metastasis to peripancreatic organs or lymph nodes, liver metastasis, and distant metastasis have occurred. It is called the king of cancer. The most widely used in traditional chemotherapy for pancreatic cancer are cytotoxic anti-tumo...

Examples

example 1

[0058]Amino acid composition, solid-phase chemical synthesis, chromatographic purification and mass spectrometry identification

[0059]1) Analysis of the amino acid composition of sp2: 10.2 mg sample was weighed and was dissolved with 7 mL of 6N HCl, and hydrolyzed at 110° C. under nitrogen protection for 22 hours. The reaction solution was transferred to a 10 mL volumetric flask after cooling, and was made to volume. 0.2 mL of the solution was taken and blown dry with nitrogen at 55° C.; 1 mL of distilled water was added and dried, and repeated three times. The dried product was dissolved thoroughly with 1.2 mL of deionized water (pH was adjusted with 0.02 mol / L HCl) and mixed well. It was filtered with 0.45 μM filter membrane, and injected 20 μL for computer testing (Hitachi L-8900 amino acid analyzer).

[0060]2) Solid-phase chemical synthesis, purity detection and molecular weight confirmation of sp2:

[0061]The solid-phase chemical synthesis of sp2 used the polypeptide Fmoc solid-phas...

example 2

Cell Proliferation Inhibition Test

[0064]Cells were digested and counted to prepare a cell suspension with a concentration of 1×105 cells / mL. 100 μL of the cell suspension was added to each well of a 96-well plate (1×104 cells per well); the 96-well plate was placed in a 37° C., 5% CO2 incubator for 24 h; 100 μL of the corresponding drug-containing medium was added to each well, and a negative control group, a menstruum control group, and a positive control group were set up at the same time with 5 replicate wells in each group; the 96-well plate was placed in a 37° C., 5% CO2 incubator for 72 h; 10 μL of CCK-8 solution was added to each well, and the culture plate was incubated in the incubator for 4 h; the OD value at 450 nm was measured with a microplate reader to calculate the inhibitory rate and IC50 value of sp2 on BxPC-3 tumor cell lines. The evaluation standard ploted different concentrations of the same sample versus the tumor cell inhibition rate to obtain a dose-effect cur...

example 3

[0065]In vivo efficacy evaluation of sp2 on human pancreatic cancer in situ, human cervical cancer, human ovarian cancer, human osteosarcoma, and human poorly differentiated pregastric cancer subcutaneously in nude mice[0066]I. Cell lines: human cervical cancer cell line SiHa, human ovarian cancer cell line A2780, human pancreatic cancer in situ BxPC-3, human poorly differentiated pregastric cancer BGC-823, and human osteosarcoma cell line MG-63 were cultured in the RPMI-1640 medium containing 10% fetal bovine serum.[0067]II. Modeling method: subcutaneous injection of cell line, tumor formation in the armpit[0068]III. Experimental steps:[0069](i)Cell Preparation Stage

[0070]1. The MG-63 cell lines in a good state of recovery was inoculated into a T75 cell culture flask and cultured at 37° C., 5% CO2.

[0071]2. The medium was changed every 2-3 days, and when the cell healing degree reached about 80%, a 1:3 passage was performed, and about 20 bottles were needed.

[0072]3. After the number...

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