CN104623628A - Application of micro-molecule polypeptide LZ1 - Google Patents
Application of micro-molecule polypeptide LZ1 Download PDFInfo
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- CN104623628A CN104623628A CN201510015916.9A CN201510015916A CN104623628A CN 104623628 A CN104623628 A CN 104623628A CN 201510015916 A CN201510015916 A CN 201510015916A CN 104623628 A CN104623628 A CN 104623628A
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Abstract
The invention belongs to the technical field of polypeptide drugs in biochemistry and particularly relates to application of micro-molecule polypeptide LZ1 in preparation of anti-tumor drugs. The LZ1 provided by the invention is an active polypeptide which is manually designed and synthesized. The LZ1 comprises 15 amino acid residues, the molecular weight is 2228.77Da and the isoelectric point is 12.05. The LZ1 provided by the invention represents a remarkable anti-tumor activity in vitro in form of inducing apoptosis and is non-toxic to normal cells, and similarly represents a good anti-tumor activity in vivo, so that the LZ1 can be applied to preparing anti-tumor drugs. The polypeptide is short in sequence, convenient to manually synthesize and easy to produce in large scale, and has a broad application prospect.
Description
Technical field:
The invention belongs to the polypeptide drugs technical field in biochemistry, be specifically related to a kind of micromolecule polypeptide LZ1 and preparing the application in antitumor drug.
Background technology:
Malignant tumor has become the principal disease threatening human health at present, the trend increased year by year is all presented at China's malignancy disease sickness rate and mortality rate, " 2014 Chinese tumor registration annual report " is presented at the newly-increased Malignant Tumor Case 3,090,000 of China in 2010, death 1,960,000.Malignancy disease not only serious threat the health of the mankind, needs to drop into a large amount of manpowers and fund to the treatment of malignant tumor simultaneously, drastically influence economic construction and social development.Therefore, finding and developing antitumor drug or method is safely and effectively everybody questions of common interest, is also the most important study hotspots of current scientific circles.
The Drug therapy of malignant tumor is the important component part in combined therapy of tumour, and wherein, tumor cytotoxicity series antineoplastic medicament uses maximum important point of penetration being also current antitumor drug and developing clinically.Scientist achieved a series of important achievement in research in antitumor drug exploitation in recent years, and the application of new type antineoplastic medicine also effectively extends the life span of cancer patient clinically.But the attention of people is also caused gradually about the series of problems occurred in antitumor drug use procedure, as: antitumor drug poor selectivity, large to normal cellulotoxic side effect, easily produce drug resistance in antitumor drug use procedure.Micromolecule natural polypeptides is extensively present in nature, and because it exists composition simply, molecular weight is little, and feature easily of synthesizing shows wide application prospect.But also there is subproblem in micromolecule polypeptide cancer therapy drug in Clinical practice process, as: anticancer mechanism is indefinite, and internal metabolism is fast, and poor stability, exists immunogenicity, there is cytotoxicity to normal cell.For these shortcomings of natural polypeptides, can transform it while reservation natural polypeptides antitumaous effect, strengthen its stability, weaken natural polypeptides to Normocellular lethal effect, this is the important directions of peptide medicament research.
Summary of the invention:
A kind of micromolecule polypeptide LZ1 is the object of the present invention is to provide to prepare the application in antitumor drug.
LZ1 of the present invention is the straight chain antibacterial peptide of artificial design and synthesis, comprises 15 amino acid residues, and molecular weight is 2228.77Da, and isoelectric point, IP is 12.05.The complete sequence of LZ1 antibacterial peptide is as follows: valine-lysine-arginine-Trp-Ly-lysine-Trp-Trp-arginine-lysine-Trp-Ly-lysine-tryptophan-valine-NH
2.
Beneficial effect of the present invention is: LZ1 polypeptide is synthetic, has molecular weight little, and synthetic is convenient; Polypeptide LZ1 optionally killing tumor cell, does not act on normal cell, and the mechanism of this polypeptide to tumor cytotoxicity effect is inducing apoptosis of tumour cell; This polypeptide has obvious inhibitory action to the growth of solid tumor in body, can prepare the application in antitumor drug.
Accompanying drawing illustrates:
Fig. 1 is that the micromolecule polypeptide LZ1 of variable concentrations is to the effect schematic diagram of hepatoma carcinoma cell Huh-7, breast cancer cell HCC1806, breast cancer cell MCF-7 and cervical cancer cell Hela.
Fig. 2 is that the micromolecule polypeptide LZ1 of variable concentrations is to the effect schematic diagram of people's normal control cells embryonic kidney cells HEK-293.
Fig. 3 is micromolecule polypeptide LZ1 to the apoptosis situation schematic diagram of this cell detected by flow cytometry means after hepatoma carcinoma cell Huh-7 effect.
In Fig. 4 nude mouse that to be micromolecule polypeptide LZ1 induce breast cancer cell HCC1806, the inhibitory action of implanted solid tumor growth, contrasts with normal saline and cisplatin respectively.Wherein, Fig. 4 A is micromolecule polypeptide LZ1 and to the change in volume situation schematic diagram impinging upon nude mice solid tumor after administration different number of days, Fig. 4 B is the size comparison diagram of micromolecule polypeptide LZ1 and contrast administration nude mice solid tumor after 28 days, and Fig. 4 C is the weight comparison diagram of micromolecule polypeptide LZ1 and contrast administration nude mice solid tumor after 28 days.* represents p<0.01.
In Fig. 5 nude mouse that to be micromolecule polypeptide LZ1 induce hepatoma carcinoma cell Huh-7, the inhibitory action of implanted solid tumor growth, contrasts with normal saline and cisplatin respectively.Wherein, Fig. 5 A is micromolecule polypeptide LZ1 and to the change in volume situation schematic diagram impinging upon nude mice solid tumor after administration different number of days, Fig. 5 B is the size comparison diagram of micromolecule polypeptide LZ1 and contrast administration nude mice solid tumor after 28 days, and Fig. 5 C is the weight comparison diagram of micromolecule polypeptide LZ1 and contrast administration nude mice solid tumor after 28 days.* represents p<0.01.
Detailed description of the invention:
Elaborate to the present invention below in conjunction with accompanying drawing, the effect of embodiment is only explain and non-limiting the present invention.
Embodiment 1: the preparation of micromolecule polypeptide LZ1
I, the chemical synthesis process of antibacterial peptide LZ1: according to the aminoacid sequence of our design, synthesize its complete sequence, by HPLC reversed phase column chromatography desalting and purifying with automatic Peptide synthesizer (433A, Applied Biosystems).
II, molecular weight determination adopts Matrix Assisted Laser Desorption ionization time of flight mass spectrometry (MALDI-TOF).
III, the golden antibacterial peptide LZ1 high-efficient liquid phase chromatogram HPLC method of purification identifies its purity, molecular weight determination adopts Matrix Assisted Laser Desorption ionization time of flight mass spectrometry (MALDI-TOF), isoelectric focusing electrophoresis measures isoelectric point, IP, measures amino acid sequence structure with automatic Protein Sequencer.
Antibacterial peptide LZ1 comprises altogether 15 amino acid residues, and molecular weight is 2228.77Da, and isoelectric point, IP is 12.05.The complete sequence of LZ1 antibacterial peptide is as follows: valine-lysine-arginine-Trp-Ly-lysine-Trp-Trp-arginine-lysine-Trp-Ly-lysine-tryptophan-valine-NH2 (C-holds amidatioon).
Embodiment 2: micromolecule polypeptide LZ1 tests the effect of hepatoma carcinoma cell Huh-7, breast cancer cell HCC1806, breast cancer cell MCF-7 and cervical cancer cell Hela
Collect respectively and be in hepatoma carcinoma cell Huh-7, the breast cancer cell HCC1806 of exponential phase, breast cancer cell MCF-7 and cervical cancer cell Hela makes cell suspension, adjustment cell density to 2 × 10
6individual/ml, often kind of cell enters 96 orifice plates according to the volume kind in 200 μ l/ holes, cell at 37 DEG C, the CO of 5%
2condition under spend the night adherent.Within second day, add for often kind of cell the micromolecule polypeptide LZ1 that final concentration is 50 ~ 300 μ g/ml respectively, wherein use the normal saline of equal volume in contrast.Cell culture is after 24 hours, and every hole adds the MTT solution of 20 μ l5mg/ml, continues to cultivate 4h.Cultivation terminates culture medium in rear sucking-off hole, and every hole adds the dimethyl sulfoxide of 200 μ l, 96 orifice plates is put low speed shake 10min abundant dissolved cell intercrystalline thing on shaking table.The absorbance of OD490nm place 96 orifice plate is detected by microplate reader, note arranging zeroing hole, control wells, result is as shown in Figure 1: the form that micromolecule polypeptide LZ1 relies on gradient in 50 ~ 300 μ g/ml concentration ranges has the effect of obvious Developing restraint to hepatoma carcinoma cell Huh-7, breast cancer cell HCC1806, breast cancer cell MCF-7 and cervical cancer cell Hela, wherein the inhibitory action of this polypeptide to hepatoma carcinoma cell Huh-7 and breast cancer cell HCC1806 is the strongest.
Embodiment 3: micromolecule polypeptide LZ1 tests the effect of human normal cell line embryonic kidney cells HEK-293
Collect the embryonic kidney cells HEK-293 being in exponential phase and make cell suspension, adjustment cell density to 2 × 10
6individual/ml, enters 96 orifice plates according to the volume kind in 200 μ l/ holes, cell at 37 DEG C, the CO of 5%
2condition under spend the night adherent.Within second day, add the micromolecule polypeptide LZ1 that final concentration is 50 ~ 300 μ g/ml, wherein use the normal saline of equal volume in contrast.Cell culture is after 24 hours, and every hole adds the MTT solution of 20 μ l 5mg/ml, continues to cultivate 4h.Cultivation terminates culture medium in rear sucking-off hole, and every hole adds the dimethyl sulfoxide of 200 μ l, 96 orifice plates is put low speed shake 10min abundant dissolved cell intercrystalline thing on shaking table.Detect the absorbance of OD490nm place 96 orifice plate by microplate reader, note arranging zeroing hole, control wells, result as shown in Figure 2: micromolecule polypeptide LZ1 does not have obvious growth inhibited effect to embryonic kidney cells HEK-293 in 50 ~ 300 μ g/ml concentration ranges.
Embodiment 4: micromolecule polypeptide LZ1 is on the impact of hepatoma carcinoma cell Huh-7 apoptosis
Collect the hepatoma carcinoma cell Huh-7 being in exponential phase and make cell suspension, adjustment cell density to 2 × 10
6individual/ml, enters 6 orifice plates according to the volume kind in 2ml/ hole, cell at 37 DEG C, the CO of 5%
2condition under spend the night adherent.Within second day, add the micromolecule polypeptide LZ1 that final concentration is 12.5 ~ 200 μ g/ml, wherein use the normal saline of equal volume in contrast.Cell culture is after 24 hours, first cell suspension made in cell trypsinization, then uses the apoptosis situation by flow cytomery hepatoma carcinoma cell Huh-7 after propidium iodide (PI)/Annexin V apoptosis detection kit dyeing.In normal cell, Phosphatidylserine is only distributed in inside cell membrane, Phosphatidylserine in the early stage cell membrane of apoptosis is by turning on one's side laterally in adipose membrane, Annexin V is a kind of albumen with Phosphatidylserine with high-affinity, and the Phosphatidylserine therefore exposed by detecting outside judges the apoptosis that cell is early stage.PI is a kind of nucleic acid dye, can not pass normal cell membrane, and in early apoptosis of cells process, PI dyeing is for negative.Result is as shown in Figure 3: in the concentration range of 12.5 ~ 200 μ g/ml, along with the increase of peptide concentration, the change of hepatoma carcinoma cell Huh-7PI stained positive ratio is little, but Annexin V positive staining ratio increases gradually, the form that namely micromolecule polypeptide LZ1 relies on gradient induces hepatoma carcinoma cell Huh-7 apoptosis.
Embodiment 5: the impact of implanted solid tumor growth in the nude mouse that micromolecule polypeptide LZ1 induces breast cancer cell HCC1806
Take the logarithm the breast cancer cell HCC1806 of trophophase, aseptically, by 1 × 10
7the amount of/only/0.1ml be inoculated in BALB/cA nude mice hind leg on the right side of axillary fossa subcutaneous.After tumor mass grows 8 days, measure tumor footpath, according to tumorous size random packet, often organize 6, grouping day is 0 day, and next day starts administration.
Gross tumor volume (tumor volume, TV), computing formula is: TV=1/2 × a × b
2
Wherein a, b represent length and width respectively.
Administration is grouped as follows: saline control group: the normal saline of equal volume; Cisplatin matched group: 2mg/kg; Micromolecule polypeptide LZ1 administration group: 4mg/kg.
Administering mode is as follows: within every two days, be administered once, by the mode administration of tail vein injection, and successive administration 28 days.
Administration terminates rear execution mice, separates solid tumor, measures tumor weight.
Result is as shown in Figure 4: the growth of solid tumor in the nude mouse that micromolecule polypeptide LZ1 can obviously suppress breast cancer cell HCC1806 to induce, namely micromolecule polypeptide LZ1 can play good antitumor action in vivo.
Embodiment 6: the impact of implanted solid tumor growth in the nude mouse that micromolecule polypeptide LZ1 induces hepatoma carcinoma cell Huh-7
Take the logarithm the hepatoma carcinoma cell Huh-7 of trophophase, aseptically, by 1 × 10
7the amount of/only/0.1ml be inoculated in BALB/cA nude mice hind leg on the right side of axillary fossa subcutaneous.After tumor mass grows 8 days, measure tumor footpath, according to tumorous size random packet, often organize 6, grouping day is 0 day, and next day starts administration.
Gross tumor volume (tumor volume, TV), computing formula is: TV=1/2 × a × b
2
Wherein a, b represent length and width respectively.
Administration is grouped as follows: saline control group: the normal saline of intravenous injection equal volume; Cisplatin matched group: 2mg/kg; Micromolecule polypeptide LZ1 administration group: 4mg/kg.
Administering mode is as follows: within every two days, be administered once, by the mode administration of tail vein injection, and successive administration 28 days.
Administration terminates rear execution mice, separates solid tumor, measures tumor weight.
Result is as shown in Figure 5: the growth of solid tumor in the nude mouse that micromolecule polypeptide LZ1 can obviously suppress hepatoma carcinoma cell Huh-7 to induce, namely micromolecule polypeptide LZ1 can play good antitumor action in vivo.
Claims (1)
1. molecular weight is 2228.77Da, isoelectric point, IP is 12.05, comprise the micromolecule polypeptide LZ1 of 15 amino acid residues is preparing the application in antitumor drug.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104974228A (en) * | 2015-06-15 | 2015-10-14 | 四川合泰新光生物科技有限公司 | Small molecule polypeptide ZY4 and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102924574A (en) * | 2012-10-26 | 2013-02-13 | 苏州康尔生物医药有限公司 | Antibacterial peptide LZ1 and application of antibacterial peptide in preparation of antibacterial medicament |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102924574A (en) * | 2012-10-26 | 2013-02-13 | 苏州康尔生物医药有限公司 | Antibacterial peptide LZ1 and application of antibacterial peptide in preparation of antibacterial medicament |
Non-Patent Citations (2)
| Title |
|---|
| ZHIYE ZHANG ET AL.: "A Small Peptide with Therapeutic Potential for Inflammatory Acne Vulgaris", 《PLOS ONE》 * |
| 陈巍 等: "抗菌肽———一类新型抗肿瘤药物", 《海峡药学》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104974228A (en) * | 2015-06-15 | 2015-10-14 | 四川合泰新光生物科技有限公司 | Small molecule polypeptide ZY4 and application thereof |
| WO2016201972A1 (en) * | 2015-06-15 | 2016-12-22 | 四川合泰新光生物科技有限公司 | Small-molecule polypeptide zy4 and application thereof |
| JP2018521041A (en) * | 2015-06-15 | 2018-08-02 | 四川合泰新光生物科技有限公司Ｓｉｃｈｕａｎ Ｈｅｔａｉ Ｓｙｎｌｉｇｈｔ Ｂｉｏｔｅｃｈ Ｌｔｄ | Low molecular polypeptide ZY4 and use thereof |
| EP3309167A4 (en) * | 2015-06-15 | 2018-12-19 | Sichuan Hetai Synlight Biotech Ltd. | Small-molecule polypeptide zy4 and application thereof |
| US10208088B1 (en) | 2015-06-15 | 2019-02-19 | Sichuan Synlight Biotech Ltd. | Low molecular polypeptide ZY4 and applications thereof |
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