CN117362408A - Antitumor polypeptide and preparation method and application thereof - Google Patents
Antitumor polypeptide and preparation method and application thereof Download PDFInfo
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- CN117362408A CN117362408A CN202311311939.5A CN202311311939A CN117362408A CN 117362408 A CN117362408 A CN 117362408A CN 202311311939 A CN202311311939 A CN 202311311939A CN 117362408 A CN117362408 A CN 117362408A
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- tumor
- tumor polypeptide
- polypeptide
- esophageal cancer
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- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 75
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- 230000000259 anti-tumor effect Effects 0.000 title claims abstract description 52
- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- 206010030155 Oesophageal carcinoma Diseases 0.000 claims abstract description 39
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 claims abstract description 38
- 229960004316 cisplatin Drugs 0.000 claims abstract description 37
- 208000000461 Esophageal Neoplasms Diseases 0.000 claims abstract description 31
- 201000004101 esophageal cancer Diseases 0.000 claims abstract description 31
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- 208000017897 Carcinoma of esophagus Diseases 0.000 description 5
- 229930192392 Mitomycin Natural products 0.000 description 5
- NWIBSHFKIJFRCO-WUDYKRTCSA-N Mytomycin Chemical compound C1N2C(C(C(C)=C(N)C3=O)=O)=C3[C@@H](COC(N)=O)[C@@]2(OC)[C@@H]2[C@H]1N2 NWIBSHFKIJFRCO-WUDYKRTCSA-N 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 5
- 201000005619 esophageal carcinoma Diseases 0.000 description 5
- 229960004857 mitomycin Drugs 0.000 description 5
- 230000008685 targeting Effects 0.000 description 5
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 230000002401 inhibitory effect Effects 0.000 description 4
- IYMAXBFPHPZYIK-BQBZGAKWSA-N Arg-Gly-Asp Chemical compound NC(N)=NCCC[C@H](N)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(O)=O IYMAXBFPHPZYIK-BQBZGAKWSA-N 0.000 description 3
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Classifications
-
- 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
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
The invention discloses an anti-tumor polypeptide and a preparation method and application thereof. The amino acid sequence of the antitumor polypeptide comprises a sequence shown as SEQ ID NO. 1. The invention develops an anti-tumor polypeptide which has a killing effect on esophageal cancer cells and low toxic and side effects, can be applied to esophageal cancer treatment, and further discovers that the anti-tumor polypeptide can further improve the treatment effect by being combined with cisplatin.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and relates to an anti-tumor polypeptide and a preparation method and application thereof.
Background
Esophageal cancer is a type of digestive tract malignant tumor with higher invasiveness, the traditional treatment mode of esophageal cancer has poor curative effect, and in recent years, continuous breakthrough of targeted drug treatment on malignant tumor brings new treatment options for esophageal cancer patients, and development of novel targeted drugs gradually attracts attention.
A peptide-drug conjugate is a single molecule that can exert multiple functions simultaneously in a drug delivery system, such as in vivo drug distribution, targeted release, and bioactive functions. Such a molecule can be regarded as an integrated delivery system and is therefore referred to as a molecular delivery system. Peptide-drug conjugates include tumor targeting peptides, payloads, and linkers. The tumor targeting peptide specifically recognizes membrane receptors on tumor cells, improves the targeting treatment effect of the medicine and reduces toxic and side effects. As in CN115590975a, a mitomycin-RGD peptide conjugate is disclosed, comprising mitomycin, a linker and an RGD peptide; the mitomycin and RGD peptide are coupled through the linker, the mitomycin is transported to tumor tissues through the RGD peptide, the targeting property of the mitomycin is increased, and the toxic and side effects on normal cells are reduced while the antitumor activity of the mitomycin is maintained. CN113769086a discloses a targeted therapeutic drug, a preparation method and application thereof, wherein the targeted therapeutic drug comprises an active targeted unit, a signal molecule and an antitumor drug molecule; the active targeting unit, the signal molecule and the antitumor drug molecule are sequentially connected through chelation reaction and/or chemical coupling reaction. The targeted therapeutic drug can actively target tumor cells over-expressed by the neuropeptide YY3 receptor, and kill cancer cells through chemical drug treatment, photo-thermal treatment, photodynamic treatment, sonodynamic treatment or magnetothermal treatment.
In conclusion, the novel polypeptide applicable to tumor treatment and the corresponding treatment strategy are developed, and the method expands tumor treatment means and has important significance for the tumor treatment field.
Disclosure of Invention
Aiming at the defects and actual demands of the prior art, the invention provides an anti-tumor polypeptide, a preparation method and application thereof, in particular to a polypeptide applicable to esophageal cancer treatment and application thereof.
In order to achieve the above purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides an anti-tumor polypeptide, the amino acid sequence of which comprises the sequence shown in SEQ ID NO. 1.
The invention develops an anti-tumor polypeptide which has a killing effect on esophageal cancer cells and low toxic and side effects, can be applied to esophageal cancer treatment, and further discovers that the anti-tumor polypeptide can further improve the treatment effect by being combined with cisplatin.
SEQ ID NO.1：
FQAALSRKVAKLVHFLLLKYRAREPVTKAEMLGSVVGNWQYFFPVIFSKASDSLQLVFGIELMEVDPIGHVYIFATCLGLSYDGLLGDNQIMPKTGFLIIILAIIAKEGDCAPEEKIWEELSVLEVFEGREDSIFGDPKKLLTQYFVQENYLEYRQVPGSDPACYEFLWGPRALIETSYVKVLHHMVKISGGPRISYPLLHEWALREGEE。
It will be appreciated that mutants having a similar function to the polypeptide of the present application, obtained by using mutation means commonly used in the art, such as insertion, deletion or substitution, on the basis of the polypeptide sequence of the present invention, are also suitable for use in the technical scheme of the present invention.
In a second aspect, the invention provides a nucleic acid molecule encoding an anti-tumour polypeptide according to the first aspect.
In a third aspect, the present invention provides a recombinant vector comprising a nucleic acid molecule according to the second aspect.
In a fourth aspect, the invention provides a recombinant cell comprising a nucleic acid molecule according to the second aspect.
In a fifth aspect, the present invention provides a method for preparing the anti-tumor polypeptide according to the first aspect, wherein the preparation method comprises:
inserting the encoding gene of the anti-tumor polypeptide in the first aspect into an expression vector to obtain a recombinant vector, introducing the recombinant vector into a host cell for culture, collecting the cell, and purifying to obtain the anti-tumor polypeptide.
Preferably, the expression vector comprises a plasmid.
Preferably, the plasmid comprises a pCS2 plasmid.
Preferably, the host cell comprises a HEK293T cell.
It will be appreciated that based on the known amino acid sequence of the anti-tumor polypeptide, the anti-tumor polypeptide may be prepared according to expression and purification methods commonly used in the art.
In a sixth aspect, the present invention provides the use of an anti-tumour polypeptide according to the first aspect for the manufacture of an anti-tumour medicament, said tumour comprising an oesophageal carcinoma.
In a seventh aspect, the invention provides a pharmaceutical composition comprising an anti-tumour polypeptide according to the first aspect.
Preferably, the pharmaceutical composition further comprises cisplatin.
In the invention, the combination of the antitumor polypeptide and cisplatin can be used for remarkably improving the treatment effect.
Preferably, the pharmaceutical composition is a single compound formulation or a combination of two separate formulations.
Preferably, the pharmaceutical composition further comprises pharmaceutically acceptable pharmaceutical excipients.
Preferably, the pharmaceutical excipients comprise any one or a combination of at least two of carriers, diluents, binders, wetting agents, disintegrants, emulsifiers, co-solvents, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH regulators, antioxidants, bacteriostats or buffers.
In an eighth aspect, the invention provides the use of an anti-tumor polypeptide according to the first aspect or a pharmaceutical composition according to the seventh aspect for the preparation of an esophageal cancer cell growth inhibitor or an esophageal cancer cell apoptosis inducer.
In a ninth aspect, the present invention provides the use of an anti-tumour polypeptide according to the first aspect or a pharmaceutical composition according to the seventh aspect for the preparation of an oesophageal carcinoma cell growth inhibitor or oesophageal carcinoma cell apoptosis inducer for non-disease diagnosis and/or treatment purposes.
In the invention, the anti-tumor polypeptide can inhibit the growth of esophageal cancer cells/induce the apoptosis of esophageal cancer cells, so the result shows that the anti-tumor polypeptide can be used as an esophageal cancer cell growth inhibitor or an esophageal cancer cell apoptosis inducer for non-disease diagnosis and/or treatment, and can be used in the scientific research field, such as researching more esophageal cancer cell growth and metabolic mechanisms or behaviors, screening medicines for treating esophageal cancer and the like.
Compared with the prior art, the invention has the following beneficial effects:
the invention develops an anti-tumor polypeptide which has a killing effect on esophageal cancer cells and low toxic and side effects, can be applied to esophageal cancer treatment, and further discovers that the anti-tumor polypeptide can further improve the treatment effect by being combined with cisplatin.
Drawings
FIG. 1 shows pCS2-MAGEA6 ΔIDR -strep plasmid map;
FIG. 2 is a schematic diagram of MAGEA6 ΔIDR Peptide SDS gel electrophoresis verifies that the size is 35kDa;
FIG. 3A is MAGEA6 ΔIDR Cytotoxicity test results of peptides in combination with cisplatin as a human esophageal cancer cell KYSE 150: the purified proteins were separated into a control group and a cisplatin group (40. Mu.M), the abscissa represents the action concentration of the purified proteins, which were 0. Mu.M, 0.01. Mu.M, and 0.05. Mu.M, respectively, and the ordinate represents the cell viability of KYSE 150;
FIG. 3B is MAGEA6 ΔIDR Peptide in combination with cisplatin as a result of KYSE150 for human esophageal cancer cells, half maximal inhibitory concentration (IC 50 )；
FIG. 4A is MAGEA6 ΔIDR Results of toxicity test of peptides in combination with cisplatin for human esophageal carcinoma organoids: the abscissa represents different treatment groups and the ordinate represents organoid viability;
FIG. 4B is a schematic diagram of MAGEA6 ΔIDR Peptide used in combination with cisplatin as a graph for human esophageal carcinoma organoids, half maximal inhibitory concentration (IC 50 )；
FIG. 5 is MAGEA6 ΔIDR Peptide used as results for in vitro transplantation tumor model of C57BL/6N nude mice in combination with cisplatin, KYSE150 human esophageal cancer cells (2×10) were subcutaneously injected in the right inguinal region of C57BL/6N nude mice 6 Six (100) μl) was administered 6 days after (n=5 per group), group 1 was treated with physiological saline, group 2 was treated with cisplatin, and group 3 was treated with MAGEA6 ΔIDR Peptide treatment, panel 4 MAGEA6 ΔIDR Peptide and cisplatin combination treatment. Cisplatin concentration of 3mg/kg, MAGEA6 was injected intraperitoneally into mice of cisplatin treatment group and combination group ΔIDR Mice of the peptide group and the combination group were intraperitoneally injected with 3. Mu.M/kg (body weight) of a physiological saline solution every other day, mice of the physiological saline group were intraperitoneally injected with physiological saline every other day, tumors were taken at 24 th day, the length and width of the tumors were measured with calipers, and tumor volume was calculated as length×width 2 /2(mm 3 ) (n=5 per group).
Detailed Description
The technical means adopted by the invention and the effects thereof are further described below with reference to the examples and the attached drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting thereof.
The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or equipment used were conventional products available for purchase through regular channels, with no manufacturer noted.
Example 1
The antitumor polypeptide was prepared in this example.
Construction of the plasmid shown in FIG. 1, anti-tumor polypeptide (MAGEA 6 ΔIDR ) The fragment is derived from KYSE150 cDNA, the specific nucleic acid sequence is shown as SEQ ID NO.2, and MAGEA6 with Strep-Tag II is prepared by ClonExpress homologous recombination technology based on a cloning strategy of standard PCR ΔIDR Ligation to the pCS2 vector.
SEQ ID NO.2：
cgccattctgcctggggacgtcggagcaagcttgatttaggtgacactatagaatacaagctacttgttctttttgcaggatctgccaccatggctagcgcagcactcagtaggaaggtggccaagttggttcattttctgctcctcaagtatcgagccagggagccggtcacaaaggcagaaatgctggggagtgtcgtcggaaattggcagtacttctttcctgtgatcttcagcaaagcttccgattccttgcagctggtctttggcatcgagctgatggaagtggaccccatcggccacgtgtacatctttgccacctgcctgggcctctcctacgatggcctgctgggtgacaatcagatcatgcccaagacaggcttcctgataatcatcctggccataatcgcaaaagagggcgactgtgcccctgaggagaaaatctgggaggagctgagtgtgttagaggtgtttgaggggagggaagacagtatcttcggggatcccaagaagctgctcacccaatatttcgtgcaggaaaactacctggagtaccggcaggtccccggcagtgatcctgcatgctatgagttcctgtggggtccaagggccctcattgaaaccagctatgtgaaagtcctgcaccatatggtaaagatcagtggaggacctcgcatttcctacccactcctgcatgagtgggctttgagagagggggaagagtggagtcatccccaattcgaaaaactcgaggcatcaatgcagaagctgatctcagaggaggacctgtaatgatctagaactatagtgagtcgtattacgtagatccagacatgataagatacattgatgagtttggacaaaccacaactagaatgcagtgaaaaaaatgctttatttgtgaaatttgtgatgctattgctttatttgtaaccattataagctgcaataaacaagttaacaacaacaattgcattcattttatgtttcaggttcagggggaggtgtgggaggttttttaattcgcggccgcggcgccaatgcattgggcccggtacccagcttttgttccctttagtgagggttaattgcgcgcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaa
gctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctt
tctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttc
agcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagca
gccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggcta
cactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggc
aaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagat
cctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaag
gatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttacc
aatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataac
tacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttat
cagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaat
tgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtc
acgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaa
aagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactg
cataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtat
gcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatca
ttggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacc
caactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaaggg
aataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatga
gcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctaaatt
gtaagcgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctcattttttaaccaataggccgaaatcggcaaaatc
ccttataaatcaaaagaatagaccgagatagggttgagtgttgttccagtttggaacaagagtccactattaaagaacgtgg
actccaacgtcaaagggcgaaaaaccgtctatcagggcgatggcccactacgtgaaccatcaccctaatcaagttttttgg
ggtcgaggtgccgtaaagcactaaatcggaaccctaaagggagcccccgatttagagcttgacggggaaagccggcg
aacgtggcgagaaaggaagggaagaaagcgaaaggagcgggcgctagggcgctggcaagtgtagcggtcacgctg
cgcgtaaccaccacacccgccgcgcttaatgcgccgctacagggcgcgtcccattcgccattcaggctgcgcaactgtt
gggaagggcgatcggtgcgggcctcttcgctattacgccagtcgaccatagccaattcaatatggcgtatatggactcatg
ccaattcaatatggtggatctggacctgtgccaattcaatatggcgtatatggactcgtgccaattcaatatggtggatctgg
accccagccaattcaatatggcggacttggcaccatgccaattcaatatggcggacttggcactgtgccaactggggagg
ggtctacttggcacggtgccaagtttgaggaggggtcttggccctgtgccaagtccgccatattgaattggcatggtgcca
ataatggcggccatattggctatatgccaggatcaatatataggcaatatccaatatggccctatgccaatatggctattggc
caggttcaatactatgtattggccctatgccatatagtattccatatatgggttttcctattgacgtagatagcccctcccaatg
ggcggtcccatataccatatatggggcttcctaataccgcccatagccactcccccattgacgtcaatggtctctatatatgg
tctttcctattgacgtcatatgggcggtcctattgacgtatatggcgcctcccccattgacgtcaattacggtaaatggcccgc
ctggctcaatgcccattgacgtcaataggaccacccaccattgacgtcaatgggatggctcattgcccattcatatccgttct
cacgccccctattgacgtcaatgacggtaaatggcccacttggcagtacatcaatatctattaatagtaacttggcaagtaca
ttactattggaaggacgccagggtacattggcagtactcccattgacgtcaatggcggtaaatggcccgcgatggctgcc
aagtacatccccattgacgtcaatggggaggggcaatgacgcaaatgggcgttccattgacgtaaatgggcggtaggcgtgcctaatgggaggtctatataagcaatgctcgtttagggaac。
Under standard tissue culture conditions (37 ℃,5.0% co) 2 48 hours), the MAGEA 6-. DELTA.IDR-Strep-Tag II protein was overexpressed in HEK293T cells (mimicking mammalian protein modification). Cells were collected and then lysed according to the following formulation: 25mM Tris pH 7.4, 150mM NaCl, 0.5% TritonX-100, 1mM EDTA and 10% glycerol in admixture with protease inhibitor.
Preparation of anti-tumor polypeptide Using general affinity chromatography Strep-Tactin System, cells were spin-lysed at 4℃for 20 min, then at 10000gCentrifuge for 20 minutes. The protein was filtered through a 0.22 μm filter. Using Strep-The soluble protein was purified by the XT column (2-5012-001, iba) and eluted in the following buffers: 100mM Tris-Cl, 150mM NaCl, 1mM EDTA, 50mM biotin, pH 8 (2-1043-000, iba, or PBS (C10010500 BT, gibco), 500mM biotin, pH 8. Purified proteins were concentrated using an amicon ultra-4 filtration adsorption column (Merck), and verified by Coomassie blue staining (FIG. 2) to give the correct size.
Example 2
This example performs a cytotoxicity experiment.
KYSE150 cells were counted and plated in RPMI 1640 medium containing 10% fetal bovine serum to 24 well plates (5X 10 per well) 3 Individual cells). Cells were randomly assigned to the following groups: EXAMPLE 1 preparation of anti-tumor polypeptide (MAGEA 6 ΔIDR Peptide) was added to physiological saline group (CON) and 40. Mu.M cisplatin (DDP) at 0. Mu.M, 0.01. Mu.M, 0.05. Mu.M, respectively, and placed in 5% CO 2 Cells were counted and collected by incubation in an incubator at 37℃for 48 hours.
For IC 50 Cell experiments, IC using CCK8 kit 50 And (5) measuring. KYSE150 was seeded into 96-well plates at 2000 cells per well for 24 hours and treated with PBS and gradient concentrations of cisplatin or anti-tumor polypeptide for 48 hours. CCK8 was then added to the medium for 1 hour. CCK8 signal was detected at 450nm with a microplate reader and the results are shown in FIGS. 3A and 3B for MAGEA6 at concentrations of 0.01. Mu.M and 0.05. Mu.M ΔIDR The peptide has a certain effect of killing KYSE150 esophageal cancer cells, and the concentration of MAGEA6 is 0.01 mu M and 0.05 mu M ΔIDR After the peptide is combined with cisplatin, the activity rate of KYSE150 esophageal cancer cells is obviously lower than that of cisplatin alone. Half maximal Inhibitory Concentration (IC) of drug 50 ) Experiments show that MAGEA6 ΔIDR Peptide in combination with cisplatin as KYSE150, cisplatin IC for human esophageal cancer cells 50 Significantly reduced. Thus, with MAGEA6 ΔIDR The peptide and cisplatin combined use significantly improves the sensitivity of esophageal cancer cells to cisplatin.
Organoid toxicity testIC of ESCC organoids using quantification of the vital cell metabolism marker ATP using 3D cell viability assay kit (G9681, promega) 50 And (5) testing. Reagents were prepared overnight at 4 ℃. After equilibration of the organoids in 96-well plates to 25℃for about 30 minutes, 100. Mu.L of 3D reagent was added to each organoid well, and the contents were vigorously mixed for 5 minutes to lyse the cells. The 96-well plates were incubated at 25℃for an additional 25 minutes to stabilize luminescence. The luminescence intensity was recorded in the LUM full wavelength mode using a microplate reader, and the results are shown in FIGS. 4A and 4B for MAGEA6 at a concentration of 0.1. Mu.M ΔIDR After the peptide is combined with cisplatin, the human esophageal carcinoma organoid activity is obviously lower than that of a cisplatin group alone. Half maximal Inhibitory Concentration (IC) of drug 50 ) Experiments show that MAGEA6 ΔIDR Peptide and cisplatin combination for human esophageal carcinoma device, cisplatin IC 50 Significantly reduced. Thus, with MAGEA6 ΔIDR The peptide and cisplatin combined use significantly increase the sensitivity of esophageal carcinoma organoids.
Example 3
In vivo polypeptide therapy assays were performed in this example.
KYSE150 cells (purchased from ATCC) expressing MAGEA6 (WT) or MAGEA6-sgRNA (MAGEA 6-/-) were subcutaneously injected in the right inguinal region of nude mice from the center of animals of university of south medical science at 6 to 8 weeks of age (2X 10) 6 100 μl) was used for tumor formation 4 to 6 days later, and the length and width of the tumor was measured every 3 days using calipers. Tumor volume was calculated as length x width 2/2 (mm) 3 )。
Mice were randomly assigned to four treatment groups (number of mice per group 5): saline-treated group (CON), cisplatin (DDP) treated group, preparation of antitumor polypeptide (MAGEA 6) in example 1 ΔIDR Peptide (3. Mu.M/kg)) treatment group and cisplatin combined anti-tumor polypeptide treatment group (MAGEA 6) ΔIDR +DDP (3 mg/kg)). The anti-tumor polypeptide treatment group and cisplatin combined anti-tumor polypeptide treatment group mice are injected with 3 mu M/kg polypeptide every other day around tumor (the control group is injected with physiological saline), the cisplatin treatment group and the combined group cisplatin administration concentration is 3mg/kg, the tumor volume is measured after 24 days of treatment, the result is shown in fig. 5, both the anti-tumor polypeptide and cisplatin prepared in example 1 can inhibit tumor growth, and the anti-tumor polypeptide and cisplatin prepared in example 1 can be used in combinationThe treatment effect is improved in one step.
In conclusion, the invention develops the anti-tumor polypeptide which has the effect of killing the esophageal cancer cells and has low toxic and side effects, can be applied to esophageal cancer treatment, and in addition, the anti-tumor polypeptide can be combined with cisplatin to further improve the treatment effect, so that a new scheme and a new idea are provided for esophageal cancer treatment.
The applicant states that the detailed method of the present invention is illustrated by the above examples, but the present invention is not limited to the detailed method described above, i.e. it does not mean that the present invention must be practiced in dependence upon the detailed method described above. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.
Claims (10)
1. An anti-tumor polypeptide, which is characterized in that the amino acid sequence of the anti-tumor polypeptide comprises a sequence shown as SEQ ID NO. 1.
2. A nucleic acid molecule encoding the anti-tumor polypeptide of claim 1.
3. A recombinant vector comprising the nucleic acid molecule of claim 2.
4. A recombinant cell comprising the nucleic acid molecule of claim 2.
5. A method of producing an anti-tumor polypeptide according to claim 1, comprising:
inserting the encoding gene of the anti-tumor polypeptide in claim 1 into an expression vector to obtain a recombinant vector, introducing the recombinant vector into a host cell for culturing, collecting cells, and purifying to obtain the anti-tumor polypeptide.
6. The method of producing an antitumor polypeptide according to claim 5, wherein the expression vector comprises a plasmid;
preferably, the plasmid comprises a pCS2 plasmid;
preferably, the host cell comprises a HEK293T cell.
7. The use of an anti-tumor polypeptide according to claim 1 in the preparation of an anti-tumor medicament;
the tumor includes esophageal cancer.
8. A pharmaceutical composition comprising the anti-tumor polypeptide of claim 1.
9. The pharmaceutical composition of claim 8, wherein the pharmaceutical composition further comprises cisplatin;
preferably, the pharmaceutical composition is a single compound formulation or a combination of two separate formulations;
preferably, the pharmaceutical composition further comprises pharmaceutically acceptable pharmaceutical excipients;
preferably, the pharmaceutical excipients comprise any one or a combination of at least two of carriers, diluents, binders, wetting agents, disintegrants, emulsifiers, co-solvents, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH regulators, antioxidants, bacteriostats or buffers.
10. Use of an anti-tumor polypeptide according to claim 1 or a pharmaceutical composition according to claim 8 or 9 for the preparation of an esophageal cancer cell growth inhibitor or an esophageal cancer cell apoptosis inducer.
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