CN116715734A - Cyclic polypeptide and application thereof in preparation of antitumor drugs - Google Patents
Cyclic polypeptide and application thereof in preparation of antitumor drugs Download PDFInfo
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- CN116715734A CN116715734A CN202310468127.5A CN202310468127A CN116715734A CN 116715734 A CN116715734 A CN 116715734A CN 202310468127 A CN202310468127 A CN 202310468127A CN 116715734 A CN116715734 A CN 116715734A
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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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- 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
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Molecular Biology (AREA)
- Veterinary Medicine (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Public Health (AREA)
- Gastroenterology & Hepatology (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Zoology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
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- Physics & Mathematics (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses a cyclic polypeptide and application thereof in preparing antitumor drugs. The amino acid sequence of the cyclic polypeptide is as follows: CKGKFKRPPLKRVRMSADAMLC. The invention also provides a preparation method of the cyclic polypeptide: the DNA sequence for encoding the cyclic polypeptide is cloned into a prokaryotic expression vector in a traceless way, then escherichia coli DH5 alpha is transformed, the expression is induced by IPTG, and then the label is removed by SUMO protease treatment, so that the cyclic polypeptide is obtained. The prepared cyclic polypeptide has improved enzymolysis stability and longer half-life, i.e. longer retention time in vivo. The invention also discovers through experiments that the cyclic polypeptide can inhibit the growth and proliferation of various tumor cells, so that the cyclic polypeptide can be used for resisting tumors.
Description
Technical Field
The invention relates to the field of biological medicine, in particular to a cyclic polypeptide and application thereof in preparing an anti-tumor medicament.
Background
Liver cancer is one of the six most common cancers worldwide, with hepatocellular carcinoma (HCC) being the most prevalent subtype leading to liver cancer death. Despite advances in diagnosis and therapy, most HCC patients have a late survival of less than 1 year. However, there is no specific drug for HCC in clinic at present, and therefore, the development of new therapeutic drugs has been a hot spot.
The polypeptide medicine refers to peptide with specific therapeutic effect extracted from chemical synthesis, gene recombination or animals and plants, and is a specific application of the polypeptide in the field of medicine. Compared with small molecular medicines, the polypeptide medicine has the advantages of high bioactivity, high targeting property, high specificity, high efficiency and safety, and less side effect on human body because the polypeptide itself is a compound consisting of amino acids and the metabolite is amino acid. Compared with protein medicines, the polypeptide medicine has the advantages of better stability, high purity, low production cost, low immunogenicity or no immunogenicity and the like. Therefore, based on the unique advantages, the polypeptide medicament is widely applied to the treatment fields of diabetes, tumors, hepatitis and the like, for example, in the aspect of tumor treatment, the water-soluble nine peptide leuprorelin can stimulate the pituitary to secrete gonadotrophin, and is used for treating or relieving various sex hormone-dependent diseases such as prostate cancer and the like. In addition, the cilengitide with wide application in tumor treatment can inhibit the signal transduction process mediated by integrin by blocking the combination of integrin and extracellular matrix, and further block the proliferation, survival and migration of cells, thereby playing an anti-tumor role.
Disclosure of Invention
The primary object of the present invention is to overcome the disadvantages and shortcomings of the prior art and to provide a cyclic polypeptide.
The invention also aims to provide application of the cyclic polypeptide in preparing antitumor drugs.
The aim of the invention is achieved by the following technical scheme:
a cyclic polypeptide having the amino acid sequence shown below: CKGKFKRPPLKRVRMSADAMLC (SEQ ID NO. 1).
The DNA sequence of the cyclic polypeptide is shown as SEQ ID NO. 2.
A recombinant expression vector comprising the DNA sequence of the above-described cyclic polypeptide.
A recombinant bacterium is prepared by transforming competent cells of escherichia coli with the recombinant expression vector.
The preparation method of the cyclic polypeptide comprises the following steps:
(1) The DNA sequence of the annular polypeptide is cloned into a prokaryotic expression vector pET-N-His-PreScission-SUMO in a traceless manner to obtain a prokaryotic expression plasmid;
(2) And (3) converting the prokaryotic expression plasmid obtained in the step (1) into escherichia coli, performing isopropyl-beta-D-thiogalactoside (IPTG) induced expression, and performing SUMO protease treatment to remove the tag to obtain the cyclic polypeptide.
The escherichia coli in the step (2) is escherichia coli DH5 alpha.
The induction concentration of isopropyl-beta-D-thiogalactoside (IPTG) in the step (2) is 1mmol/L.
The conditions of induction described in step (2) are preferably: induction was carried out at 37℃for 4h.
The preparation method of the cyclic polypeptide further comprises the step of further purifying the cyclic polypeptide obtained in the step (2); the purification is realized by the following steps: after isopropyl-beta-D-thiogalactoside (IPTG) is induced to be expressed, ultrasonic sterilization is carried out, supernatant is taken, metal chelating affinity chromatography and ion exchange chromatography are sequentially utilized to purify, SUMO-His labels are removed through SUMO enzyme treatment, and metal chelating affinity chromatography is utilized to purify, so that purified cyclic polypeptide is obtained.
The application of the cyclic polypeptide in preparing antitumor drugs.
The tumor comprises liver cancer.
The application of the cyclic polypeptide in preparing medicines for inhibiting the growth and/or proliferation of liver cancer cells.
The effective concentration of the cyclic polypeptide is 12.5-100 mu mol/L; preferably 50. Mu. Mol/L.
The liver cancer cells comprise at least one of human liver cancer cells SMMC-7721, hepG2, huh7, MHCC97H and MHCC 97L; preferably human hepatoma cell MHCC97L.
The medicament may contain one or at least two pharmaceutically acceptable carriers.
The carrier is preferably a sustained release agent, an excipient, a filler, a binder, a wetting agent, a disintegrating agent, an absorption enhancer, an adsorption carrier, a surfactant, a lubricant, or the like.
The medicine is in the form of decoction, tablet, pill, capsule, injection (such as powder injection), granule, oral liquid or syrup.
Compared with the prior art, the invention has the following advantages and effects:
1. the cyclic polypeptide (named ctSAIF) has the advantages of improved enzymolysis stability, longer half-life and longer in vivo retention time.
2. The cyclic polypeptide ctSAIF has remarkable inhibition effect on various tumor cells including human liver cancer cell lines (7721, hepG2, huh7 and MHCC97H, MHCC L).
3. The cyclic polypeptide ctSAIF of the present invention inhibits the development of liver cancer transplants in mice.
4. The cyclic polypeptide ctSAIF can be synthesized through bioengineering, does not destroy ecological environment, is easy to realize industrialization, and is a very promising antitumor drug resource.
5. The cyclic polypeptide ctSAIF has an anti-tumor effect at the cellular level and the animal level, so that the ctSAIF can be used for preparing anti-tumor medicines for treating liver cancer.
Drawings
FIG. 1 is a diagram showing the concept of the ctSAIF polypeptide of the present invention; wherein A is SAIF in vitro serum degradation experimental HPLC; b is SAIF in vitro serum degradation curve; c is the total ion flow spectrum in SAIF in vitro serum degradation experiments; d is an amino acid sequence summary table.
FIG. 2 is a diagram showing the process of synthesizing ctSAIF polypeptide of the present invention by bioengineering; wherein, A is SDS-PAGE result graph before and after induction (lane 1: marker; lane 2: whole bacteria before induction; lane 3: whole bacteria after induction; lane 4: supernatant of the lysate of the bacteria after induction; lane 5: precipitation of the lysate of the bacteria after induction); b is SDS-PAGE result before and after purification (lane 1: thallus broken liquid, lane 2: ni column flow-through liquid, lanes 3, 4: ni column two elution peaks, lane 5:30Q flow-through liquid, lane 6, 7:30Q two elution peaks, lane 8: protein molecular weight standard); c is SDS-PAGE result before and after purification (lane 1: marker; lane 2: B2C-SUMO; lane 3: mix after digestion; lane 4: ni column flow-through; lane 5: ni column elution peak); d is a mass spectrogram result of ctSAIF; e is the in vitro serum degradation curve of cyclic peptides ctSAIF and SAIF.
FIG. 3 is a graph showing the results of the inhibition of the growth of liver cancer cell lines by ctSAIF polypeptide of the present invention in vitro; wherein, A is the influence of two polypeptides on the activity of human liver cells 7701 and human liver cancer cell lines (7721, MHCC97H, hepG, huh7, MHCC 97L); b is the inhibitory effect of two polypeptides on the clonal formation of human liver cells 7701, human liver cancer cells (7721, mhc 97H, hepG, huh7, mhc 97L); c is a statistical histogram of the cell colony.
FIG. 4 is a graph showing the results of inhibiting liver cancer metastasis with ctSAIF polypeptide of the present invention in mice; wherein A is a tumor size visual image; b is a tumor volume curve; c is tumor weight 14 days after SAIF administration; d is the weight change of nude mice during drug treatment; e is a nude mouse tumor section Ki67 stain.
Detailed Description
The present invention will be described in further detail with reference to examples, but embodiments of the present invention are not limited thereto. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art. The test methods for specific experimental conditions are not noted in the examples below, and are generally performed under conventional experimental conditions or under experimental conditions recommended by the manufacturer. The reagents and starting materials used in the present invention are commercially available unless otherwise specified.
Example 1: ctSAIF polypeptide design thought and production method
1.1 Experimental design
When in vitro serum stability analysis is carried out on polypeptide SAIF (sequence KEIDDLNLKVFDLKGKFKRPPLKRVRMSADAML, synthesized by Shanghai Yao Biotechnology Co., ltd.) (method is 1.4), the stability is not good, the half-life period is only 2-3 h, but the HPLC map shows a plurality of new peaks with drifting retention time, which are suspected to be degradation peaks, and the new peaks still exist at 12h, so that the stability of the degradation peaks is suspected to be possibly improved (figures 1A, 1B and 1C); in this regard, we performed mass spectrometry analysis to obtain a degradation peak which remained at a high level when incubated with in vitro serum for 12h, and from the primary mass spectrum it was found that the molecular weight was 2395.38, we named this sequence as ctSAIF, we enumerated the molecular weight of all possible degradation peptides based on the SAIF sequence, and further found that the corresponding amino acid sequence at this molecular weight was: CKGKFKRPPLKRVRMSADAMLC (fig. 1D).
1.2 preparation of ctSAIF
1.2.1 summary
The DNA sequence (GTGAGCAGATTGGAGTGCAAGGGCAAGTTCAAGAGGCCCCCCCTGAAGAGGGTGAGGATGAGCGCCGACGCCATGCTGTGCGATCCGAATTCGAGCT) was obtained from the amino acid sequence of ctSAIF (SEQ ID NO. 2), and after the chemical synthesis of this DNA sequence was committed by Shanghai Biotechnology Co., ltd, it was cloned tracelessly into the prokaryotic expression vector pET-N-His-SUMO (disclosed in the literature: xie Q, yao S, chen X, xu L, peng W, zhang L, zhang Q, liang X-F, hong A: A polypeptide from shark troponin I can inhibit angiogenesis and tumor growth. Molecular biology reports 2012,39 (2): 1493-1501.). The method is a prokaryotic expression plasmid for expressing target proteins with N-terminal containing His tag and SUMO tag, and is used for transforming escherichia coli DH5 alpha, inducing expression (IPTG final concentration 1mM, temperature 37 ℃ C., induction 4 h) by isopropyl-beta-D-thiogalactoside (IPTG), obtaining fusion protein ctSAIF-SUMO-His, removing tag by SUMO protease (purchased from Shanghai Biyun Tian biological Co., ltd., product number P2312S) treatment, purifying, realizing accurate expression of polypeptide, and verifying by utilizing mass spectrum.
1.2.2 procedure and results
As can be seen from lanes 2 and 3 of FIG. 2A, after transformation of a vector containing the ctSAIF sequence into E.coli DH 5. Alpha. A large amount of protein having a molecular weight of about 21kDa was expressed after induction compared with the control without IPTG induction. After resuspension of the cells (50 mM Tris-HCl,150mM NaCl,1mM ethylenediamine tetraacetic acid (EDTA); pH 7.5), after sonication, most of the inducer proteins were present in soluble form in the supernatant, as can be seen from lanes 4 and 5 of FIG. 2A.
Loading the bacterial supernatant on a metal chelating affinity chromatography Ni Sepharose 6Fast Flow, and after chromatographic elution (eluent: 20mM Tris,300mM imidazole, pH 8.0), loading the bacterial supernatant on an ion exchange chromatography Source 30Q and eluting (eluent: 20mM Tris,300mM sodium chloride, pH 8.0); as can be seen from FIG. 2B, lane 6, a single ctSAIF-SUMO-His band with a molecular weight of about 21kDa was obtained. As a result of removing the SUMO-His tag by SUMO enzyme treatment and digesting for 16 hours at 4℃after purification, as shown in FIG. 2C, it was found that ctSAIF-SUMO-His was separated into about 18kDa SUMO-His (lane 2) and about 3kDa single-chain ctSAIF (lane 3), and finally, the enzymatic hydrolysis mixture was subjected to metal chelate affinity chromatography Ni Sepharose 6Fast Flow (eluent: 20mM Tris,300mM imidazole, pH 8.0) again, and the Flow-through solution (Flow-through solution: 20mM Tris, pH 8.0) was collected (lane 4) to obtain a relatively pure single-chain ctSAIF. All purification procedures were run at room temperature using an AKTAavant instrument, cat No. 28930842, all columns purchased from glaiser biotechnology, inc, with Ni Sepharose 6Fast Flow packing No. 17531805,Source 30Q packing No. 17127302 and Flow rates of 1 milliliter per minute.
1.3 preparation of cyclic polypeptide ctSAIF
In order to obtain cyclic peptide, the single-chain ctSAIF is subjected to oxidation treatment to dehydrogenate and oxidize the first and the last cysteines to form disulfide bonds, and the specific steps are as follows: dimethyl sulfoxide (DMSO) was added to the purified single-stranded ctSAIF solution to a final concentration of 5% (v/v), the pH was adjusted to 8.5 with a 1M NaOH solution, and the solution was magnetically stirred at room temperature for 8 hours, during which time the free cysteine residue was monitored using an Ellman reagent until no free cysteine residue was detected, and oxidation was complete.
Subsequent mass spectrometry of the oxidized product confirmed whether or not dimers were formed between the molecules of ctSAIF, which showed a molecular weight of 2534.20, and no dimers were formed between the molecules of ctSAIF (fig. 2D), indicating that cyclic polypeptide ctSAIF had been cyclized.
1.4 in vitro stability experiments of Cyclic polypeptide ctSAIF
Rat serum (serum now taken from sd rats of 8 weeks old, purchased from guangdong pharmaceutical biotechnology limited) was uniformly mixed with polypeptide at a ratio of 9:1 (w/v) to give a polypeptide concentration of 1mg/mL; incubating the polypeptide-serum mixture in a 37 ℃ water bath for different times; after a predetermined time, adding pure trifluoroacetic acid to each sample to give a final trifluoroacetic acid concentration of 10% (v/v) in the mixture; centrifuging at 13000rpm for 10min after shaking, and collecting 10 μl of supernatant in HPLC sample loading bottle;
HPLC-UV analysis (instrument model Waters ACQUITY Arc Bio) was performed in the manner shown in Table 1, using a C18 analytical column (Thermo Acclaim 300C 18), determining a target peak from the retention time, recording the peak area thereof, and making a degradation curve;
according to the pretreatment method and chromatographic method of table 1, trifluoroacetic acid TFA was changed to formic acid, and analysis was performed using high resolution mass spectrometry (AB SCIEX 500R) to obtain a total ion flow diagram and a primary mass spectrum, and the instrument calculated the molecular weight of the main peak according to the charge-to-mass ratio.
Results are shown in fig. 2E: the half-life of ctSAIF is extended from about 2h to about 6h of SAIF.
TABLE 1
| Time (min) | Solution a:0.1% (v/v) TFA+water | Solution B:0.1% (v/v) TFA+acetonitrile |
| 0min | 95％ | 5％ |
| 27min | 75％ | 25％ |
| 28min | 5％ | 95％ |
| 33min | 5％ | 95％ |
| 40min | 95％ | 5％ |
| 45min | 95％ | 5％ |
Example 2: effect of Cyclic polypeptide ctSAIF on liver cancer cell growth in vitro
2.1 Experimental materials
And (3) cells: since the invention aims at inhibiting tumor generation, five human liver cancer cell lines SMMC-7721 (abbreviated as 7721), hepG2, huh7, MHCC97H (abbreviated as 97H) and MHCC97L (abbreviated as 97L) are selected, and 6 cells are purchased from ATCC cell libraries by taking normal liver cell QSG-7701 (abbreviated as 7701) as a control.
Reagent: the cyclic polypeptides ctSAIF, SAIF polypeptide, DMEM medium prepared in example 1.
2.2 Experimental methods
Taking cells with good growth condition and in logarithmic phase, inoculating into 96-well plate at 2500 cells per wellPlacing at 37deg.C and 5% (v/v) CO 2 Culturing in an incubator for 24 hours. The old medium was then removed and the cells were starved by treatment with DMEM medium (containing 0.5% (v/v) fetal bovine serum) for 24 hours. The cyclic polypeptide ctSAIF was diluted to various concentrations (12.5, 25, 50, 100. Mu.M) in DMEM medium containing 0.5% (v/v) fetal bovine serum, and 100. Mu.L was added to each well. Culturing in incubator at 37deg.C for 48 hr, removing culture medium, adding 100 μl/well of CCK-8 working solution, and placing at 37deg.C and 5% (v/v) CO 2 Incubating in incubator for 1h. Finally, the absorbance of each well at 450nm is detected by a multifunctional enzyme-labeled instrument. The experiment was repeated three times with no polypeptide drug added as a blank control and SAIF polypeptide added as a control.
2.3 experimental results
To determine if ctSAIF has the same inhibitory effect on tumors as SAIF, we performed the analysis using hepatoma cells as a cell model. As a result, as shown in FIG. 3, the inhibition of human HCC cell proliferation by SAIF and ctSAIF cyclopeptides was first confirmed by cell proliferation assay, and it was found that after five HCC cell lines (7721, hepG2, huh7, MHCC97H, MHCC L) were treated with different concentrations of SAIF or ctSAIF cyclopeptides for 48 hours, both SAIF and ctSAIF cyclopeptides decreased cell viability in a concentration-dependent manner (FIG. 3A). Meanwhile, the clonogenic assay (fig. 3B) also shows that SAIF and B2 peptides have a long-term inhibitory effect on the proliferation of HCC cells. In addition, human normal hepatocyte line 7701 was used to initially evaluate toxicity of SAIF and ctSAIF cyclic peptides, indicating that neither polypeptide was significantly toxic to normal hepatocyte lines.
Example 3: in vivo antitumor effect of cyclic polypeptide ctSAIF
3.1 Experimental materials
Animals: BALB/c-nude mice (average body weight 25g, 12 total) at 5 weeks of age were purchased from Guangdong Kangdong Biotechnology Co., ltd., product number D000521.
Reagent: the cyclic polypeptide ctSAIF and SAIF polypeptide prepared in example 1.
3.2 Experimental methods
Male BALB/c-nude mice were fed feed and purified water at room temperature in SPF laboratories. MHCC97L cells (1X 10 in 0.1mL physiological saline) 7 Individual cells) were subcutaneously injected under the axilla of 5-week-old male BALB/c-nude. Tumor volume and body weight were measured every 2 days and tumor volume was calculated using the following formula: (short diameter) 2× (long diameter)/2. When the tumor volume reaches about 50mm 3 At this time, these mice were randomly divided into 3 groups: model group (as control), SAIF treated group (25 mg/kg) and ctSAIF cyclopeptide treated group (25 mg/kg). SAIF and ctSAIF cyclic peptides were administered by tail vein injection at a volumetric dose of 0.1mL/20 g. Tumors were collected for histopathological examination, sacrificed 14 days after dosing.
3.3 experimental results
As a result, as shown in fig. 4, a significant decrease in average tumor volume (fig. 4A and 4B) and average tumor weight (fig. 4C) was observed after treatment with ctSAIF compared to the control group. No significant change in body weight occurred in each group of nude mice during the treatment (fig. 4D). Meanwhile, ki-67 staining of tumor tissue further demonstrated that both SAIF and ctSAIF cyclic peptides were able to inhibit tumor cell growth (FIG. 4E).
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (10)
1. A cyclic polypeptide characterized by the amino acid sequence shown below: CKGKFKRPPLKRVRMSADAMLC.
2. The DNA sequence of the cyclic polypeptide of claim 1, wherein: the nucleotide sequence is shown as SEQ ID NO. 2.
3. A recombinant expression vector, characterized in that: a DNA sequence comprising the cyclic polypeptide of claim 2.
4. A recombinant bacterium, characterized in that: prepared by transforming E.coli competent cells with the recombinant expression vector of claim 3.
5. The method for producing a cyclic polypeptide according to claim 1, comprising the steps of:
(1) A DNA sequence of the cyclic polypeptide of claim 2 is tracelessly cloned into a prokaryotic expression vector pET-N-His-PreScission-SUMO to obtain a prokaryotic expression plasmid;
(2) And (3) converting the prokaryotic expression plasmid obtained in the step (1) into escherichia coli, carrying out isopropyl-beta-D-thiogalactoside induced expression, and then carrying out SUMO protease treatment to remove the tag to obtain the cyclic polypeptide.
6. The use of the cyclic polypeptide of claim 1 in the preparation of an antitumor drug.
7. The use according to claim 6, characterized in that: the tumor is liver cancer.
8. The use of the cyclic polypeptide of claim 1 for the preparation of a medicament for inhibiting the growth and/or proliferation of liver cancer cells.
9. The use according to claim 8, characterized in that: the effective concentration of the cyclic polypeptide is 12.5-100 mu mol/L.
10. The use according to claim 9, characterized in that: the effective concentration of the cyclic polypeptide is 500 mu mol/L.
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