Antitumor polypeptide and application thereof
阅读说明：本技术 抗肿瘤多肽及其用途 (Antitumor polypeptide and application thereof ) 是由 张倩 于 2021-07-29 设计创作，主要内容包括：本发明涉及生物医药领域,具体地,本发明涉及一种抗肿瘤多肽及其用途。本发明提供一种抗肿瘤多肽,其包括MDX-(1)VDQSAVGFEYQGX-(2)TEX-(3)HA SQX-(4)GX-(5)TX-(6)X-(7)VQX-(8)EPAPGAPMGX-(9)VTAT或与其具有至少90％序列同一性的氨基酸序列；其中,X-(1)、X-(6)、X-(9)选自R或Q；X-(2)、X-(3)、X-(4)、X-(5)、X-(7)、X-(8)选自K或Q。该多肽显著抑制人肺癌、肝癌、胶质瘤、乳腺癌、结肠癌细胞增殖,并且显著抑制了肿瘤细胞克隆形成能力和细胞迁移能力,同时显著抑制小鼠成瘤模型生长。(The invention relates to the field of biological medicines, in particular to an anti-tumor polypeptide and application thereof. The invention provides an anti-tumor polypeptide which comprises MDX 1 VDQSAVGFEYQGX 2 TEX 3 HA SQX 4 GX 5 TX 6 X 7 VQX 8 EPAPGAPMGX 9 VTAT or an amino acid sequence having at least 90% sequence identity thereto; wherein, X 1 、X 6 、X 9 Is selected from R or Q; x 2 、X 3 、X 4 、X 5 、X 7 、X 8 Is selected from K or Q. The polypeptide can obviously inhibit the cell proliferation of human lung cancer, liver cancer, glioma, breast cancer and colon cancer, and can obviously inhibit the clone formation energy of tumor cellsForce and cell migration ability, and simultaneously obviously inhibits the growth of a mouse tumor model.)
1. An anti-tumor polypeptide, wherein the amino acid sequence of said anti-tumor polypeptide comprises MDX1VDQSAVGFEYQG X2TEX3HASQX4GX5TX6X7VQX8EPAPGAPMGX9VTAT or an amino acid sequence having at least 90% sequence identity thereto;
wherein, X1、X6、X9Each independently selected from R or Q;
X2、X3、X4、X5、X7、X8each independently selected from K or Q.
2. The anti-tumor polypeptide of claim 1, wherein the anti-tumor polypeptide comprises at least one amino acid sequence selected from the group consisting of amino acid sequences shown in SEQ ID NOS: 1-7.
3. A nucleic acid molecule encoding the anti-tumor polypeptide of claim 1 or 2.
4. A pharmaceutical composition comprising the anti-tumor polypeptide of claim 1 or 2 and/or the nucleic acid molecule of claim 3.
5. The pharmaceutical composition of claim 4, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
6. The pharmaceutical composition of claim 4, wherein the pharmaceutical composition is in the form of a tablet, injection, spray, capsule or coated pellet.
7. Use of the anti-tumor polypeptide of claim 1 or 2, the nucleic acid molecule of claim 3, the pharmaceutical composition of any one of claims 4-6 for the preparation of a medicament for the treatment of cancer.
8. The use according to claim 7, wherein the cancer is selected from at least one of lung cancer, liver cancer, colon cancer, glioma, breast cancer.
Technical Field
The invention relates to the field of biological medicines, in particular to an anti-tumor polypeptide and application thereof.
Background
The polypeptide is a compound formed by connecting one or more amino acids together by peptide bonds, and is generally formed by dehydration condensation of 10-100 amino acid molecules, and the molecular weight of the compound is less than 10000 Da. The polypeptide is an active group for the protein to play a role, is a bioactive substance related to various cell functions in an organism and participates in regulating various physiological functions, so the polypeptide has very important development value in clinical application.
The source of active polypeptide is divided into natural active polypeptide, artificially modified polypeptide based on natural product and artificially synthesized active polypeptide. The natural polypeptide has wide source, and can be divided into animal bioactive polypeptide and plant polypeptide. With the development of science and technology, natural polypeptides have not been the only source of polypeptides, but instead, genetically recombinant polypeptides and chemically synthesized polypeptides.
Methods for the synthesis of bioactive peptides include liquid phase synthesis, solid phase synthesis, enzymatic synthesis, and combinatorial biosynthesis. The liquid phase synthesis has 2 strategies of gradual synthesis and fragment synthesis, is convenient and quick for synthesizing the polypeptide with less amino acids, has high purity and can be synthesized in large quantity. Solid phase synthesis is a method in which the C-terminus of an amino acid is immobilized on an insoluble resin, and then the amino acids are condensed in sequence. This method simplifies the work-up procedure for each reaction step, and has a high yield, with the disadvantages that the intermediate products in each reaction step cannot be purified, and the final product must be purified by a reliable separation means. Both liquid phase synthesis and solid phase synthesis belong to chemical synthesis, and 90% of polypeptide drugs developed and marketed at present are chemically synthesized, so that the application is wide. The enzymatic synthesis is a biosynthesis method which utilizes certain specific enzymes to catalyze reactions, has mild reaction conditions and strong stereospecificity, and has the defects of many adverse reactions and easy denaturation and inactivation of the enzymes. Combinatorial biosynthesis is the manipulation of genes encoding enzymes in the metabolic pathways of microorganisms to obtain new products. The method has the problems of high cost, easy formation of improper folding and posttranslational modification of eukaryotic gene expression and the like, thereby preventing the wide application of the method.
Currently, local cancers, while successfully treated with surgery and radiation therapy, are still the first choice for conventional treatment of advanced or metastatic tumors. The chemotherapy drugs have the defects of low selectivity, large adverse reaction, multi-drug resistance and the like, so the use of the chemotherapy drugs is limited to a great extent. Although the monoclonal antibody tumor targeted therapy solves the problem of targeting, the monoclonal antibody tumor targeted therapy has large protein molecular weight, high immunogenicity, easy generation of allergy and immunological cross reaction, high selling price due to high research and development cost, high research and development difficulty, limited productivity and the like, common cancer patients are difficult to popularize and use, and meanwhile, the monoclonal antibody drugs have the problems of single dosage form, most of administration modes are intravenous injection or instillation, oral administration cannot be realized, and the like, so that the drug application is very inconvenient.
Compared with small molecular chemical drugs, the anti-tumor polypeptide has higher affinity and stronger specificity to target tumors, has low adverse reaction, and can also increase the sensitivity of the tumors to other treatment methods; compared to antibodies, they are more easily penetrated into tissues due to their small volume; can be chemically synthesized, and can be chemically modified by various means, which is helpful for designing and researching novel active polypeptide.
The novel anti-tumor active polypeptide has the characteristics of high affinity, strong specificity and low adverse reaction, and most of the novel anti-tumor active polypeptide also has the property of selectively targeting tumor cells, so the novel anti-tumor active polypeptide has very important development value in clinical application and has better prospect as an anti-tumor compound. However, the currently known antitumor polypeptides are limited in kind and further enrichment is required.
Disclosure of Invention
The present invention is directed to solving, at least to some extent, one of the technical problems in the related art. The invention provides an anti-tumor polypeptide, which can obviously inhibit the cell proliferation of human lung cancer, liver cancer, glioma, breast cancer and colon cancer, obviously inhibit the clonogenic capacity of tumor cells, obviously inhibit the cell migration capacity of human lung cancer, liver cancer and glioma, and obviously inhibit the growth of a mouse tumor-forming model, and particularly has more obvious effect of inhibiting the lung cancer. The polypeptide can be used as an effective component for a medicament for treating cancer.
In view of this, the first aspect of the present invention provides an anti-tumor polypeptide. According to an embodiment of the invention, the amino acid sequence of the anti-tumor polypeptide comprises MDX1VDQSAVGFEYQGX2TEX3HASQX4GX5TX6X7VQX8EPAPGAPMGX9VTAT or an amino acid sequence having at least 90% sequence identity thereto;
wherein, X1、X6、X9Each independently selected from R or Q;
X2、X3、X4、X5、X7、X8each independently selected from K or Q.
Based on tumorigenesis characteristics and treatment difficulties, the inventor creatively discovers an anti-tumor polypeptide with an amino acid sequence shown as above, and discovers that the polypeptide can remarkably inhibit the proliferation of human lung cancer, liver cancer, glioma, breast cancer and colon cancer cells, remarkably inhibit the clonogenic capacity of tumor cells, remarkably inhibit the migration capacity of human lung cancer, liver cancer and glioma cells, and remarkably inhibit the growth of a mouse tumorigenic model, especially the effect of the polypeptide on inhibiting lung cancer is more obvious by performing in vivo and in vitro experiments.
According to an embodiment of the present invention, the above-mentioned anti-tumor polypeptide may further comprise at least one of the following additional technical features:
according to an embodiment of the invention, the anti-tumor polypeptide comprises at least one of the amino acid sequences shown in SEQ ID NO 1-7.
The amino acid sequence shown in SEQ ID NO. 1 is:
MDQVDQSAVGFEYQGKTEKHASQKGKTRKVQKEPAPGAPMGRVTAT
the amino acid sequence shown in SEQ ID NO. 2 is:
MDRVDQSAVGFEYQGKTEKHASQKGKTRKVQKEPAPGAPMGQVTAT
the amino acid sequence shown in SEQ ID NO. 3 is:
MDRVDQSAVGFEYQGKTEKHASQKGKTQKVQKEPAPGAPMGRVTAT
the amino acid sequence shown in SEQ ID NO. 4 is:
MDQVDQSAVGFEYQGKTEKHASQKGKTQQVQKEPAPGAPMGQVTAT
the amino acid sequence shown in SEQ ID NO. 5 is:
MDRVDQSAVGFEYQGKTEKHASQQGQTRKVQKEPAPGAPMGRVTAT
the amino acid sequence shown in SEQ ID NO. 6 is:
MDRVDQSAVGFEYQGQTEQHASQKGKTRKVQKEPAPGAPMGRVTAT
the amino acid sequence shown in SEQ ID NO. 7 is:
MDQVDQSAVGFEYQGKTEKHASQKGKTRQVQQEPAPGAPMGRVTAT
based on tumorigenesis characteristics and treatment difficulties, the inventor finds that the amino acid sequences shown in SEQ ID NO 1-7, then researches the antitumor functions of the polypeptide and the polypeptide composition, and shows that the polypeptide and the polypeptide composition have important anticancer functions of inhibiting tumor cell activity and inhibiting the growth of a mouse tumorigenic model at in vivo and in vitro levels. The invention provides experimental basis for developing new targets for tumor treatment, and has very important development and application prospect for clinical treatment of tumors such as lung cancer, liver cancer, colorectal cancer, glioma and the like.
In a second aspect, the invention provides a nucleic acid molecule. According to an embodiment of the invention, the nucleic acid molecule encodes the anti-tumor polypeptide of the first aspect.
In a third aspect, the invention provides a pharmaceutical composition. According to an embodiment of the invention, the pharmaceutical composition comprises the anti-tumor polypeptide of the first aspect and/or the nucleic acid molecule of the second aspect.
The pharmaceutical composition provided by the invention can contain any one, two, three, four, five, six or seven of the amino acid sequences shown in SEQ ID NO. 1-7.
According to an embodiment of the present invention, the above pharmaceutical composition may further comprise at least one of the following additional technical features:
according to an embodiment of the invention, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
According to an embodiment of the present invention, the pharmaceutical composition is in the form of a tablet, injection, spray, capsule or coated pill.
In a fourth aspect, the present invention provides the use of the anti-tumor polypeptide of the first aspect, the nucleic acid molecule of the second aspect, or the pharmaceutical composition of the third aspect, in the manufacture of a medicament. According to an embodiment of the invention, the medicament is for the treatment of cancer.
According to an embodiment of the invention, the cancer is selected from at least one of lung cancer, liver cancer, colon cancer, glioma, breast cancer.
The invention has the beneficial effects that:
compared with the prior art, the invention creatively discovers the anti-tumor polypeptide through an anti-tumor gene therapy approach based on the tumorigenic characteristics and the treatment difficulty, then researches the anti-tumor function of the polypeptide or the polypeptide composition, and shows in vivo and in vitro levels through the treatment of the polypeptide or the polypeptide composition. The invention provides experimental basis for developing new targets for tumor treatment, and has very important development and application prospect for clinical treatment of tumors such as lung cancer, liver cancer, colorectal cancer, glioma and the like.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1 shows the results of CCK-8 experiments in SEQ ID NO 1-7 and control group in lung cancer cell H1299, liver cancer cell HepG2, glioma cell U87, breast cancer cell MCF7 and colon cancer cell HCT116, respectively, where "Con" is the control group;
FIG. 2 is a graph showing the results of the colony formation experiment in different treatment groups, wherein "Con" is a control group;
FIG. 3 shows a graph of the results of the cell scratch-healing experiments in different treatment groups;
FIG. 4 shows the tumor size of subcutaneous tumors of nude mice in different treatment groups;
FIG. 5 shows the statistics of tumor volume in different treatment groups, wherein "Con" is the control group,
in the drawings, SEQ ID No. 1 and SEQ ID No. 1 refer to the same amino acid sequence.
Detailed Description
The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.
Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "a plurality" means at least two, e.g., two, three, etc., unless specifically limited otherwise.
The term "optionally" is used for descriptive purposes only and is not to be construed as indicating or implying relative importance. Thus, a feature defined as "optionally" may or may not explicitly include the feature.
According to a particular embodiment of the invention, the invention provides an anti-tumour polypeptide, the amino acid sequence of which comprises MDX1VDQSAVGFEYQGX2TEX3HASQX4GX5TX6X7VQX8EPAPGAPMGX9VTAT or an amino acid sequence having at least 90% sequence identity thereto;
wherein, X1、X6、X9Each independently selected from R or Q;
X2、X3、X4、X5、X7、X8each independently selected from K or Q.
"having at least 90% sequence identity" means that the amino acid sequence thereof has 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% similarity to the aforementioned amino acid sequence.
"independently selected" means that X1-X9The amino acids that can be selected do not interfere with each other.
According to an embodiment of the invention, the anti-tumor polypeptide is selected from at least one of the amino acid sequences shown in SEQ ID NO 1-7.
The amino acid sequence shown in SEQ ID NO. 1 is:
MDQVDQSAVGFEYQGKTEKHASQKGKTRKVQKEPAPGAPMGRVTAT
the amino acid sequence shown in SEQ ID NO. 2 is:
MDRVDQSAVGFEYQGKTEKHASQKGKTRKVQKEPAPGAPMGQVTAT
the amino acid sequence shown in SEQ ID NO. 3 is:
MDRVDQSAVGFEYQGKTEKHASQKGKTQKVQKEPAPGAPMGRVTAT
the amino acid sequence shown in SEQ ID NO. 4 is:
MDQVDQSAVGFEYQGKTEKHASQKGKTQQVQKEPAPGAPMGQVTAT
the amino acid sequence shown in SEQ ID NO. 5 is:
MDRVDQSAVGFEYQGKTEKHASQQGQTRKVQKEPAPGAPMGRVTAT
the amino acid sequence shown in SEQ ID NO. 6 is:
MDRVDQSAVGFEYQGQTEQHASQKGKTRKVQKEPAPGAPMGRVTAT
the amino acid sequence shown in SEQ ID NO. 7 is:
MDQVDQSAVGFEYQGKTEKHASQKGKTRQVQQEPAPGAPMGRVTAT
the anti-tumor polypeptide of the present invention is not limited to a single polypeptide, but includes any combination of these polypeptides. For example, the amino acid sequences of the antitumor polypeptides are the amino acid sequences shown in SEQ ID NO. 1 and SEQ ID NO. 3, or the amino acid sequences of the antitumor polypeptides are the amino acid sequences shown in SEQ ID NO. 1 and SEQ ID NO. 5, or the amino acid sequences of the antitumor polypeptides are the amino acid sequences shown in SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, etc.
The anti-tumor polypeptide or polypeptide composition provided by the invention can be obtained by a solid phase synthesis mode or a molecular cloning technology to obtain an expression vector for expressing the polypeptide, so that the anti-tumor polypeptide or polypeptide composition is obtained.
The inventor researches the anti-tumor function of the amino acid sequences shown in SEQ ID NO. 1-7, and shows that the polypeptide and the polypeptide composition have important anti-cancer functions of inhibiting the activity of tumor cells and inhibiting the growth of a mouse tumor model at in vivo and in vitro levels. The invention provides experimental basis for developing new targets for tumor treatment, and has very important development and application prospect for clinical treatment of tumors such as lung cancer, liver cancer, colorectal cancer, glioma and the like.
According to a particular embodiment of the invention, there is provided a nucleic acid molecule. The nucleic acid molecule encodes MDX1VDQSAVGFEYQGX2TEX3HASQX4GX5TX6X7VQX8EPAPGAPMGX9VTAT or an amino acid sequence having at least 90% sequence identity thereto, wherein X1、X6、X9Each independently selected from R or Q; x2、X3、X4、X5、X7、X8Each independently selected from K or Q. The nucleic acid molecule may be DNA or RNA.
According to a specific embodiment of the present invention, there is provided a pharmaceutical composition comprising the above antitumor polypeptide and/or the above nucleic acid molecule.
The pharmaceutical composition may be a polypeptide drug comprising the above antitumor polypeptide, or may be a nucleic acid drug comprising the above nucleic acid molecule expressing the antitumor polypeptide. The pharmaceutical composition can also be a combination of an anti-tumor polypeptide and a nucleic acid molecule expressing the anti-tumor polypeptide.
The pharmaceutical composition provided by the invention can contain any one, two, three, four, five, six or seven of the amino acid sequences shown in SEQ ID NO. 1-7.
According to a particular embodiment of the invention, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
According to a specific embodiment of the present invention, the pharmaceutical composition may be in the form of a tablet, injection, spray, capsule or coated pill. The dosage form of the pharmaceutical composition of the present invention is not limited thereto, and may be changed to other dosage forms in the pharmaceutical field as needed.
According to a specific embodiment of the present invention, the above antitumor polypeptide, nucleic acid molecule encoding antitumor polypeptide, pharmaceutical composition can be used for preparing a medicament for treating cancer. The cancer is at least one selected from lung cancer, liver cancer, colon cancer, glioma, and breast cancer.
In order to explore the tumor inhibiting effect of the antitumor polypeptide found by the inventor, the following related researches are carried out:
the in vitro verification research of the function of the polypeptide for resisting lung cancer, liver cancer, colon cancer and glioma; the influence of the peptide fragment sequence on the cell proliferation capacity is researched through a CCK8 and clone formation experiment; the influence of the peptide fragment sequence on the cell migration capacity is studied by scratch test.
The polypeptide is found to obviously inhibit the cell proliferation of human lung cancer, liver cancer, glioma, breast cancer and colon cancer, obviously inhibit the clonogenic capacity of tumor cells, obviously inhibit the cell migration capacity of human lung cancer, liver cancer and glioma, and obviously inhibit the growth of a mouse tumor-forming model, especially the effect of the polypeptide on inhibiting lung cancer is more obvious.
It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples do not specify particular techniques or conditions, and are carried out according to techniques or conditions described in literature in the art (for example, refer to molecular cloning, a laboratory Manual, third edition, scientific Press, written by J. SammBruke et al, Huang Petang et al) or according to product instructions. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
EXAMPLE 1 preparation of polypeptide working solutions
Polypeptide SEQ ID NO 1-7 is synthesized by Kinry Biotech company, and peptide fragments with different sequences are respectively added into 0.01MPBS to prepare 10X peptide fragment working solution (the concentration is 500 ng/mL).
The polypeptide SEQ ID NO 1-7 has the sequence shown in the following table 1:
TABLE 1
Numbering
Sequence of
SEQ ID NO:1
MDQVDQSAVGFEYQGKTEKHASQKGKTRKVQKEPAPGAPMGRVTAT
SEQ ID NO:2
MDRVDQSAVGFEYQGKTEKHASQKGKTRKVQKEPAPGAPMGQVTAT
SEQ ID NO:3
MDRVDQSAVGFEYQGKTEKHASQKGKTQKVQKEPAPGAPMGRVTAT
SEQ ID NO:4
MDQVDQSAVGFEYQGKTEKHASQKGKTQQVQKEPAPGAPMGQVTAT
SEQ ID NO:5
MDRVDQSAVGFEYQGKTEKHASQQGQTRKVQKEPAPGAPMGRVTAT
SEQ ID NO:6
MDRVDQSAVGFEYQGQTEQHASQKGKTRKVQKEPAPGAPMGRVTAT
SEQ ID NO:7
MDQVDQSAVGFEYQGKTEKHASQKGKTRQVQQEPAPGAPMGRVTAT
Example 2 experimental verification of CCK8
The effect of different polypeptides of SEQ ID NO:1-7 on cell proliferation was investigated using the CCK-8 detection kit (Dojindo). Briefly, cells were plated at 2X 10 per well3Individual cell concentrations were seeded into 96-well plates and incubated with polypeptide for 24, 48, 72h, respectively. At the indicated time points, 10. mu.L of CCK-8 assay reagent was added and the plates were rewarmingAnd (5) breeding for 4 h. Absorbance at 450nm was measured using a SpectraMax M5 microplate reader (Molecular Devices). Lung cancer cell H1299, liver cancer cell HepG2, glioma cell U87, breast cancer cell MCF7 and colon cancer cell HCT116 were detected respectively, and the control group was PBS. The results are shown in FIG. 1, which shows that in the control group of 5 tumor cells without the polypeptide of the present invention, the tumor cells proliferated most rapidly, while 7 polypeptides of SEQ ID NO 1-7 inhibited tumor proliferation to different extents. The 7 polypeptides of SEQ ID NO. 1-7 are shown to have the obvious effect of inhibiting the cell proliferation of human lung cancer, liver cancer, glioma, breast cancer and colon cancer, and the peptide segments inhibit the cell proliferation of tumor to different degrees.
The statistical results of the CCK-8 experiments are shown in the following Table 2, wherein the percentage shown in the table is the inhibition rate of different polypeptides in different tumor cells compared with the control group:
TABLE 2
Example 3 Soft agar formation experiment
The working solution of the peptide fragment of SEQ ID NO:1-7 obtained in example 1 was diluted (concentration: 50ng/mL), cells were treated, tumor cells in logarithmic phase (lung cancer cell H1299, liver cancer cell HepG2, glioma cell U87) were collected and prepared into cell suspension, and PBS was used as a control. Adding the cell suspension with the adjusted concentration into each culture bottle, then adding agar into each culture bottle, and uniformly mixing. The plates were plated and cell agar suspension was added to each well and each group was added. The cell agar suspension is left to solidify at room temperature. The plates were then transferred to an incubator at 37 ℃. After 14 days of culture, the cell colonies were visually observed and counted. The results are shown in figure 2, and it can be clearly seen that the tumor cell clonogenic capacity is inhibited in the peptide segment group of SEQ ID NO 1-7, which shows that the peptide segment of SEQ ID NO 1-7 has the ability of significantly inhibiting the proliferation of human lung cancer, liver cancer and glioma cells, and the peptide segment inhibits the tumor cell clonogenic capacity to different degrees.
Example 4 cell scratch-healing experiment (scratch-healing)
And scribing a line at the back of the 6-hole plate by 0.5-1 cm, and transversely penetrating through the hole. Each hole passes through at least 5 lines. Then, cells (lung cancer cells H1299, liver cancer cells HepG2 and glioma cells U87) treated by diluting the working solution of the peptide fragment of SEQ ID NO:1-7 (with the concentration of 50ng/mL) are inoculated into a 6-well plate, and the inoculation number is 5-10 x 105And (4) cells. On the next day, the gun head is used, compared with the straight ruler, the gun head is perpendicular to the transverse line scratch on the back as much as possible, and the gun head is not inclined. The cells were rinsed 3 times with PBS, the scraped cells were removed, serum free medium (Gibco) was added, and 37 degrees 5% CO was added2And (5) an incubator for culture. Samples were taken at 0, 6, 12, and 24 hours, photographed, and counted for cell migration ability, with PBS as a control group. The results are shown in figure 3, and the results show that the polypeptides of SEQ ID NO. 1-7 of the invention have the capability of obviously inhibiting the migration of human lung cancer, liver cancer and glioma cells, and different peptide segments have the capability of inhibiting the migration of tumor cells to different degrees.
EXAMPLE 5 nude mice subcutaneous tumor formation experiment
Subcutaneous tumorigenicity experiments were performed in nude mice (5 weeks old) and divided into: control group (PBS) and polypeptide treated group (SEQ ID NO:1-7 peptide fragment), each group containing 5 of the following peptides, were treated by the following method: cells from different groups were collected after trypsinization. Cell counts were performed after one wash of serum-free medium Gibco. Suspending the counted cells in a serum-free medium to adjust the cell density to 5X 107The volume is ready for use. Inoculating H1299 and HepG2 cells under the skin of experimental nude mice, breeding nude mice of different groups in cages after inoculation, feeding water and feed regularly, and replacing padding regularly. After the tumor formation, the tumor body was injected at a concentration of 20mg/kg from day 3, day 7 and day 10, respectively, and the mice were sacrificed for 30 days, photographed, tumor tissue was peeled off and the tumor body size was calculated. The results are shown in fig. 4 and 5, fig. 4 shows the tumor size of subcutaneous tumor formation of nude mice in different treatment groups, fig. 5 shows the statistical result of the tumor volume in different treatment groups, and the results show that the polypeptide of SEQ ID nos. 1-7 has the effect of remarkably inhibiting the growth of a mouse tumor formation model, and especially the effect of the polypeptide in inhibiting lung cancer is more obvious.
Tumor size statistics are shown in table 3 below, where the percentages shown are the percent reduction in tumor volume compared to the control group.
TABLE 3
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Sequence listing
<110> Shanghai Kanji biological medicine science and technology Co., Ltd
<120> antitumor polypeptide and use thereof
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Met Asp Gln Val Asp Gln Ser Ala Val Gly Phe Glu Tyr Gln Gly Lys
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Thr Glu Lys His Ala Ser Gln Lys Gly Lys Thr Arg Lys Val Gln Lys
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Glu Pro Ala Pro Gly Ala Pro Met Gly Arg Val Thr Ala Thr
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Met Asp Arg Val Asp Gln Ser Ala Val Gly Phe Glu Tyr Gln Gly Lys
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Thr Glu Lys His Ala Ser Gln Lys Gly Lys Thr Arg Lys Val Gln Lys
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Glu Pro Ala Pro Gly Ala Pro Met Gly Gln Val Thr Ala Thr
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Met Asp Arg Val Asp Gln Ser Ala Val Gly Phe Glu Tyr Gln Gly Lys
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Met Asp Arg Val Asp Gln Ser Ala Val Gly Phe Glu Tyr Gln Gly Gln
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Met Asp Gln Val Asp Gln Ser Ala Val Gly Phe Glu Tyr Gln Gly Lys
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