CN107056887B

CN107056887B - Polypeptide and application thereof in preparing medicine for treating and preventing tumors

Info

Publication number: CN107056887B
Application number: CN201610921217.5A
Authority: CN
Inventors: 胡卓伟; 花芳; 李珂
Original assignee: Individual
Current assignee: Individual
Priority date: 2015-10-22
Filing date: 2016-10-21
Publication date: 2020-10-16
Anticipated expiration: 2036-10-21
Also published as: CN107056887A; WO2017067510A1

Abstract

The invention discloses a polypeptide specifically binding to TRB3 and application thereof in preparing a medicament for treating and preventing tumors. The amino acid sequence of the polypeptide is shown by replacing two or more than two amino acids in the amino acid sequence shown in the sequence table SEQ ID No.8 with unnatural amino acids which enable other side chains to be connected. The polypeptide can be specifically combined with TRB3, so that the interaction between TRB3 and P62 protein is blocked, and the polypeptide can be applied to the preparation of medicines for treating and preventing tumors. The prepared medicine has the advantages of obvious curative effect, less toxic and side effect and safe use in treating tumor diseases.

Description

Polypeptide and application thereof in preparing medicine for treating and preventing tumors

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a polypeptide and application thereof in preparing a medicament for treating and preventing tumors.

Background

TRB3 (Tribbeles Homologue 3) is one of the Tribbeles homologous protein family members, and is involved in regulating cell proliferation, migration and morphogenesis during development. TRB3 is a pseudo kinase protein family member, has a joint protein-like function, and is involved in the assembly of various protein complexes. Multiple studies suggest that TRB3 can interact with various transcription factors, ubiquitin ligases, type II BMP receptors on cell membranes and MAPK, PI3K signal pathway member proteins, and participate in regulation of glycolipid metabolism, adipocyte differentiation, apoptosis, stress, and the like. Recently, various evidences suggest that TRB3 exhibits high expression in various tumor cell lines and human tumor tissues, and plays an important promoting role in the development of tumors. The research finds that the TRB3 inhibits the autophagy activity of cells and promotes the proliferation and metastasis of tumor cells by interacting with the autophagy wagon protein p 62. It can be seen that targeting the interaction between TRB3 and p62 is a potential target for tumor treatment. Therefore, the research and development of the substance for blocking the interaction between TRB3 and P62 protein have good prospect of drug development and development inhibition of tumor.

Protein-protein interactions (PPIs) play important roles in many biological processes, such as cell proliferation, growth, differentiation and programmed death. Many potential therapeutic targets in human disease are primarily protein-protein interactions. During protein-protein interactions, the alpha helix and beta sheet secondary structures are the major interface units involved in PPIs. In recent years, the chemical synthesis of highly active and highly selective synthetic polypeptide drugs has become a new research focus. However, the binding ability of polypeptides to affected proteins is very weak, and ordinary linear polypeptides cannot permeate cell membranes and are easily hydrolyzed by proteases. Therefore, the polypeptide target drugs have a plurality of defects at present. From the above, it is highly desirable to obtain a highly active and highly selective synthetic polypeptide drug targeting the interaction between TRB3 and p 62.

Disclosure of Invention

The invention aims to solve the technical problem of providing a polypeptide specifically binding to TRB3 and application thereof in preparing a medicament for treating and preventing tumors, aiming at the current situation that a high-activity and high-selectivity synthetic polypeptide medicament targeting the interaction between TRB3 and p62 is absent at present.

Through intensive research and repeated experiments, the inventors of the present invention found that the polypeptide A2 (amino acid sequence is shown in SEQ ID No.8 of the sequence table) targeting TRB3 to interact with p62 protein has low specific binding capacity and biological stability with TRB 3. It is directly related to the inability of polypeptide a2 to stably form the alpha helix conformation required for activity in solution. Therefore, the inventors have conducted targeted studies and experiments, and found that if an amino acid residue at a specific position in the polypeptide a2 is replaced with an unnatural amino acid to which a side chain can be connected, such as S-pentenylalanine (S5), the modified polypeptide has a stable secondary structure of an α -helix, so that the modified polypeptide has extremely high affinity, stability against enzymatic hydrolysis, and cell-penetrating property, i.e., the α -helix stability, TRB3 binding capacity, and metabolic stability are improved, and proliferation and metastasis of various tumor cells are inhibited. Experiments prove that the modified polypeptide can be applied to preparing medicaments for treating and preventing tumors. Based on the research work of the inventor, the invention provides the following technical scheme.

One of the technical schemes provided by the invention is as follows: a polypeptide specifically binding to TRB3, wherein the amino acid sequence of the polypeptide is shown by replacing two or more than two amino acids in the amino acid sequence shown in the sequence table SEQ ID No.8 with unnatural amino acids with connectable side chains.

The unnatural amino acid to which the other side chain is attached is an unnatural amino acid which is conventional in the art, and preferably S-pentenylalanine (S5).

Preferably, in the polypeptide, the number of the substituted amino acids is two, and the positions of the substituted amino acids are i-th position and i + 3-th position respectively, or i-th position and i + 4-th position, wherein i is more than or equal to 1 and less than or equal to 7, and i is a positive integer.

More preferably, the amino acid sequence of the polypeptide is as shown in any one of sequence tables SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6 and SEQ ID No. 7.

Wherein, the amino acid sequence shown in SEQ ID No.1-SEQ ID No.7 can be appropriately substituted, deleted or added, as long as the modified amino acid sequence can still be specifically combined with TRB3 and the activity before modification is kept.

The second technical scheme provided by the invention is as follows: application of a polypeptide specifically binding TRB3 in preparing a medicament for treating and/or preventing tumors.

The tumor is a tumor which is conventional in the art. Preferably liver cancer, lung cancer, breast cancer, intestinal cancer or leukemia. Wherein the liver cancer is the conventional liver cancer in the field, and preferably primary liver cancer or secondary liver cancer. The lung cancer is conventional lung cancer in the field, and preferably small cell lung cancer or non-small cell lung cancer. The breast cancer is conventional breast cancer in the field, and preferably non-invasive breast cancer, early invasive breast cancer, invasive specific type breast cancer or invasive non-specific type breast cancer. The intestinal cancer is conventional intestinal cancer in the art, preferably colon cancer or rectal cancer. The leukemia is a leukemia conventional in the art, preferably a lymphocytic leukemia or a non-lymphocytic leukemia.

Such prevention is conventional in the art and preferably means preventing or reducing the development of a tumor after use in the presence of a potential tumor agent. The treatment is conventional in the art and preferably means reducing the extent of the tumor, or curing the tumor to normalize it, or slowing the progression of the tumor.

The third technical scheme provided by the invention is as follows: an anti-tumor pharmaceutical composition, which contains the polypeptide specifically binding to TRB3 as an active ingredient.

The active component is a compound with the function of preventing or treating tumors. In the pharmaceutical composition, the polypeptide specifically binding to TRB3 can be used as an active ingredient alone or together with other compounds having antitumor activity.

The administration route of the pharmaceutical composition of the present invention is preferably injection administration or oral administration. The injection administration preferably comprises intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection and the like. The pharmaceutical composition is various dosage forms which are conventional in the field, preferably in the form of solid, semisolid or liquid, and can be aqueous solution, non-aqueous solution or suspension, and more preferably tablet, capsule, granule, injection or infusion, etc.

Preferably, the pharmaceutical composition of the present invention further comprises one or more pharmaceutically acceptable carriers. The medicinal carrier is a conventional medicinal carrier in the field, and can be any suitable physiologically or pharmaceutically acceptable medicinal auxiliary material. The pharmaceutical adjuvant is conventional in the field, and preferably comprises pharmaceutically acceptable excipient, filler or diluent and the like. More preferably, the pharmaceutical composition comprises 0.01-99.99% of the protein and 0.01-99.99% of a pharmaceutical carrier, wherein the percentage is the mass percentage of the pharmaceutical composition.

Preferably, the pharmaceutical composition is administered in an effective amount, which is an amount that alleviates or delays the progression of the disease, degenerative or damaging condition. The effective amount can be determined on an individual basis and will be based in part on the consideration of the condition to be treated and the result sought.

On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.

The reagents and starting materials used in the present invention are commercially available.

The positive progress effects of the invention are as follows: the polypeptide can be specifically combined with TRB3 to block the interaction between TRB3 and P62 protein, so that the polypeptide can be applied to the preparation of medicines for treating and preventing tumors. The prepared medicine has the advantages of obvious curative effect, less toxic and side effect and safe use in treating tumor diseases.

Drawings

FIG. 1 shows that the binding ability of the polypeptides A2, S1, S2, S3, S4, S5, S6 and S7 to TRB3 protein was verified by the surface plasmon resonance method. Wherein FIG. 1(A) is a kinetic profile of polypeptide A2 and TRB3 proteins; FIG. 1(B) is a graph showing the binding kinetics of the polypeptide S1 and TRB3 proteins; FIG. 1(C) is a graph showing the binding kinetics of the polypeptide S2 and TRB3 proteins; FIG. 1(D) is a graph showing the binding kinetics of the polypeptide S3 and TRB3 proteins; FIG. 1(E) is a graph showing the binding kinetics of the polypeptide S4 and TRB3 proteins; FIG. 1(F) is a graph showing the binding kinetics of the polypeptide S5 and TRB3 proteins; FIG. 1(G) is a graph showing the binding kinetics of polypeptide S6 and TRB3 protein; FIG. 1(H) is a graph showing the binding kinetics of the polypeptide S7 and TRB3 proteins. The abscissa in FIG. 1 represents the reaction time in seconds. The ordinate represents the reaction intensity of the reaction chip surface with the polypeptide, and the unit is RU.

FIG. 2 is a graph of the interaction of polypeptides A2, S1, S2, S3, S4, S5, S6 and S7 with TRB3 and p62 proteins. Wherein a shows that a2, S1, S2 and S3 interfere with the interaction of TRB3 with P62 protein; wherein B shows that S4, S5, S6 and S7 interfere with the interaction of TRB3 with P62 protein. "one" indicates the input, i.e., the amount of protein of TRB3 protein and P62 protein in the cell lysate; "two" indicates the amounts of TRB3 protein and P62 protein contained in the output, i.e., after precipitation with the P62 antibody.

FIG. 3 is a graph showing the results of polypeptides S1, S2, S3, S4, S5, S6 and S7 inhibiting the growth of hepatoma cell HepG 2. The abscissa is the time of administration in days. The ordinate is the number of cells in ten thousand. The control was polypeptide A2.

FIG. 4 is a graph showing the results of polypeptides S1, S2, S3, S4, S5, S6 and S7 inhibiting the growth of lung cancer cell A549. The abscissa is the time of administration in days. The ordinate is the number of cells in ten thousand. The control was polypeptide A2.

FIG. 5 is a graph showing the results of polypeptides S1, S2, S3, S4, S5, S6 and S7 inhibiting the growth of breast cancer cells MDA-MB-231. The abscissa is the time of administration in days. The ordinate is the number of cells in ten thousand. The control was polypeptide A2.

FIG. 6 is a graph showing the results of polypeptides S1, S2, S3, S4, S5, S6 and S7 inhibiting the growth of HCT-8 cells in intestinal cancer. The abscissa is the time of administration in days. The ordinate is the number of cells in ten thousand. The control was polypeptide A2.

FIG. 7 is a graph showing the results of polypeptides S1, S2, S3, S4, S5, S6 and S7 inhibiting the growth of leukemia cell K562. The abscissa is the time of administration in days. The ordinate is the number of cells in ten thousand. The control was polypeptide A2.

FIG. 8 is a graph showing the results of polypeptides A2, S1, S2, S3, S4, S5, S6 and S7 inhibiting the migration of hepatoma cell HepG 2. The ordinate is the ratio of the repaired area after cell scratching in percent. The control was polypeptide A2. Denotes p < 0.001.

FIG. 9 is a graph showing the results of polypeptides A2, S1, S2, S3, S4, S5, S6 and S7 inhibiting migration of lung cancer cells A549. The ordinate is the ratio of the repaired area after cell scratching in percent. The control was polypeptide A2. Denotes p < 0.001.

FIG. 10 is a graph of the results of polypeptides A2, S1, S2, S3, S4, S5, S6, and S7 inhibiting breast cancer cell MDA-MB-231 migration. The ordinate is the ratio of the repaired area after cell scratching in percent. The control was polypeptide A2. Denotes p < 0.001.

FIG. 11 is a graph showing the results of polypeptides A2, S1, S2, S3, S4, S5, S6 and S7 inhibiting migration of HCT-8 cells from intestinal cancer cells. The ordinate is the ratio of the repaired area after cell scratching in percent. The control was polypeptide A2. Denotes p < 0.001.

FIG. 12 is a graph showing the results of polypeptides A2, S1, S2, S3, S4, S5, S6 and S7 inhibiting the formation of leukemia cell K562 clone. The ordinate is the number of colony formations in units of counts. The control was polypeptide A2. Denotes p < 0.001.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

Unless otherwise specified, the PBS solution used in the examples refers to a phosphate buffer solution having a concentration of 0.1M, pH and a value of 7.2.

The room temperature in the examples is a room temperature conventional in the art, and preferably 15 to 30 ℃.

The experimental results are expressed by mean value plus or minus standard error, and the significant difference is considered to be existed by comparing that p is less than 0.05 and p is less than 0.01 through parameter or nonparametric variance test.

EXAMPLE 1 Synthesis of the polypeptide

The amino acid sequence of the polypeptide A2 is shown in a sequence table SEQ ID No. 8. The polypeptide A2 was synthesized and purified by Beijing Saibaosheng Gene technology, Inc.

Two unnatural amino acids S-pentenylalanine (S5) were introduced for solid phase polypeptide chain synthesis. And after the synthesis of the solid-phase polypeptide chain is finished, performing olefin metathesis (RCM) cyclization by using ruthenium as a catalyst to obtain the target polypeptide. Finally, the target polypeptide is cleaved from the resin and purified. The above-mentioned steps for solid phase peptide chain synthesis and purification are carried out by Zhongji peptide Biochemical Co., Ltd. Wherein, two S-pentenoic alanines are inserted into the i, i +3 position or the i, i +4 position in the amino acid sequence of the polypeptide A2, so as to obtain the modified polypeptide with different sequences (the amino acid sequence is shown in the sequence table SEQ ID No.1-SEQ ID No.7), and the specific insertion sites are as follows:

s1: S5-Gly-Trp-S5-Thr-Arg-Leu-Leu-Gln-Thr-Lys; the position i and i +3 of the insertion site, wherein i is 1;

s2: Gly-S5-Trp-Leu-Thr-S5-Leu-Leu-Gln-Thr-Lys; the position i and i +4 of the insertion site, wherein i is 2;

s3: Gly-Gly-S5-Leu-Thr-Arg-S5-Leu-Gln-Thr-Lys; the position i and i +4 of the insertion site, wherein i is 3;

s4: Gly-Gly-Trp-S5-Thr-Arg-Leu-S5-Gln-Thr-Lys; the position i and i +4 of the insertion site, wherein i is 4;

s5: Gly-Gly-Trp-Leu-S5-Arg-Leu-Leu-S5-Thr-Lys; the position i and i +4 of the insertion site, wherein i is 5;

s6: Gly-Gly-Trp-Leu-Thr-S5-Leu-Leu-Gln-S5-Lys; the position i and i +4 of the insertion site, wherein i is 6;

s7: Gly-Gly-Trp-Leu-Thr-Arg-S5-Leu-Gln-Thr-S5; the position i and i +4 of the insertion site, i-7.

Example 2 detection of the binding Capacity of the polypeptide to TRB3 protein by surface plasmon resonance

The surface plasmon resonance experiment was performed in a surface plasmon resonance instrument Biacore T200, and the procedure was performed according to the specification of the surface plasmon resonance instrument Biacore T200. The method comprises the following specific steps:

1. the purified TRB3 protein (from RD) was coupled to a CM5 chip (from GE) via an amino group, unbound protein was removed by elution at a flow rate of 10. mu.L/min, and the chip surface was equilibrated for 2 hours. The specific steps of amino coupling, elution and equilibration are described in the relevant specification of the chip CM5, GE.

2. 250 μ L of the S1-S7 and A2 polypeptide fragments prepared in example 1 at different concentrations (800, 400, 200, 50, 12.5, 6.25 and 3.125nM) were injected automatically and the whole surface plasmon resonance experiment was performed at 25 ℃. The buffer used was HBS-EP buffer [0.01M HEPES, 0.15M NaCl, 3mM EDTA and 0.005% (w/w) surfactant ]. Binding curves of different concentrations of the polypeptide to TRB3 were simulated using Biacore T200 self-contained analysis software, and the results are shown in FIGS. 1(A) - (H) and Table 1. Fig. 1(a) - (H) and table 1 show that the affinity of peptides S1, S2, S3, S4, S5, S6 and S7 to TRB3 protein is significantly higher than the affinity of polypeptide a2 to TRB3 protein.

TABLE 1 affinity test of the polypeptides S1-S7 and A2 with TRB3 protein

Example 3 circular dichroism method for detecting alpha helix rate of polypeptide

The alpha helix rate of the polypeptide was measured by circular dichroism spectroscopy (purchased from Jasco, Japan). The polypeptides A2, S1, S2, S3, S4, S5, S6 and S7 prepared in example 1 were dissolved in PBS solution, and the on-machine concentration of the circular dichroism spectroscopy instrument was adjusted to 1mg/mL, and the results are shown in Table 2. Table 2 shows that the α -helix rates of the polypeptides S1, S2, S3, S4, S5, S6 and S7 are significantly higher than that of the polypeptide a2, and since the α -helix secondary structure of the polypeptide mediates the binding of the polypeptide to TRB3 protein, the improvement of the α -helix rate of the polypeptide S1-S7 is related to the increase of the binding ability thereof to TRB3 protein, and the proliferation and metastasis of various tumor cells. Wherein, the alpha helix rate refers to the percentage of the number of peptide fragments which maintain the alpha helix of the secondary structure to the total number of peptide fragments.

TABLE 2 circular dichroism method for determining alpha helix rate of polypeptide

Polypeptide name

A2

S1

S2

S3

S4

S5

S6

S7

α spiral rate

0.87％

42.1％

39.6％

50.2％

47.8％

46.0％

41.6％

45.0％

Example 4 method of co-immunoprecipitation to verify that the polypeptide inhibits binding of protein p62 to TRB3 at the cellular level

Wherein, the related reagents of the co-immunoprecipitation are as follows:

lysate a: 0.6057g Tris base, 1.7532g NaCl, 0.1017g MgCl₂·6H₂O, 0.0742g EDTA, 10mL glycerol and 10mL 10% (v/v) NP40, adding deionized water to 150mL, adjusting pH to 7.6 with HCl, diluting to 191mL, mixing well, filtering with 0.45 μm filter membrane, and storing at 4 ℃.

Lysate B: 200. mu.L of 2 M.beta. -phosphoglycerol, 4mL of 2.5M NaF, 2mL of 100mM PMSF, 200. mu.L of 1M DTT, and 200. mu.L each of Leu, Pep, and Apr at 1mg/mL in a total volume of 9mL, and stored at-20 ℃. Wherein 2M beta-glycerophosphate, 2.5M NaF, 100mM PMSF, 1M DTT and 1mg/mL Leu, Pep and Apr are stored in mother liquor, except that the mother liquor of PMSF is prepared by using methanol as solvent, the rest is water as solvent. When the lysate B is used, the lysate B is unfrozen, and the lysate B is added into the lysate A according to the volume ratio of 1:100 of the lysate B to the lysate A and uniformly mixed.

Co-immunoprecipitation lotion: 1% (v/v) NP40, 150mM NaCl, 20mM Hepes, pH 7.510% (v/v) glycerol and 1mM EDTA.

Protein A/G Plus-Agarose is available from Santacruz, USA.

The specific operation steps are as follows:

1. liver cancer HepG2 cells (purchased from the institute of basic medicine of Chinese academy of medical sciences) were plated 90mm²After the cells are attached to the culture dish, 1mg/mL of the polypeptide A1, S1, S2, S3, S4, S5, S6 or S7 prepared in example 1 is added respectively, and the cells are collected after being incubated for 12 hours in an incubator at 37 ℃.

2. The lysate A and the lysate B were prepared into 10mL of lysate at a volume ratio of 100:1, 550. mu.L of lysate was added to lyse the cells collected in step 1, and total proteins in the cells were harvested and each histone was adjusted to the same concentration. Each group of proteins was used as a control, with 200. mu.g each.

3. 200. mu.g of each of the histones from step 2 was taken, and 2. mu. G P62 antibody (from Sigma) was added, while 10. mu.L of Protein A/G Plus-Agarose (from Santa Cruze) was added and resuspended thoroughly, and slowly shaken with rotation at 4 ℃ overnight. Centrifuge at 3000rpm for 5min at 4 ℃ and carefully aspirate the supernatant. 0.5mL of co-immunoprecipitation washing solution was added, mixed well, left to stand in an ice bath for 1min, centrifuged at 3000rpm for 30 seconds at 4 ℃ and the supernatant carefully aspirated. Washing was repeated 5 times, and left for 5min before the final centrifugation. Carefully remove the supernatant, add 30. mu.L of 2 XSDS gel loading buffer, mix well, denature for 3min at 95 ℃ and quickly transfer to an ice bath to cool. Centrifuging at 12000rpm for 2min at room temperature to obtain supernatant as precipitated protein sample, and performing SDS-polyacrylamide gel electrophoresis on part or all of the protein sample. The results are shown in FIG. 2. The results in FIG. 2 demonstrate that the interaction capacity of the TRB3/p62 protein for the polypeptides S1, S2, S3, S4, S5, S6 and S7 is significantly higher than that of the polypeptide A2.

Example 5 cell count experiments demonstrated that polypeptides S1, S2, S3, S4, S5, S6, and S7 can inhibit the growth of tumor cells

The specific operation steps are as follows:

1. liver cancer cells HepG2 (purchased from the institute of basic medicine of Chinese medical science institute), lung cancer cells A549 (purchased from the institute of basic medicine of Chinese medical science institute), colon cancer cells HCT-8 (purchased from the institute of basic medicine of Chinese medical science institute), breast cancer cells MDA-MB-231 (purchased from the institute of basic medicine of Chinese medical science institute) and leukemia cells K562 (purchased from the institute of basic medicine of Chinese medical science institute) in logarithmic growth phase were collected, and the cell concentration was adjusted to prepare a cell suspension of 15 ten thousand/mL.

2. 1mL of the cell suspension prepared in step 1 was added to a 12-well plate and cultured (wherein a culture medium for HepG2, A549, HCT-8 and MDA-MB-231 cells was DMEM medium and a culture medium for K562 cells was 1640 medium, all purchased from Invitrogen; culture temperature was 37 ℃ and volume of the culture medium was 1mL), and after 12 hours, a new culture medium was replaced, and 1. mu.g/mL of the polypeptides S1, S2, S3, S4, S5, S6 and S7 prepared in example 1 was added. Passages were performed every other day and counted. Growth curves were plotted after 12 days of culture. The results of the experiment are shown in FIGS. 3-7. FIGS. 3-7 illustrate that the polypeptides S1, S2, S3, S4, S5, S6, and S7 are more capable of inhibiting the growth of tumor cells than A2. Wherein, the inhibition level of the polypeptide S1 on the growth of lung cancer cells is up to 4 times, the inhibition level of the polypeptide S2 on the growth of liver cancer and colon cancer cells is up to 3 times, the inhibition level of the polypeptide S3 on the growth of lung cancer cells is up to 3 times, the inhibition level of the polypeptide S4 on the growth of breast cancer cells is up to 3 times, the inhibition level of the polypeptide S5 on the growth of lung cancer cells is up to 3 times, the inhibition level of the polypeptide S6 on the growth of leukemia cells is up to 3 times, and the inhibition level of the polypeptide S7 on the growth of breast cancer cells is up to 3 times.

Example 6 cell scarification experiment demonstrated that polypeptides a1, S1, S2, S3, S4, S5, S6, and S7 inhibit healing after tumor cell scarification

The specific operation steps are as follows:

1. firstly, a marking pen is used at the back of the 6-hole plate, a straight ruler is used for drawing a transverse line, and the transverse line penetrates through the through hole.

2.5 × 10 was added to each well separately⁵The tumor cells are attached after being cultured in a DMEM medium at 37 ℃ in an incubator overnight. The tumor cells are in logarithmic growth phaseThe liver cancer cell HepG2, the lung cancer cell A549, the colon cancer cell HCT-8 and the breast cancer cell MDA-MB-231.

3. The tip is used for scratching the ruler on the next day, and is perpendicular to the transverse line at the back as much as possible.

4. The cells were washed 3 times with PBS, the scraped cells were removed, and new medium was added while adding 1. mu.g/mL of the polypeptides A1, S1, S2, S3, S4, S5, S6 and S7 prepared in example 1.

5. Then put into 5% (v/v) CO at 37 DEG C₂The incubator was used for cultivation, and samples were taken after 24 hours and photographed. The results of the experiments are shown in FIGS. 8-11 and tables 3-6, where the damage repair area ratio refers to the ratio of the area of the damage after repair to the area of the damage before repair, in percent. The results in tables 3-6 show that the larger the lesion repair area ratio, the stronger the migration ability of tumor cells and the stronger the healing ability after cell scratching. Thus the polypeptides S1, S2, S3, S4, S5, S6 and S7 can reduce the healing capacity after tumor cell scoring.

TABLE 3 Polypeptides S1-S7 and A2 inhibit the migration of hepatoma cells HepG2

Polypeptide name	Area ratio of damage repair
		A2 (control)	79.4±1.05
S1	22.6±0.33
		S2	18.7±0.51
S3	19.8±0.69
		S4	12.1±0.47
S5	13.3±0.31
		S6	29.1±0.41
S7	20.0±0.35

TABLE 4 Polypeptides S1-S7 and A2 inhibit the migration of Lung cancer cells A549

TABLE 5 Polypeptides S1-S7 and A2 inhibit the migration of colon cancer cells HCT-8

Polypeptide name	Area ratio of damage repair
		A2 (control)	67.7±2.33
S1	24.5±1.97
		S2	18.2±2.30
S3	12.7±2.37
		S4	12.6±2.29
S5	12.5±2.60
		S6	12.1±1.92
S7	11.2±1.01

TABLE 6 Polypeptides S1-S7 and A2 inhibit the migration of breast cancer cells MDA-MB-231

Polypeptide name	Area ratio of damage repair
		A2 (control)	91.3±3.22
S1	36.7±2.91
		S2	20.3±2.11
S3	10.5±1.92
		S4	21.7±3.35
S5	20.4±4.47
		S6	10.4±1.96
S7	17.9±1.05

Example 7 cloning experiments demonstrated that polypeptides A1, S1, S2, S3, S4, S5, S6, and S7 inhibit clonogenic leukemia cells

The operation steps are as follows:

1. laying agar at the lower layer: 5% (w/w) agar was boiled in a water bath to completely melt, cooled to 50 ℃, and 9 times the volume of a 37 ℃ pre-warmed 1640 culture solution (purchased from Invitrogen corporation) was added thereto, mixed well, added to a 24-well plate (0.8 mL per well), and solidified at room temperature for use.

2. Laying upper agar: to 9.4mL of the cell suspension was added 0.6mL of 5% (w/w) agar at 50 ℃ and mixed well, and then added 0.8mL of the agar-plated 24-well plate. Solidifying at room temperature. The number of cells per well was 100. The preparation method of the cell suspension comprises the following steps: leukemia cells K562 were diluted with 1640 medium and adjusted to a concentration of 132 cells/mL.

3. The cells obtained in step 2 were incubated in 1640 medium at 37 ℃ for 3 weeks in an incubator, and the number of formed colonies was counted.

The results are shown in fig. 12 and table 7. The results in table 7 demonstrate that the polypeptide S1-S7 significantly increased the level of inhibition of leukemia cell clonogenic relative to polypeptide a 2.

TABLE 7 Polypeptides S1-S7 and A2 inhibit clonogenic leukemia cells

Polypeptide name	Clone number of formation
		A2 (control)	110±2.94
S1	53±5.7
		S2	47±2.3
S3	27±5.5
		S4	30±5.4
S5	34±6.1
		S6	29±5.3
S7	19±2.1

Example 8 subcutaneous tumor growth experiments demonstrated that polypeptides A2, S1, S2, S3, S4, S5, S6, and S7 inhibit tumor cell growth in mice

The operation steps are as follows:

1. experiment consumables and reagents: sterilized EP tube 1.5mL, 15mL centrifuge tube, tip, filter screen (100 mesh), absorbent cotton ball, forceps holder, alcohol cotton ball, sterile 1mL syringe, 500mL beaker (sterilized, irradiated with UV), PBS (filtered), pancreatin, serum.

2. Experimental animals and groups: 80 male nude mice (purchased from Beijing Wittingle laboratory animals Co., Ltd.) at 4-6 weeks were randomly divided into 8 groups: groups A2, S1, S2, S3, S4, S5, S6 and S7, 10 each.

3. Cell preparation, which comprises subjecting tumor cells cultured by adherence to pancreatin digestion, sucking pancreatin after reaching pancreatin digestion time (at the time, the cell state should be single cell and the adherence can not be removed), terminating with PBS containing 1% serum according to 2 mL/dish, blowing down the cells, transferring into 15mL centrifuge tube, centrifuging 1200 for 5min, discarding supernatant, resuspending PBS, sieving once with 100 mesh sieve, counting cells, adjusting cell final concentration to 2.5 × 10⁷The tumor cells are liver cancer cells HepG2, lung cancer cells A549, colon cancer cells HCT-8 and breast cancer cells MDA-MB-231 in logarithmic growth phase, suspended leukemia cells K562 are directly collected into a 15mL centrifuge tube, the centrifuge is carried out for 5min at 1200 turns, supernatant is discarded, PBS is resuspended, the suspension is filtered by a 100-mesh filter screen once, the cells are counted, and the final concentration of the cells is adjusted to be 2.5 × 10⁷/mL。

4. Tumor cell inoculation, inoculation 5 × 10⁶One tumor cell (cell suspension 200. mu.l) was subcutaneously placed in the left upper abdomen and near the underarm of nude mice.

5. And (3) observing the growth of the tumor: tumor cells were treated with the polypeptide one week after subcutaneous injection (5mg/kg body weight twice weekly) and tumor size was recorded with a vernier caliper.

The results are shown in tables 8-12, in which the p-values of the polypeptides S1-S7 are less than 0.001 compared with the control A2. The larger tumor volume indicates faster tumor growth, and thus the polypeptides S1, S2, S3, S4, S5, S6 and S7 can inhibit tumor cell growth in mice.

TABLE 8 Polypeptides S1-S7 and A2 inhibit growth of hepatoma cell HepG2 in mice

TABLE 9 Polypeptides S1-S7 and A2 inhibit growth of colon cancer cell HCT-8 in mice

Polypeptide name	Tumor volume (mm)³)
		A2 (control)	1754±107.9
S1	583.4±68.2
		S2	501.8±81.9
S3	488.8±62.4
		S4	496.5±81.7
S5	606.4±74.3
		S6	511.1±54.6
S7	619.8±75.5

TABLE 10 Polypeptides S1-S7 and A2 inhibit the growth of breast cancer cells MDA-MB-231 in mice

Polypeptide name	Tumor volume (mm)³)
		A2 (control)	2379.4±165.4
S1	666.1±74.3
		S2	718.7±67.5
S3	570.8±69.4
		S4	730.1±84.7
S5	737.3±83.1
		S6	629.5±54.1
S7	598.8±75.9

TABLE 11 Polypeptides S1-S7 and A2 inhibit the growth of Lung cancer cell A549 in mice

TABLE 12 Polypeptides S1-S7 and A2 inhibit the growth of leukemia cell K562 in mice

Polypeptide name	Tumor volume (mm3)
		A2 (control)	1996.4±1.05
S1	572.6±63.9
		S2	638.9±75.1
S3	699.8±69.0
		S4	722.1±94.7
S5	703.3±73.6
		S6	693.1±54.8
S7	589.9±75.2

The results of the above examples show that the polypeptide of the present invention has significant anti-tumor effect, and can be used as an active ingredient for preparing anti-tumor drugs.

It should be understood that various changes and modifications can be made by those skilled in the art after reading the above disclosure, and equivalents also fall within the scope of the invention as defined by the appended claims.

Claims

1. A polypeptide specifically binding to TRB3 is characterized in that the amino acid sequence of the polypeptide is shown in any one of sequence tables SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6 and SEQ ID No. 7.

2. Use of the polypeptide that specifically binds TRB3 according to claim 1 in the preparation of a medicament for the treatment of a tumor.

3. The use according to claim 2, wherein the tumor is liver cancer, lung cancer, breast cancer, intestinal cancer or leukemia.

4. The use of claim 3, wherein the liver cancer is primary or secondary liver cancer; the lung cancer is small cell lung cancer or non-small cell lung cancer; the breast cancer is non-invasive breast cancer, early invasive breast cancer, invasive special type breast cancer or invasive non-special type breast cancer; the intestinal cancer is colon cancer or rectal cancer; the leukemia is lymphocytic leukemia or non-lymphocytic leukemia.

5. An anti-tumor pharmaceutical composition comprising the polypeptide of claim 1 that specifically binds TRB 3.

6. The pharmaceutical composition of claim 5, further comprising one or more pharmaceutically acceptable carriers.

7. The pharmaceutical composition according to claim 5, which comprises the polypeptide that specifically binds to TRB3 according to claim 1 as an active ingredient alone; alternatively, it contains the polypeptide specifically binding to TRB3 according to claim 1 together with other compounds having antitumor activity as active ingredients.