An anti-tumor dual-target polypeptide and its preparation method and application

Abstract

The present invention discloses an anti-tumor dual-target polypeptide and a preparation method and application thereof, wherein the dual-target polypeptide targets integrin and VEGR targets respectively and satisfies one or more of the following conditions: (1) it is a 17-26 peptide; (2) it includes an amino acid sequence as shown in SEQ ID NO: 12; (3) it includes an amino acid sequence as shown in SEQ ID NO: 13. The dual-target polypeptide provided by the present invention has good anti-tumor activity, can significantly inhibit HUVEC cell proliferation, reduce HUVEC cell tube nodes, significantly reduce the number of tubes, has no significant effect on the tube length, and can also reduce HUVEC cell adhesion; at the same time, it can significantly reduce the tube area and significantly inhibit liver cancer tumor growth.

Description

[0001] This application claims the priority of Chinese patent application No. 2022117366310 filed on December 30, 2022. This application cites the entire text of the above Chinese patent application. Technical Field

[0002] The present invention belongs to the field of biomedicine, and specifically relates to an anti-tumor dual-target polypeptide and a preparation method and application thereof. Background Art

[0003] The growth process of tumors can be divided into two stages: the prevascular stage and the vascular stage. In the prevascular stage, due to lack of blood supply, the tumor is dormant, with a diameter of 1-2mm and no disease. In the vascular stage, due to sufficient blood supply, the tumor volume increases rapidly, infiltration and metastasis occur, and angiogenesis is induced. Tumor angiogenesis refers to the process of microvascular growth induced by tumor cells and the establishment of blood circulation in the tumor. The process of tumor angiogenesis is regulated by angiogenesis-promoting factors and inhibitors. Angiogenesis inhibitors indirectly inhibit the growth of tumor cells by inhibiting the formation of blood vessels in tumor tissues and cutting off blood supply. The use of angiogenesis inhibitors to treat tumors has the advantages of a broad anti-tumor spectrum, no toxic side effects, and no drug resistance. Angiogenesis inhibitors cannot directly kill tumor cells, and the tumor will recur after discontinuation of the drug. Therefore, angiogenesis inhibitors are mainly used as adjuvant drugs for chemotherapy drugs, used in conjunction with chemotherapy drugs to reduce the dosage of chemotherapy drugs and reduce the toxicity of chemotherapy drugs.

[0004] Various major clinical diseases, including malignant tumors, cardiovascular and cerebrovascular diseases, etc., are usually the result of multiple factors. For example, the occurrence and development of tumors involve multiple signal molecules, multiple pathological links and multiple gene effects. Therefore, it is destined that a single mechanism and a single target are difficult to treat tumors. This is also the main reason for the drug resistance and failure of drugs that work in a single pathway. Multi-target drugs can act on multiple pathological links and multiple pathogenesis of the same disease at the same time to produce synergistic effects, thereby improving the efficacy of drugs. At present, clinical treatment drugs have also used a large number of drug combination schemes with different targets and different mechanisms, and have achieved good results, which are significantly better than monotherapy and have become the main scheme for clinical treatment of tumors. For example, in the treatment schemes for the top five tumors with the highest mortality in my country, the first-line treatments for gastric cancer and esophageal cancer are all combined drugs. Although combined drugs show good advantages in efficacy, the strategy of combined drugs often has defects such as complex dose setting, uncontrollable pharmacokinetic properties, and drug-drug interactions. In contrast, single-molecule multi-target drugs have the advantages of better efficacy, more reasonable pharmacokinetic properties, and low toxicity.

[0005] Malignant tumors are a type of disease that seriously threatens human health and life. Finding new, highly effective, and low-toxic anti-tumor drugs is a hot topic in medical research and development at home and abroad. Small molecule peptides have the characteristics of small molecular weight, low toxicity, high activity, and easy penetration of tumor cells. They can be administered in a variety of ways, are easily absorbed in multiple ways, and are not easy to develop drug resistance. Protein macromolecule drugs have large molecular weights, complex structures, are not easy to penetrate cells, and are immunogenic. Small molecule peptide structures are easy to modify and have low artificial synthesis costs. Due to their small relative molecular mass (Mr), high activity, and low toxicity, they have important value in the clinical treatment of tumors. Summary of the invention

[0006] In order to overcome the technical problem that the prior art lacks small molecule dual-target polypeptide drugs with anti-tumor activity, the present invention provides an anti-tumor dual-target polypeptide and its preparation method and application. The anti-tumor dual-target polypeptide has good tumor inhibition activity, for example, it can inhibit the proliferation of human umbilical vein endothelial cells (HUVEC cells), tumor cell migration and HUVEC cell adhesion. It has good clinical application potential. The present invention discloses a dual-target polypeptide, which has the activity of inhibiting angiogenesis and inhibiting tumor cell proliferation, and is safe and effective in treating tumors, and can be used as an anti-tumor drug.

[0007] In order to solve the above technical problems, the first aspect of the present invention provides a polypeptide, wherein the polypeptide targets two targets, integrin and VEGR, respectively, and satisfies one or more of the following conditions:

[0008] (1) is a 17-26 peptide;

[0009] (2) comprising the amino acid sequence as shown in SEQ ID NO: 12; wherein X in the amino acid sequence is D or N;

[0010] (3) comprising the amino acid sequence as shown in SEQ ID NO: 13; wherein X in the amino acid sequence is F or G.

[0011] In some preferred embodiments, the amino acid sequence shown in SEQ ID NO: 12 is located at the C-terminus of the polypeptide, and the amino acid sequence shown in SEQ ID NO: 13 is located at the N-terminus of the polypeptide.

[0012] Preferably,

[0013] The polypeptide further comprises an amino acid sequence as shown in SEQ ID NO: 14 at the N-terminus of SEQ ID NO: 12, or,

[0014] The polypeptide further comprises an amino acid sequence as shown in SEQ ID NO: 15 at the N-terminus of SEQ ID NO: 12; or

[0015] The polypeptide comprises two amino acid sequences as shown in SEQ ID NO: 13, preferably two amino acid sequences as shown in SEQ ID NO: 13 are sequentially connected. In certain embodiments, the amino acid sequence of the polypeptide is shown in any one of SEQ ID NO: 1-11.

[0016] The second aspect of the present invention provides an isolated nucleic acid, wherein the nucleic acid encodes the polypeptide as described in the first aspect of the present invention.

[0017] The third aspect of the present invention provides a recombinant expression vector, which comprises the nucleic acid described in the second aspect of the present invention.

[0018] The fourth aspect of the present invention provides a transformant, wherein the transformant comprises the nucleic acid as described in the second aspect of the present invention or the recombinant expression vector as described in the third aspect of the present invention.

[0019] The fifth aspect of the present invention provides a polypeptide vaccine, which comprises one or more polypeptides as described in any one of the first aspect of the present invention.

[0020] The sixth aspect of the present invention provides a nucleic acid vaccine comprising one or more nucleic acids as described in the second aspect of the present invention.

[0021] The seventh aspect of the present invention provides a method for preparing a polypeptide, comprising culturing the transformant under conditions suitable for the growth of the transformant as described in the fourth aspect of the present invention and fermenting it to produce the polypeptide as described in the first aspect of the present invention.

[0022] The eighth aspect of the present invention provides the use of the polypeptide as described in the first aspect of the present invention, the nucleic acid as described in the second aspect of the present invention, or the transformant as described in the fourth aspect of the present invention in the preparation of a drug for inhibiting angiogenesis activity and / or inhibiting tumor cell proliferation activity. Preferably, the cell is a human umbilical vein endothelial cell.

[0023] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0024] The reagents and raw materials used in the present invention are commercially available.

[0025] The positive and progressive effects of the present invention are:

[0026] The anti-tumor activity of the polypeptide provided by the present invention was verified by in vitro experiments, including HUVEC cell proliferation inhibition test, HUVEC cell tube formation ability influence test, HUVEC cell adhesion test, and in vitro tumor cell migration Transwell test.

[0027] The results of the HUVEC cell proliferation inhibition test showed that polypeptides 1-11 can significantly inhibit HUVEC cell proliferation. The results of the HUVEC cell tube formation ability test showed that polypeptides 1-11 can reduce tube formation nodes, significantly reduce the number of tubes, have no significant effect on the tube length, and can significantly reduce the tube area. The polypeptides provided by the present invention can reduce HUVEC cell adhesion. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The inhibitory ability of the polypeptide on VEGF-induced HUVEC proliferation.

[0029] Figure 2 Representative images of lumen formation in some polypeptide HUVECs, phase contrast microscopy (10 times).

[0030] Figure 3 Representative images of lumen formation in some polypeptide HUVECs, phase contrast microscopy (10 times).

[0031] Figure 4 The effect of peptides on the tube-forming ability of HUVEC cells, Figure 4 Part A is for quantification of branch length of HUVECs; Figure 4 Part B is the cell branch.

[0032] Figure 5 The effect of peptides on HUVEC adhesion.

[0033] Figure 6 The figure shows the effect of different concentrations of peptide 4 on HUVEC adhesion.

[0034] Figure 7 This is the effect of the peptide on HCT-116 cell adhesion.

[0035] Figure 8 Representative images of the inhibitory effect of the peptide on the migration ability of colon cancer cells HCT-116, phase contrast microscopy.

[0036] Fig. 9 It is the average migration distance of HCT-116 cells under the action of different peptides relative to the negative control group. DETAILED DESCRIPTION

[0037] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0038] The reagent materials used in the examples are shown in Table 1:

[0039] Table 1 Reagents and Materials

[0040]

[0041] The instruments and equipment used in the examples are shown in Table 2:

[0042] Table 2 Instruments and equipment

[0043]

[0044]

[0045] Table 3 Specific sequences of polypeptides

[0046] SEQ ID NO: Peptide name Peptide sequence Peptide Type 1 Peptide 1 FRGDSFQPVLHLVALNS 17 peptides 2 Peptide 2 FRGDSGGGGFQPVLHLVALNS 21 peptides 3 Peptide 3 FRGDSPAPAPFQPVLHLVALNS 22 peptides 4 Peptide 4 GRGDSFQPVLHLVALNS 17 peptides 5 Peptide 5 GRGDSPKFQPVLHLVALNS 19 peptides 6 Peptide 6 GRGDSGRGDSFQPVLHLVALNS 22 peptides 7 Peptide 7 GRGDSPKGRGDSPKFQPVLHLVALNS 26 peptides 8 Peptide 8 GRGDSFQPPVLHLVALDS 18 peptides 9 Peptide 9 GRGDSWQPPGLHLVALNS 18 peptides 10 Peptide 10 GRGDSPKFQPPVLHLVALDS 20 peptides 11 Peptide 11 GRGDSPKWQPPGLHLVALNS 20 peptides 12 Peptide fragment 1 <![CDATA[LHLVALX1S, where X1 is D or N]]> - 13 Peptide fragment 2 <![CDATA[X2RGDS, where X2 is F or G]]> - 14 Peptide fragment 3 <![CDATA[FQPX3V, where X3 is P or does not exist]]> - 15 Peptide fragment 4 WQP -

[0047] Among them, polypeptides 1-11 correspond to TB1041 (abbreviated as P41), TB1042 (abbreviated as P42), TB1043 (abbreviated as P43), TB1050 (abbreviated as P50), TB1051 (abbreviated as P51), TB1052 (abbreviated as P52), TB1053 (abbreviated as P53), TB1054 (abbreviated as P54), TB1055 (abbreviated as P55), TB1056 (abbreviated as P56) and TB1057 (abbreviated as P57), respectively.

[0048] Example 1 Screening test of the inhibitory effect of polypeptides on HUVEC cell proliferation

[0049] Test materials

[0050] 1.1 Test substances

[0051] Table 4 Information of test substance and reference substance

[0052]

[0053]

[0054] TB01(SEQ ID NO:16):RGDRGDMHSHRDFQPVLHLVALNSPLSGGM

[0055] 1.2 Preparation of test substance / reference substance

[0056] 1.2.1 Test article preparation

[0057] Preparation method: Accurately weigh 8 mg of the test substance, add 1 mL of injection water to fully dissolve, prepare a working solution with a concentration of 8 mg / mL, and then dilute it for use. When using, add human umbilical vein endothelial cell complete culture medium to dilute to the intended use concentration.

[0058] Storage conditions and precautions: Transport and transfer on ice, avoid light.

[0059] 1.2.2 Preparation of reference substances

[0060] 1.2.2.1TB01 Preparation

[0061] Preparation method: Add 1 mL of 5% glucose injection to 8 mg of TB01 to prepare a working solution with a concentration of 8 mg / mL, and then dilute it for use. When using, add human umbilical vein endothelial cell complete culture medium to dilute to the intended use concentration.

[0062] 1.3 Cells: HUVEC cells

[0063] Cell recovery, culture, and passaging

[0064] Take 1 tube of HUVEC cryopreserved cells and revive them gently and quickly in a 37°C water bath. When the cryopreservation solution in the tube melts until only an ice crystal with a diameter of about 2 mm remains, stop the water bath. Continue to shake the cryopreserved tube until the ice crystal melts. Centrifuge at 250×g for 4 minutes and remove the supernatant after centrifugation. Add 2mL of complete culture medium and gently blow the cell pellet to fully disperse and mix. Inoculate the cells into a T25 culture flask. Add sufficient human umbilical vein endothelial cell complete culture medium. The total amount of human umbilical vein endothelial cell complete culture medium in a T25 culture flask should not be less than 5mL. Shake the cells and place them in a CO2 incubator at 37°C, 5% CO2, and saturated humidity. Observe the cell status on the second day of recovery and replace with fresh human umbilical vein endothelial cell complete culture medium or pass it on. After that, the complete culture medium of human umbilical vein endothelial cells was replaced every 2 days until the cells grew to 90% confluence, which was then required to be passaged (usually the ratio of human umbilical vein endothelial cells for passage was 1:3, and they grew to a confluence suitable for passage within 72 hours).

[0065] 1.4 Inhibitory effect of peptide on VEGF165 proliferation

[0066] HUVECs were treated with peptide 1-11 (100 μg / ml), Avastin and Cilengitide (1 μg / ml) for 72 h, and CellTiter- Reagent was used to detect cell viability.

[0067] (1) HUVEC cells in the logarithmic growth phase were obtained, and the cells were digested with 0.25% trypsin, and the digestion was terminated with complete culture.

[0068] (2) HUVEC complete medium and HUVEC basal medium were mixed at a ratio of 1:30 and named solution A. HUVECs were washed once with solution A and the cell number and viability were counted.

[0069] (3) Adjust the cell density. Take 50 μl of the cell suspension and inoculate it into a 96-well plate with solution A. The final cell density is 5 × 103 / well. Set up 3 parallel wells in each group.

[0070] (4) Pre-mix the peptide and recombinant human VEGF165 with solution A to prepare a premix, and incubate at room temperature for 30 min; add 50 ul of the premix into a 96-well plate containing HUVEC cells (so that the final concentration is 12.5 ng / mL VEGF165 and the final concentration is 100 μg / ml of the test substance).

[0071] (5) After incubation at 37°C, 5% CO2 for 72 h, the cell fluorescence value was measured using CellTiter-Glo (Beijing Promega Biotechnology Co., Ltd., batch number: G7572). That is, 100 μL of CellTiter-Glo was added to 100 μL of the culture medium containing the cells. 2.0 Reagent; mix the contents on an orbital shaker for 2 minutes to induce cell lysis.

[0072] (6) Incubate the culture plate at room temperature for 10 min to stabilize the luminescent signal.

[0073] (7) Detect the fluorescence value on an ELISA instrument, record the data, and calculate the cell proliferation inhibition rate (IR). Proliferation inhibition rate (%) = (1-OD value of the experimental group / OD value of the control group) × 100%.

[0074] 1.5 Results

[0075] The results are as follows Figure 2 As shown, compared with the positive control group, peptides 1-11 have a significant inhibitory effect on HUVEC, among which peptides 1-4 and peptide 6 can significantly inhibit the cell proliferation promoted by VEGF.

[0076] Example 2 Experiment on the effect of polypeptide on HUVEC cell tube formation ability

[0077] The materials are the same as in Example 1.

[0078] Cells: HUVEC cryopreserved cells.

[0079] Reagents and equipment: The main reagents and equipment required for this example are shown in Tables 1 and 2.

[0080] 2.1 Cell recovery and culture

[0081] Same as Example 1

[0082] 2.2 HUVEC cell tube formation assay

[0083] Test steps:

[0084] (1) Precool the 24-well plate in a -20°C refrigerator for 30 min. Before the experiment, Matrigel must be fully thawed on ice for 30 min.

[0085] (2) Mix HUVEC complete medium and HUVEC basal medium in a ratio of 1:9, named solution B, and pre-cool in a refrigerator.

[0086] (3) Place the 24-well plate on ice, mix pre-cooled solution B and matrix gel at a ratio of 1:1, add 300 μL Matrigel to each well, and incubate in a 37°C incubator for 45 min.

[0087] (4) HUVEC cells in the logarithmic growth phase were taken, the culture medium was discarded, and the cells were washed twice with PBS; and the cells were digested with 0.25% trypsin.

[0088] (5) Centrifuge at 1000 rpm for 5 min, take 1 mL of complete culture medium for human umbilical vein endothelial cells, resuspend the cells, obtain a cell suspension, and count the cells under a microscope.

[0089] (6) Resuspend the cells to a final concentration of 1×10 5 cells / mL, 500 μl of cell suspension mixed with the test substances was added to a 24-well plate (final concentration of the test substances and control substances was 200 μg / mL, bevacizumab was 25 μg / mL).

[0090] (7) Culture the cells at 5% CO2 and 37°C for 4-6 h, take photos under a microscope, and observe the angiogenesis.

[0091] 2.3 Experimental Results

[0092] like Figure 2 , Figure 3 and Figure 4 As shown, Figure 4 Part A is for quantification of branch length of HUVECs; Figure 4 The B part is the cell branch. The above results show that polypeptides 1-3, polypeptide 8 and polypeptide 11 have a significant inhibitory effect on HUVEC tube formation, can reduce tube formation nodes, significantly reduce the number of tubes, have no significant effect on tube length, and reduce the tube area.

[0093] Example 3: Effect of polypeptide on HUVEC cell adhesion

[0094] The test materials are the same as those in Example 1.

[0095] Cells: HUVEC cryopreserved cells.

[0096] Reagents and equipment: The main reagents and equipment required for this example are shown in Tables 1 and 2.

[0097] 3.1 Cell recovery and culture

[0098] Same as Example 1

[0099] 3.2 HUVEC cell adhesion assay

[0100] Test steps:

[0101] (1) Coating of 96-well culture plates at room temperature: Incubate with 50 μl of 5 μg / ml Vitronectin at room temperature for 1 h.

[0102] (2) BSA immersion: aspirate the coating solution, wash twice with PBS, add 150 μl of sterile-filtered 1% BSA and incubate the plate at 37°C for 1 h.

[0103] (3) Washing with culture medium: Wash the plate twice with serum-free culture medium.

[0104] (4) Preparation of cell suspension: Digest the cells with 0.25% trypsin, terminate the digestion with serum-containing medium, wash the cells with serum-free medium and resuspend them, count the cells, and prepare the cell suspension with medium containing a specific concentration of the test drug.

[0105] (5) Seed plate: 100 μl of cell suspension (4×10 4 cells per well) and plated in a pre-treated 96-well plate.

[0106] (6) After incubation in a 37°C incubator for 2 h, non-adherent cells were gently washed once with PBS.

[0107] (7) Observe and take photos under a microscope.

[0108] (8) Use CCK8 to measure the cell OD value. Add 10 μl of CCK8 solution to each well and shake the plate manually several times.

[0109] (9) The culture plate was incubated in an incubator for 2 hours and the absorbance at 450 nm was measured using an ELISA reader.

[0110] (10) Calculate the inhibition rate of peptide on fibronectin adhesion. The inhibition rate of peptide on cell adhesion at different concentrations = (OD value of control group - OD value of test group) / OD value of test group × 100%.

[0111] 3.3 Experimental Results

[0112] like Figure 5 As shown, the polypeptides of the present invention can reduce HUVEC cell adhesion, and in particular, it is shown that polypeptide 4 and polypeptide 2 can significantly inhibit HUVEC adhesion to glass protein coating.

[0113] 3.4 Effects of different concentrations of peptides on HUVEC cell adhesion

[0114] Different concentrations of polypeptide 4 (10 μg / ml, 30 μg / ml and 100 μg / ml) were used to determine its effect on HUVEC cell adhesion, and the determination steps were the same as 3.2.

[0115] The results are as follows Figure 6 As shown, Figure 6 Part A represents the microscopic images of different concentrations (100×); Figure 6 Part B is the relative absorbance of adherent HUVEC cells measured by CCK8 method; Figure 6 Part C shows the adhesion curve of HUVEC cells under the action of different concentrations of P50. *p<0.05 is the comparison between P50 (30μg / ml) and the control group (CTL), ****p<0.0001 is the comparison between P50 (100μg / ml) and the control group (CTL). The results show that peptide 4 can significantly inhibit the adhesion of HUVEC, and its IC50 is 68.57μg / ml.

[0116] Example 4 Effects of different peptides on HCT-116 cell adhesion

[0117] Cells were treated with peptide (100 μg / ml) and cilengitide (2.5 ug / ml) for 5-6 hours, non-adherent cells were washed off, and microscopic photography was performed. The relative absorbance of adherent cells was determined by CCK8 method or analyzed by Incucyte photography. *p<0.05 is compared with the control group (CTL), **p<0.01 is compared with the control group (CTL).

[0118] The results are as follows Figure 7 As shown, peptide 4, peptide 7 and peptide 1 can significantly inhibit the adhesion of HCT-116.

[0119] Example 5 Effect of polypeptide on the healing of tumor cell scratches

[0120] 5.1 Experimental steps:

[0121] (1) Tumor cells in the exponential growth phase were digested with 0.25% trypsin, centrifuged at 1000 rpm (room temperature) for 5 min, and the cells were counted;

[0122] (2) Adjust the cell concentration to 1×106 cells / mL using complete culture medium.

[0123] (3) Take a 12-well plate and use a marker pen to draw horizontal lines evenly on the back of the plate with a ruler, approximately every 1 cm, across the wells. Inoculate the prepared tumor cells in a volume of 1 mL / well into the 12-well plate, incubate overnight to allow the cells to adhere to the wall, and culture until the cell confluence reaches 80-90%.

[0124] (4) Discard the supernatant, use a 20 μL micropipette tip to scratch vertically in the well plate, try to scratch perpendicular to the horizontal line on the back, wash the cells 1 to 2 times with PBS, and add 1 mL / well of (1% FBS+DMEM) culture medium solution containing corresponding drug concentrations (the concentration of polypeptide 1-11 and TB01 is 200 μg / ml, and the concentration of bevacizumab and cilengitide is 1 μg / ml) to the 12-well plate with cells, set up 3 replicates for each group, and continue to culture for 24 hours.

[0125] (5) Observe the cell healing status under the microscope at 0 and 24 h after continued culture, randomly sample up and down the cross, take three points in each well, and take pictures. Use Image J software to process the scratch healing area. The calculation formula is as follows:

[0126] (6) Healing area % = (1-Ti scratch area / T0 scratch area) × 100, Ti is the scratch area at the detection time point, T0 is the initial scratch surface.

[0127] 5.2 Test results

[0128] Compare the inhibitory effects of different peptides on the migration ability of colon cancer cells HCT116. Use different drugs. Use phase contrast microscopy to observe the representative phase images of HCT-116 cells at 0 and 24h after treatment, and calculate the average migration distance of HCT-116 cells under the action of the test drugs relative to the negative control group. The results are shown in Figure 8 and Fig. 9 As shown, it can be seen that polypeptide 1, polypeptide 3, polypeptide 8 and polypeptide 11 can significantly inhibit the migration of HCT116.

Claims

1. A polypeptide, characterized in that The amino acid sequence of the polypeptide is shown in any one of SEQ ID NOs: 1-4, 6-8, and 11.

2. An isolated nucleic acid, characterized in that The nucleic acid encodes the polypeptide according to claim 1.

3. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the nucleic acid as claimed in claim 2.

4. A transformant, characterized in that The transformant comprises the nucleic acid according to claim 2 or the recombinant expression vector according to claim 3.

5. A method for preparing a polypeptide, characterized in that: The transformant according to claim 4 is cultured under conditions suitable for the growth of the transformant and fermented to produce the polypeptide according to claim 1.

6. Use of the polypeptide according to claim 1, the nucleic acid according to claim 2, or the transformant according to claim 4 in the preparation of a drug for inhibiting tumor cell proliferation activity; wherein, The tumor is colon cancer.

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