Polypeptide and application thereof in preparation of antitumor drugs

Abstract

The invention relates to a polypeptide and application thereof in preparation of antitumor drugs, and belongs to the technical field of antitumor drugs. The amino acid sequence of the polypeptide is shown as SEQ ID NO: 1. The compound can selectively inhibit CRC cells (including CT26, MC38 and HCT116), is non-toxic to other cells (including human intestinal epithelial cells CCD841 and HIEC6), and has relatively high application safety. In addition, the possibility of drug resistance of tumor cells to the polypeptide is reduced through the membrane rupture oncolytic mechanism of the polypeptide. The compound can be used for inhibiting CRC tumor cells so as to realize control and treatment of CRC.

Description

Technical Field

[0001] The present invention relates to the technical field of anti-tumor drugs, and particularly to a polypeptide and its application in the preparation of anti-tumor drugs. Background Art

[0002] Colorectal cancer (CRC) is the third most common human malignancy globally, with a high mortality rate. More than 900,000 cases die from CRC every year, and at the same time, more than 1.8 million people are diagnosed. The treatment of colorectal cancer includes biological immunotherapies such as surgery, chemotherapy, and radiotherapy. Surgical treatment is the first choice for treating colorectal cancer. Due to the high invasiveness and metastasis of CRC, the 5-year survival rate is only about 50%. 5-Fluorouracil (5-FU) is the first choice for the treatment of CRC. However, the systemic toxicity and the development of drug resistance of 5-FU severely limit its efficacy. Similarly, although preoperative radiotherapy combined with surgery is the recommended treatment mode for locally advanced colorectal cancer. However, the problem of low preoperative radiotherapy sensitivity in colorectal cancer patients also limits the treatment success rate.

[0003] Therefore, there is an urgent need for new anti-tumor drugs that can inhibit tumor growth, are not prone to drug resistance, and have low toxicity, so as to achieve the treatment of colorectal tumors. Summary of the Invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a polypeptide that can effectively inhibit tumor growth and its application in the preparation of anti-tumor drugs.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0006] In the first aspect, the present invention provides a polypeptide, which is the polypeptide with the amino acid sequence shown in SEQ ID NO: 1 and / or its derivatives.

[0007] The polypeptide of the present invention can effectively inhibit the growth of colorectal cancer tumor cells, reduce their survival rate, and is non-toxic to normal intestinal cells.

[0008] Furthermore, the derivative of the polypeptide is the phosphorylation modification of the N-terminal amino acid of the polypeptide. It is beneficial to improve the intestinal treatment effect.

[0009] In a specific embodiment of the present invention, the chemical structural formula of the derivative of the polypeptide is shown in Formula I:

[0010]

[0011] In the second aspect, the present invention also provides a preparation method of the polypeptide, including the following steps:

[0012] S1: According to the amino acid sequence order of the polypeptide, amino acids are connected to Wang resin through a peptide synthesizer to obtain peptide resin;

[0013] S2: The peptide resin in step S1 is cleaved by trifluoroacetic acid to obtain a crude polypeptide;

[0014] S3: The crude polypeptide in step S2 is purified to obtain the polypeptide.

[0015] Furthermore, in step S3, the crude polypeptide in step S2 is purified by reverse-phase high performance liquid chromatography.

[0016] Furthermore, in step S1, according to the amino acid sequence order of the polypeptide, amino acids are connected to Wang resin one by one from the C-terminus to the N-terminus.

[0017] Furthermore, the preparation method of step S1 includes the following steps:

[0018] S11: The first amino acid at the C-terminus of the polypeptide is protected by a 9-fluorenylmethoxycarbonyl group to obtain Fmoc-X. After being connected to Wang resin, the 9-fluorenylmethoxycarbonyl group is removed to obtain X-Wang resin;

[0019] S12: The second amino acid at the C-terminus of the polypeptide is protected by a 9-fluorenylmethoxycarbonyl group and a trityl group to obtain Fmoc-Y-Trt-OH, which is combined with X-Wang resin to form a peptide bond to obtain a dipeptide with protecting groups;

[0020] S13: According to steps S11 and S12, the types of corresponding amino acids are replaced according to the amino acid order of the next polypeptide sequence, and the synthesis is carried out from the C-terminus to the N-terminus of the polypeptide in turn until the preparation is completed to obtain peptide resin.

[0021] In a specific embodiment of the present invention, when preparing the derivative of the polypeptide and combining the amino acid to be phosphorylated with X-Wang resin, the used Fmoc-Y-Trt-OH is N-fluorenylmethoxycarbonyl-O-benzyl-L-phosphotyrosine (Fmoc-Tyr(HPO 3 OBzl)-OH).

[0022] Furthermore, in step S2, the peptide resin in step S1 is cleaved with a cleavage reagent composed of a mixture of trifluoroacetic acid, water and triisopropylchlorosilane.

[0023] In a specific embodiment of the present invention, the mass ratio of trifluoroacetic acid, water and triisopropylchlorosilane is 85-95:5-10:1-5, preferably 95:2.5:2.5.

[0024] Further, in step S2, the peptide resin is mixed with a cleavage reagent and reacted at 20-25°C in the dark for 1-3 h, followed by filtration to obtain a filtrate and a precipitate. The precipitate is washed with trifluoroacetic acid to obtain a wash solution. The filtrate and the wash solution are mixed, concentrated using a rotary evaporator, precipitated with anhydrous ether at -18 to -24°C for 1-3 h, centrifuged, the precipitate is collected, washed, and dried to obtain a crude polypeptide.

[0025] In a specific embodiment of the present invention, the reaction is carried out at 20°C in the dark for 2 h.

[0026] In a specific embodiment of the present invention, precipitation is carried out with anhydrous ether at -20°C for 3 h.

[0027] Further, the volume of anhydrous ether is 5-20 times, preferably 10 times, the volume of the liquid after concentration by the rotary evaporator.

[0028] Further, the centrifugation conditions are centrifugation at 2000-3000 g for 5-20 min.

[0029] In a specific embodiment of the present invention, the centrifugation conditions are centrifugation at 2500 g for 10 min.

[0030] Further, the crude polypeptide obtained in step S2 is dissolved in a 70-90% (v / v) aqueous acetonitrile solution, filtered, and eluted using a C18 normal pressure reversed-phase column. The eluent is a mixture of methanol and an aqueous sodium sulfate solution in a volume ratio of 30:70 to 70:30, the flow rate is 0.5-1 mL / min, the detection wavelength is 200-230 nm, the main peak is collected, freeze-dried to obtain an eluted product;

[0031] The eluted product is further purified using a reversed-phase C18 column. Eluent A is a 0.1-0.12% (v / v) aqueous trifluoroacetic acid solution; eluent B is a 0.1-0.12% (v / v) trifluoroacetic acid acetonitrile solution in water. The elution concentration is 25% (v / v) B to 40% (v / v) B, the elution time is 10-12 min, the flow rate is 0.5-1 mL / min, and the main peak is collected as above, freeze-dried to obtain the polypeptide.

[0032] In a specific embodiment of the present invention, the crude polypeptide obtained in step S2 is dissolved in a 90% (v / v) aqueous acetonitrile solution, filtered, and eluted using a C18 normal pressure reversed-phase column. The eluent is a mixture of methanol and an aqueous sodium sulfate solution in a volume ratio of 30:70 to 70:30, the flow rate is 1 mL / min, the detection wavelength is 220 nm, the main peak is collected, freeze-dried to obtain an eluted product;

[0033] The elution product was further purified using a reversed-phase C18 column. Eluent A was an aqueous solution of 0.1% (v / v) trifluoroacetic acid; eluent B was an aqueous acetonitrile solution of 0.1% (v / v) trifluoroacetic acid. The elution concentration was 25% (v / v) B to 40% (v / v) B, the elution time was 12 min, the flow rate was 1 mL / min. The main peak was collected as above and freeze-dried to obtain the polypeptide.

[0034] In a third aspect, the present invention also claims the use of the polypeptide in the preparation of an anti-tumor drug.

[0035] Further, the anti-tumor drug includes a drug for inhibiting tumor growth.

[0036] In a specific embodiment of the present invention, the anti-tumor drug includes an anti-colorectal cancer drug.

[0037] The polypeptide of the present invention has oncolytic activity, reduces the survival rate of CT26, MC38, and HCT116 colon cancer cells, and its oncolytic activity is further enhanced after dephosphorylation by alkaline phosphatase (ALP). Alkaline phosphatase has a higher concentration in tumor cells and the intestinal environment, and has further selectivity for the application environment. And it shows no cytotoxic effect on human intestinal epithelial cells (CCD841 and HIEC6).

[0038] In a fourth aspect, the present invention provides an anti-colorectal cancer drug containing the polypeptide.

[0039] Further, the anti-colorectal cancer drug also contains pharmaceutically acceptable additives and / or excipients and / or carriers.

[0040] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0041] The present invention provides a polypeptide for colorectal cancer (CRC), which can selectively inhibit CRC cells (including CT26, MC38, and HCT116) without toxicity to other cells (including human intestinal epithelial cells CCD841 and HIEC6), and has high application safety. In addition, the oncolytic mechanism of the polypeptide of the present invention by disrupting the cell membrane reduces the possibility of tumor cells developing drug resistance. It can be used to inhibit CRC tumor cells, thereby achieving the control and treatment of CRC. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 It is the electrospray mass spectrometry result of the polypeptide KY of the present invention.

[0043] Figure 2 It is the oncolytic activity result of the polypeptide KY of the present invention.

[0044] Figure 3 It is the in vitro safety result of the polypeptide KY of the present invention.

[0045] Figure 4 These are the in - vivo safety results of the polypeptide KY of the present invention. Among them, a is the body weight of the mice; b is the weights of the hearts, livers, kidneys, lungs and spleens of the mice; c is the blood biochemical indexes of the mice; d is the H&E staining results of the livers and kidneys of the mice.

[0046] Figure 5 These are the fluorescence electron microscopy results for detecting the oncolytic mechanism of polypeptide KY. Among them, a is the image of propidium iodide (PI) / Hoechst staining of CT26 cells (scale bar, 100 μm); b is the quantitative analysis result of the cells successfully stained with PI; c is the TEM image (scale bar, 5 μm). ***P < 0.001.

[0047] Figure 6 These are the results of the tumor size statistics of mice for detecting the in - vivo activity of polypeptide KY. Among them, a is the schematic diagram of the experimental protocol. b is the tumor volume; c is the volume of the tumor relative to that on day 0; d is the photo of the tumor obtained by dissection; e is the weight of the tumor obtained by dissection. Scale bar = 1 cm. *P < 0.05, **P < 0.01, ***P < 0.001, n.s. indicates no statistical difference. Detailed implementation manners

[0048] To better illustrate the purpose, technical solution and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. Other materials, reagents, etc. used in the embodiments can be obtained from commercial channels without special instructions.

[0049] Example 1 Design and preparation of polypeptide KY

[0050] I. Experimental methods

[0051] 1. The polypeptide KY of the present invention was designed by machine learning.

[0052] First, a random peptide library of 50 million sequences was constructed;

[0053] Subsequently, the random peptide library was filtered with multiple filters:

[0054] In the first - stage filtration, only peptides with a hydrophobic moment range of 0.5 - 0.7 and a hydrophobicity ≥ 0.5 were retained. Because among all the physicochemical properties, the hydrophobic moment is one of the properties most relevant to the antibacterial action of antibacterial peptides. Although the hydrophobic moment increases antibacterial and anticancer activities, it also increases the toxicity to non - tumor cells, so a medium threshold was selected;

[0055] The second-level filtering includes: (1) setting the number of W (tryptophan) to 2, because multiple Ws are favorable structures for promoting the peptide self-assembly process and disrupting the bacterial membrane; (2) given that there are two cationic amino acids in the sequence generator, setting the number of R (arginine) to ≥1, as R plays a more important role than K (lysine) in disrupting the bacterial cell membrane; (3) setting the number of non-cationic hydrophilic S (serine) to ≥1 to moderately reduce the percentage of hydrophobic amino acids;

[0056] The third-level filtering keeps the peptide's protein secondary structure prediction (PSSpred) value at ≥80% to support the possibility of forming an α-helix and a self-assembled structure;

[0057] After filtering, only 5,491 sequences were retained from 50 million sequences (the filtering efficiency was 99.989%) to enter the next stage of the pipeline;

[0058] Finally, through a data module containing two machine learning-based models, the steps include: (1) Acquisition of datasets for anti-cancer properties and toxicity: From the Database of Antimicrobial Activity and Structure of Peptides (DBAASP), according to custom screening conditions, polypeptide datasets for anti-cancer properties and toxicity were respectively obtained, and the IC 50Taking 128 μM as the threshold, the polypeptides in the polypeptide dataset are defined as having / without anti-cancer peptide activity or having / without toxicity; (2) Construction of an automated machine learning model: The polypeptide datasets in step (1) are respectively loaded into the automated machine learning framework AutoGluon, the polypeptide datasets are divided, the classifier is trained, the model is evaluated through 5-fold cross-validation, a trained model is obtained, and the trained model is used to predict new polypeptide data and automatically optimize the parameters; (3) Selection of the best model: AutoGluon in step (2) automatically recommends 28 models (14 anti-cancer peptide and toxicity prediction models respectively) for further evaluation, and based on the performance of the models, the best prediction models are obtained respectively; (4) Using the best prediction model in step (3) to predict anti-cancer peptide sequences from the polypeptide dataset to obtain anti-cancer peptide candidates, and a total of 97 anti-cancer peptides are obtained; (5) To improve the stability of the anti-cancer peptides as much as possible, sequences with great self-assembly potential are selected as much as possible. The sequences of the 97 anti-cancer peptides in step (4) are input into the public database AMP Prediction (dbaasp.org), and the self-assembly potential of the anti-cancer peptide sequences is predicted according to their in vitro agglutination potential, so as to select polypeptide sequences with a self-assembly prediction value greater than 400 from the 97 anti-cancer peptides, a total of 8; (6) Among the 8 polypeptide sequences in step (5), according to the difference in tyrosine Y at the N-terminus or C-terminus of the polypeptide sequence, 2 polypeptide sequences with self-assembly potential prediction values of 481.26 (amino acid sequence as shown in SEQ ID NO: 1: KSWWRKIFYFIRY) and 592.78 (amino acid sequence as shown in SEQ ID NO: 2: YKKVRSIWWYIFK) are synthesized respectively, and their C-terminal tyrosine is phosphorylated. After the polypeptide with the amino acid sequence shown in SEQ ID NO: 1 is phosphorylated by polypeptide acid, the chemical structural formula is as shown in Formula I, which is polypeptide KY. After the polypeptide with the amino acid sequence shown in SEQ ID NO: 2 is phosphorylated by acid, the chemical structural formula is as shown in Formula II, which is polypeptide YK:

[0059]

[0060] The sequences and various physicochemical parameters of polypeptide KY and polypeptide YK are shown in Table 1.

[0061] Table 1 Sequences and various physicochemical parameters of polypeptide KY and polypeptide YK

[0062]

[0063]

[0064] 2. Preparation method of polypeptide KY

[0065] In this application, polypeptide KY is synthesized successively by solid-phase chemical synthesis method. The solid-phase chemical synthesis method includes the following steps:

[0066] (1) The polypeptide KY was synthesized one by one from the C-terminus to the N-terminus according to its amino acid sequence, using a peptide synthesizer.

[0067] First, Fmoc-X (X is the first amino acid at the C-terminus of each polypeptide; Fmoc is 9-fluorenylmethoxycarbonyl, an amino protecting group) was attached to Wang resin. After removing the Fmoc group, X-Wang resin was obtained. Then, Fmoc-Y-Trt-OH (9-fluorenylmethoxycarbonyl-triphenylmethyl-Y, Y is the second amino acid at the C-terminus of each antimicrobial peptide) was combined with X-Wang resin to form a peptide bond, resulting in a dipeptide with protecting groups. The above steps of peptide bond formation were repeated, and the types of corresponding amino acids were replaced according to the amino acid sequence of the next polypeptide sequence, and the synthesis was carried out from the C-terminus to the N-terminus of the polypeptide in turn until the synthesis was completed, obtaining peptide resin. When the amino acid to be phosphorylated was combined with X-Wang resin, the Fmoc-Y-Trt-OH used was N-fluorenylmethoxycarbonyl-O-benzyl-L-phosphotyrosine (Fmoc-Tyr(HPO 3 OBzl)-OH).

[0068] (2) A cleavage reagent was added to the peptide resin in step (1), and the reaction was carried out at 20 °C in the dark for 2 h. After filtration, the filtrate was obtained; the precipitate was washed with TFA (trifluoroacetic acid) to obtain the washing solution. The washing solution was mixed with the above filtrate, concentrated by a rotary evaporator, and then 10 volumes of pre-cooled anhydrous ether were added. Precipitation was carried out at -20 °C for 3 h to precipitate a white powdery substance, which was centrifuged at 2500 g for 10 min. The precipitate was collected, washed with anhydrous ether again, and dried in vacuo to obtain the crude polypeptide. The cleavage reagent was composed of TFA, water, and TIS (triisopropylchlorosilane) mixed in a mass ratio of 95:2.5:2.5.

[0069] (3) The column was equilibrated with 0.2 mol / L sodium sulfate (adjusted to pH 7.5 with phosphoric acid) for 30 min. The polypeptide in step (2) was dissolved in a 90% (v / v) aqueous acetonitrile solution, filtered, and subjected to gradient elution using a C18 reversed-phase normal pressure column (the eluent was a mixture of methanol and 0.2 mol / L sodium sulfate aqueous solution in a volume ratio of 30:70 to 70:30), with a flow rate of 1 mL / min and a detection wavelength of 220 nm. The main peak was collected and freeze-dried to obtain the gradient elution product;

[0070] It was further purified using a reversed-phase C18 column. Eluent A was a 0.1% (w / w) aqueous TFA solution; eluent B was a 0.1% (w / w) TFA acetonitrile solution. The elution concentration was 25% (v / v) B to 40% (v / v) B, the elution time was 12 min, and the flow rate was 1 mL / min. The main peak was collected as above and freeze-dried to obtain the purified polypeptide;

[0071] (4) Identification of the polypeptide: Analyze the purified polypeptide obtained in step (3) by electrospray mass spectrometry.

[0072] II. Experimental results

[0073] As Figure 1 shown, the purity of the prepared polypeptide KY is greater than 95%.

[0074] Example 2 Detection of the IC 50 value

[0075] I. Experimental method

[0076] Dissolve polypeptide KY and polypeptide YK in PBS respectively to obtain a polypeptide KY solution and a polypeptide YK solution with a concentration of 256 μM. Add 0.32% (v / v) alkaline phosphatase ALP (1 unit / μL) to the polypeptide KY solution and the polypeptide YK solution with a concentration of 256 μM respectively. After mixing, incubate at 37 °C for at least 10 h and store overnight at -20 °C to obtain the ALP-treated polypeptide KY solution (KY-ALP) and the ALP-treated polypeptide YK solution (YK-ALP).

[0077] Use the Cell Counting Kit-8 (CCK-8, APExBIO Technology LLC, USA) to evaluate the cytotoxicity of polypeptide KY, the ALP-treated polypeptide KY solution, polypeptide YK, and the ALP-treated polypeptide YK solution with different concentrations (8, 16, 32, 64, and 128 μM) diluted with PBS against each cell line (CT26, MC38, HCT116, CCD841, and HIEC6). Culture the cells in a medium supplemented with 10% (v / v) FBS (fetal bovine serum) and culture overnight in a 96-well plate (4000 - 6000 cells / well). Use the CCK-8 detection method to evaluate the final survival rate of the cells, analyze the obtained data, make a semi-logarithmic curve of survival rate - polypeptide concentration, and calculate the IC 50 value. The cells treated with the medium are used as the control group. Each experiment is repeated at least three times, and each experiment consists of three technical parallel experiments.

[0078] II. Experimental results

[0079] As shown in Table 2, the IC 50 value of polypeptide KY against the colon cancer cell line is lower than that of polypeptide YK, indicating that the inhibitory effect of polypeptide KY on colon cancer is better than that of polypeptide YK. The IC 50The value is slightly lower than that of polypeptide YK, but this concentration far exceeds the active concentration range of polypeptide KY. The oncolytic activity of polypeptide KY is further enhanced after dephosphorylation by ALP treatment. Alkaline phosphatase has a higher concentration in tumor cells and the intestinal environment, and polypeptide KY has further selectivity for the application environment.

[0080] Table 2 IC of polypeptides against colon cancer and normal intestinal cell lines 50 Value (μM)

[0081]

[0082] Example 3 Detection of the in vitro oncolytic activity of polypeptide KY

[0083] I. Experimental method

[0084] Dissolve polypeptide KY in PBS to obtain a polypeptide KY solution with a concentration of 256 μM. Add 0.32% (v / v) alkaline phosphatase ALP (1 unit / μL) to the polypeptide KY solution with a concentration of 256 μM. After mixing, incubate at 37 °C for at least 10 h and store overnight at -20 °C to obtain the ALP-treated polypeptide KY solution.

[0085] 1. Detection of the oncolytic activity of polypeptide KY

[0086] (1) Use the Cell Counting Kit-8 (CCK-8, APExBIO Technology LLC, USA) to evaluate the cytotoxicity of polypeptide KY and ALP-treated polypeptide KY solution at different concentrations (8, 16, 32, 64, and 128 μM) diluted with PBS against each cell line (CT26, MC38, HCT116, CCD841, and HIEC6). Culture the cells in a medium supplemented with 10% (v / v) FBS (fetal bovine serum) and culture overnight in a 96-well plate (4000 - 6000 cells / well). Use the CCK-8 detection method to evaluate the final survival rate of the cells. Cells treated with the medium are used as the control group. Each experiment is repeated at least three times, and each experiment consists of three technical parallel experiments.

[0087] (2) The hemolytic activity of polypeptide KY was detected using fresh red blood cells from C57BL / 6 mice. The red blood cells were harvested after centrifugation at 1000×g for 5 min, washed twice with 10 volumes of PBS, and resuspended in PBS to a concentration of 1% (v / v) to obtain a red blood cell suspension. 50 μL of the red blood cell suspension was mixed well with 50 μL of polypeptide KY solution or ALP-treated polypeptide KY solution at different concentrations (8, 16, 32, 64, and 128 μM) dissolved in PBS, and incubated at a constant temperature in an incubator at 37 °C for 1 h; after 1 h, it was taken out, centrifuged at 1000×g for 5 min, and the supernatant was transferred to a new 96-well plate, and the OD value was measured at a wavelength of 570 nm to determine the amount of hemoglobin released. Red blood cell suspensions prepared with PBS and 0.1% (v / v) Triton X-100 were used as negative and positive controls, respectively. Each experiment was repeated at least three times, and each experiment consisted of three technical parallel experiments.

[0088] (3) Twenty-five healthy female C57BL / 6 mice (6 weeks old) were randomly assigned to 7 cages (5 mice per cage). The animal cages were placed separately in an individually ventilated cage (IVC) at a temperature of 20 - 24 °C, a humidity of 50 - 60%, with 60 air changes per hour, and a light / dark cycle of 12 / 12 h. Before the experiment, the mice were allowed to acclimatize in this facility for two days. The mice were intravenously injected with 100 μL of PBS or polypeptide KY solution at different concentrations (0, 5, and 10 mg / kg) diluted with PBS, and the change in body weight was monitored. At the end of the experiment (day 5), the mice were sacrificed by cervical dislocation and the organs were removed. The heart, liver, kidney, lung, and spleen were weighed and fixed with 4% (w / v) paraformaldehyde for H&E staining. Blood samples were collected from the orbital veins of the mice. Blood biochemical indices, including alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), blood urea nitrogen (BUN), creatine kinase (CREA), creatine kinase (CK), and low-density lipoprotein cholesterol (LDH), were measured using an automatic biochemical analyzer to reflect the damage to the liver and kidneys.

[0089] II. Experimental Results

[0090] As Figure 2 shown, polypeptide KY has oncolytic activity, reducing the survival rate of CT26, MC38, and HCT116 colon cancer cells, and its oncolytic activity is further enhanced after dephosphorylation by ALP treatment. Moreover, alkaline phosphatase has a higher concentration in tumor cells and the intestinal environment, and polypeptide KY has further selectivity for the application environment.

[0091] As Figure 3As shown, polypeptide KY did not show cytotoxicity to human intestinal epithelial cells (CCD841 and HIEC6) at the highest tested concentration. Although it caused some damage to mouse blood cells at the highest concentration of 128 μM, this concentration is much higher than its active concentrations (5 mg / kg and 10 mg / kg).

[0092] As Figure 4 shown, the in vivo safety results of mice showed that no damage occurred after 5 days of injection of polypeptide KY, indicating its safety. As Figure 4 shown in Fig. a, there was little difference in the body weights of mice injected with polypeptide KY and those injected with PBS on the 5th day; as Figure 4 shown in Fig. b, the weights of the hearts, livers, kidneys, lungs, and spleens of mice injected with polypeptide KY and those injected with PBS were basically the same; as Figure 4 shown in Fig. c, there were no significant differences in the blood biochemical indexes of mice injected with polypeptide KY and those injected with PBS; as Figure 4 shown in Fig. d, after H&E staining, the liver and kidney sections of mice injected with polypeptide KY were normal.

[0093] Example 4: Detection of the oncolytic mechanism of polypeptide KY

[0094] I. Experimental method

[0095] CT26 cells were seeded in 96-well plates at a density of 4×10 4 cells / well and cultured overnight. The KY solution treated with ALP in Example 3 was diluted with PBS to 16 μM to obtain a 16 μM KY solution treated with ALP. The 16 μM KY solution treated with ALP was added to the 1640 medium and incubated with CT26 cells overnight, and then gently washed three times with PBS. Stained with PI dye and then observed and photographed under an inverted fluorescence microscope (Nikon ECLIPSE Ti2-E).

[0096] II. Experimental results

[0097] As Figure 5 shown in Fig. a and Figure 5 Fig. b, after incubation with the KY solution treated with ALP, the non-membrane-permeable dye PI increased in CT-26 cells. As Figure 5 shown in Fig. c, after incubation with the KY solution treated with ALP polypeptide, the cell membrane was damaged.

[0098] Example 5: Detection of the in vivo activity of polypeptide KY

[0099] I. Experimental method

[0100] After 4 - 5-week-old C57BL / 6 mice were acclimated for one week, CT26 cells (1.0×10 6Construct a mouse model of colorectal cancer (CRC) bearing tumors. When the tumors appear and grow to a certain volume, the mice are randomly divided into 2 groups (5 mice in each group) for 7 consecutive days of intervention. The two groups of mice are intravenously injected with PBS and polypeptide KY solution (5 mg peptide / kg), respectively, and at the same time, ALP (0.456 units / mouse) is injected into the tumors. The body weight and tumor volume of the mice are recorded every day. After the intervention, the mice are euthanized under isoflurane anesthesia. The body weight and tumor weight of the mice are collected.

[0101] II. Experimental Results

[0102] As Figure 6 shown in a, it is a schematic diagram of the experimental protocol. Figure 6 b shows the tumor volume of the mice after treatment in different groups; Figure 6 c shows the tumor volume of the mice after treatment in different groups relative to day 0; Figure 6 d shows the photos of the tumors of the mice after treatment in different groups obtained by dissection; Figure 6 e shows the weights of the tumors of the mice after treatment in different groups obtained by dissection.

[0103] For the CRC mice treated with polypeptide KY (intratumoral injection of ALP), their tumors are relatively smaller and lighter compared to the CRC mice in the PBS injection group. The experimental results show that polypeptide KY enhances the oncolytic activity under the induction of ALP and inhibits the development of CRC tumors.

[0104] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A polypeptide, characterized in that The polypeptide is a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1 and / or its derivatives.

2. The polypeptide according to claim 1, characterized in that The polypeptide derivative is a polypeptide in which the N-terminal amino acid is phosphorylated.

3. The polypeptide according to claim 2, characterized in that The chemical structural formula of the polypeptide derivative is shown in Formula I:

4. The method for preparing the polypeptide according to any one of claims 1 to 3, characterized in that: The following steps are involved: S1: according to the amino acid sequence of the polypeptide, the amino acids are connected to Wang resin by a peptide synthesizer to obtain a peptide resin; S2: Cutting the peptide resin in step S1 with trifluoroacetic acid to obtain a crude polypeptide; S3: Purify the crude polypeptide of step S2 to obtain the polypeptide.

5. The preparation method according to claim 4, characterized in that: In step S3, the crude polypeptide of step S2 is purified by reverse phase high performance liquid chromatography.

6. Use of the polypeptide according to any one of claims 1 to 3 in the preparation of anti-tumor drugs.

7. The use according to claim 6, characterized in that: The anti-tumor drugs include drugs that inhibit tumor growth.

8. The use according to claim 6, characterized in that: The anti-tumor drugs include anti-colorectal cancer drugs.

9. An anti-colorectal cancer drug, characterized in that: The medicine contains the polypeptide according to any one of claims 1 to 3.

10. The anti-colorectal cancer drug according to claim 9, characterized in that: The anti-colorectal cancer drug also contains pharmaceutically acceptable additives and / or excipients and / or carriers.

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