Polypeptide component derived from fetal kidney for inhibiting tumor activity, and preparation method and application thereof

Abstract

The present application relates to the preparation of peptides, in particular to a polypeptide component derived from fetal kidney and its preparation method and application, which is used to solve the problems of significant deficiencies in the existing anti-tumor chemotherapy drugs in terms of efficacy, safety, drug resistance and targeting, etc. The polypeptide component derived from fetal kidney and its preparation method, which is extracted and prepared from fetal kidney, shows strong anti-tumor activity in various cancer cell lines, and can effectively inhibit the proliferation of tumor cells; and it shows low toxicity in normal cells, which is a very important characteristic in clinical application, and helps to reduce the side effects on patients during treatment; in addition, the anti-tumor effect of the polypeptide component of the present application is positively correlated with the dose, which provides the possibility for the optimization of the dose in the clinic, and the dose can be adjusted according to the specific condition of the patient to achieve the best efficacy.

Description

TECHNICAL FIELD

[0001] The present application relates to the preparation of peptides, in particular to a polypeptide component derived from fetal kidney which inhibits tumor activity and a preparation method and application thereof. BACKGROUND

[0002] The treatment methods of malignant tumors mainly include surgery, radiotherapy, chemotherapy, endocrine therapy and targeted therapy, etc. Traditional anti-tumor chemotherapy drugs are mostly cytotoxic drugs, and the efficacy of these drugs is uncertain, and is often accompanied by serious clinical adverse reactions, and is prone to cause drug resistance problems.

[0003] Bioactive peptides refer to amino acid polymers that have certain physiological functions or benefits on the life activities of biological organisms, also known as functional peptides. It is widely distributed in animals, plants and microorganisms, and is a kind of natural medicine. Small molecule anti-tumor active peptides have attracted widespread attention and research due to their strong penetration ability, low toxicity, high efficiency, easy absorption by cancer cells and other characteristics. Through research and some clinical trials, it has been confirmed that it can inhibit tumor growth and metastasis through multiple pathways. Therefore, the chemotherapy method based on polypeptide drugs has attracted great attention. Compared with traditional chemical drugs, peptides have stronger specificity to tumors, higher safety to the body, and can also increase the sensitivity of tumors to other clinical treatment methods.

[0004] At present, anti-tumor active peptides have been obtained from various organisms by artificial synthesis methods. For example, Carrera et al. obtained two polypeptide sequences homologous to the N-terminal of anti-tumor urine protein by artificial synthesis method (Carrera AA, Gallo M, Defaus S, Todorovski T, Andreu D. Topoisomeric Membrane-Active Peptides: A Review of the Last Two Decades. Pharm 2023; 15(10): 2451-2469. https: / / DOI 10.3390 / pharmaceutics15102451.), animal experiments showed that the inhibition rate of the two polypeptide sequences on human cervical cancer cells Hela was as high as 70%, and both could inhibit angiogenesis; Komatsu et al. synthesized a series of derivatives of cyclic peptide CHAP (Komatsu Y, Tomizaki K, Tsukamoto M, Kato T, Nishino NK, Sato SG, et al. Cyclic hydroxamic-acid-containing peptide 31, a potent synthetic histone deacetylase inhibitor with antitumor activity. CANCER RES 2021; 61(11): 4459-4466.), among which CHAP31 is the most active and stable compound, and nude mouse experiments showed that the polypeptide had obvious inhibitory effect on melanoma cells B16 / BL6; Laakkonen et al. obtained a nonapeptide LyP-1 by phage display technology peptide library (Laakkonen P, ME, Biliran H, Yang M, Ferrer F, Karpanen T, et al. Antitumor activity of a homing peptide that targets tumor lymphatics and tumor cells. PNAS 2022; 101(25): 9381-9386.), which accumulated in the breast cancer tumor site after intravenous injection, inducing the death of breast cancer cells. Therefore, it is very important to find anti-tumor polypeptide drugs with good activity and low toxic side effects. SUMMARY

[0005] The present application aims at solving the problems of the existing anti-tumor chemotherapy drugs in the aspects of efficacy, safety, drug resistance and targeting, and provides a polypeptide component inhibiting tumor activity derived from fetal kidney and a preparation method and application thereof.

[0006] The polypeptide component inhibiting tumor activity derived from fetal kidney has the special feature that the amino acid sequence is shown in SEQ ID NO. 1.

[0007] The polypeptide component inhibiting tumor activity derived from fetal kidney has the special feature that the amino acid sequence is shown in SEQ ID NO. 2.

[0008] The polypeptide component inhibiting tumor activity derived from fetal kidney has the special feature that the amino acid sequence is shown in SEQ ID NO. 2.

[0009] The polypeptide component inhibiting tumor activity derived from fetal kidney has the special feature that the amino acid sequence is shown in SEQ ID NO. 2.

[0010] The polypeptide component inhibiting tumor activity derived from fetal kidney has the special feature that the amino acid sequence is shown in SEQ ID NO. 2.

[0011] The polypeptide component inhibiting tumor activity derived from fetal kidney has the special feature that the amino acid sequence is shown in SEQ ID NO. 2.

[0012] Meanwhile, the present application also provides a preparation method of the polypeptide component inhibiting tumor activity derived from fetal kidney, which has the special feature that the method comprises the following steps:

[0013] Step 1, a pregnant SD female rat is executed by cervical dislocation, and the kidney tissue of the fetal rat is separated, and the cleaned kidney tissue is cut into multiple small pieces; the small pieces are added into a digestion mixed solution containing different types of collagenase for digestion for 20-30 minutes, a culture solution containing FBS and antibiotics is added to terminate the digestion process, the digested liquid is filtered, and the filtered cells are collected; the cells are inoculated, filtered again, and then fetal kidney cells are cultured;

[0014] Step 2, the fetal kidney cells cultured in step 1 are mixed with ultrapure water to obtain a fetal kidney cell suspension, N types of proteases are used to respectively perform enzymolysis treatment on N portions of the fetal kidney cell suspension, fetal kidney cell proteins are extracted, after the enzymolysis is completed, the enzymes are inactivated, and N types of protease hydrolysis protein products corresponding to the enzymes are obtained; N is greater than 2.

[0015] Step 3, the ultrafiltration equipment is cleaned and pre-wetted, and the N types of protease hydrolysis protein products obtained in step 2 are respectively subjected to ultrafiltration through the ultrafiltration equipment, and M protein components of different molecular weights of each enzymolysis protein product are obtained; M is greater than 2.

[0016] Step 4, after culturing and inoculating the tumor cells, the tumor cells are divided into N protease treatment groups according to the N proteases used in step 2, the cell survival rate of each protease treatment group is determined by using the CCK-8 method, and the determination of each protease treatment group is repeated multiple times, and multiple protein components with high tumor cell proliferation inhibition activity are obtained;

[0017] The i th protease treatment group comprises an NT grouping, a positive control grouping and M protein groupings, the M protein groupings correspond to M protein components of the enzymatic protein products corresponding to the i th protease; 1≤i≤N; the positive control grouping and the plurality of protein groupings are each provided with at least three concentrations;

[0018] Step 5, the plurality of protein components obtained in step 4 are subjected to LC-MS / MS for protein identification and quantitative analysis, and a plurality of polypeptides are obtained;

[0019] Step 6, the polypeptides obtained in step 5 are subjected to peptide activity prediction, and trained under the condition that the threshold value is 0.5, then the polypeptides with a threshold value >0.5 are screened and synthesized, and the polypeptide components with tumor inhibition activity derived from fetal kidney are obtained, and the preparation of the polypeptide components with tumor inhibition activity derived from fetal kidney is completed.

[0020] Further, the step 1 is specifically:

[0021] Step 1.1, the pregnant SD female rats are sacrificed by cervical dislocation, the fetal rats are taken out, the kidney tissues of the fetal rats are separated, the kidney tissues are placed on an ice-cold surface, the attachments on the kidney tissues are removed under a microscope, and the kidney tissues are cut into 1mm 3 pieces by dissection scissors;

[0022] Step 1.2, the small pieces are added to a preheated 4-5 times volume of digestion mixture, and are digested at 37°C for 20-30 min; the digestion is terminated by adding a complete culture medium containing 10% FBS, 1% double antibody, RPM I 160 and DMEM / F12, and is filtered through 80 mesh, 100 mesh and 400 mesh screens in sequence, and the finally filtered cells are collected;

[0023] The digestion mixture is 0.1%-0.2%(m / v) type IV collagenase and 0.05%-0.1%(m / v) type II collagenase;

[0024] Step 1.3, the cells collected in step 1.2 are resuspended, and the cells are inoculated into a cell bottle coated with 3-5μg / cm 2 type I rat tail collagen, and are cultured for at least 12 h, the cells are filtered again, the filtrate is collected, the supernatant is discarded by centrifugation, and the fetal kidney cells are cultured in an incubator.

[0025] Further, in step 2, the N is 4, and the N kinds of proteases are specifically: trypsin, alkaline protease, papain and protease.

[0026] Further, in step 3, the M is 3, and the M protein components of different molecular weights of each enzyme hydrolyzed protein product are specifically: three protein components with molecular weights of >10K, 3K-10K and <3K, respectively.

[0027] Compared with the prior art, the present application has the following beneficial effects:

[0028] (1) The polypeptide component with tumor inhibiting activity derived from fetal kidney in the present application is extracted and prepared from fetal kidney, and shows strong anti-tumor activity in various cancer cell lines, and can effectively inhibit the proliferation of tumor cells; and in normal cells, the polypeptide components (FRCP-1, FRCP-5 and FRCP-6) in the present application show low toxicity, which is a very important property in clinical application, and helps to reduce the side effects on patients during treatment; in addition, the anti-tumor effect of the polypeptide components (FRCP-1, FRCP-5 and FRCP-6) in the present application is positively correlated with the dose, which provides the possibility for dose optimization in clinic, and can adjust the dose according to the specific condition of the patient to achieve the best efficacy.

[0029] (2) The preparation method of the polypeptide component with tumor inhibiting activity derived from fetal kidney in the present application sequentially extracts and cultures fetal kidney cells, enzyme hydrolyzes fetal kidney cell extracted proteins to obtain enzyme hydrolyzed protein products, ultrafiltrates the enzyme hydrolyzed protein products to obtain protein components, functionally screens (evaluates the influence of different protein components on tumor cell proliferation to screen out protein with high inhibitory activity), protein identification and quantification, polypeptide activity prediction and synthesis, and finally obtains the polypeptide component with tumor inhibiting activity derived from fetal kidney; the preparation method of the present application not only provides a technical route for efficient extraction and screening of polypeptides with anti-tumor activity from fetal kidney cells, but also ensures the activity and functionality of the polypeptides through detailed biochemical analysis, and provides a practical research framework for discovering and developing new anti-tumor treatment methods.

[0030] (3) The present application is a method for preparing polypeptide components with tumor inhibiting activity derived from fetal kidney. The use of four different proteases (trypsin, alcalase, papain and pronase) allows a more comprehensive breakdown of proteins in fetal kidney cells. Each enzyme has a different substrate specificity, which helps to release a greater variety of proteins and polypeptides from the cell sample, increasing the diversity of potential active compounds. In addition, each enzymatic protein product is then classified by molecular weight (greater than 10K, 3K to 10K, less than 3K) using ultrafiltration equipment, so that in-depth analysis and functional testing can be performed on polypeptides of specific sizes, ensuring the accuracy and reproducibility of the separation process. The use of multiple enzymes and molecular weight classification in the present application more effectively screens polypeptides with high biological activity. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 Chromatogram of FRCP-1 in the preparation method of polypeptide components with tumor inhibiting activity derived from fetal kidney according to an embodiment of the present application;

[0032] Figure 2 Mass spectrum of FRCP-1 in an embodiment of the present application;

[0033] Figure 3 Chromatogram of FRCP-5 in an embodiment of the present application;

[0034] Figure 4 Mass spectrum of FRCP-5 in an embodiment of the present application;

[0035] Figure 5 Chromatogram of FRCP-6 in an embodiment of the present application;

[0036] Figure 6 Mass spectrum of FRCP-6 in an embodiment of the present application. DETAILED DESCRIPTION

[0037] Fetal kidney (FRCP) is kidney cells taken from the embryos of pregnant SD female rats, and has a rich pharmacological effect. The pharmacological effect of animal medicine fetal kidney has been researched since 1996, and it has been confirmed to have a series of effects such as anti-aging, enhancing the body's immunity, improving sex hormone levels, improving osteoporosis, delaying renal function failure, and correcting anemia. In recent years, it has been found that fetal kidney polypeptides have an anti-tumor effect, can inhibit the growth, proliferation, invasion and metastasis of breast cancer MCF-7 cells, but the specific polypeptide components of fetal kidney action are not yet known.

[0038] Currently, some peptide drugs have been applied to anti-tumor clinical treatment, which can be derived from different resources such as plants and animals, or synthesized. In this study, the present application identifies several bioactive fetal kidneys with strong cytotoxicity in a dose- and time-dependent manner in vitro, preferably against different types of human malignant tumor cells. Therefore, the present application can provide a new basis for designing new bioactive peptides for the clinical treatment of solid cancers.

[0039] Enzymatic hydrolysis can use active enzymes to hydrolyze specific substances at high yield and has been widely used in the preparation of bioactive peptides from animal tissues; in the present application, after collagenase digestion, four different types of enzymes are used to prepare fetal kidneys. It was found in the experiment that a certain percentage of protein components with a molecular weight <3k were produced by trypsin, papain, pronase or alkaline protease digestion, but the protein components with a molecular weight <3k produced by trypsin, papain or pronase digestion had strong cytotoxicity, preferably against several types of cancer cells. Therefore, trypsin, papain or pronase digestion is a useful method for preparing bioactive peptides from RC (collagen).

[0040] The present application will be further described below in conjunction with the accompanying drawings and exemplary embodiments.

[0041] The method for preparing polypeptide components derived from fetal kidneys with tumor inhibitory activity comprises the following steps:

[0042] Step 1, the pregnant SD female rats were executed by cervical dislocation, the kidney tissues of the fetal rats were separated, and the cleaned kidney tissues were cut into small pieces; the small pieces were added to a digestion mixture containing different types of collagenase and digested for 20-30 minutes, a culture solution containing FBS and antibiotics was added to terminate the digestion process, the digested liquid was filtered, and the cells were collected; the cells were inoculated and filtered again, and then the fetal kidney cells were cultured, specifically:

[0043] Step 1.1, the pregnant SD (Sprague-Dawley) female rats were executed by cervical dislocation, the fetal rats were taken out, the kidney tissues of the fetal rats were separated, the kidney tissues were placed on an ice-cold surface, and the attachments (kidney capsule, blood cells and connective tissue, etc.) on the kidney tissues were removed under a microscope, and then the kidney tissues were cut into small pieces with a size of about 1mm 3 left and right by using a dissecting scissors;

[0044] Step 1.2, add small pieces into the preheated 4-5 times volume of digestion mixture, and digest at 37℃ for 20-30 min; add RPM I 160 and DMEM / F12 mixed complete culture solution containing 10% FBS (fetal bovine serum) and 1% double antibody to terminate the digestion, filter through 80 mesh, 100 mesh and 400 mesh sieves in sequence, and collect the final filtered cells;

[0045] The digestion mixture is 0.1%-0.2% (m / v) type IV collagenase and 0.05%-0.1% (m / v) type II collagenase; m / v is mass / volume percentage concentration;

[0046] Step 1.3, resuspend the cells collected in step 1.2, and inoculate the cells into 3-5 μg / cm 2 The cells are coated with type I mouse tail collagen, and after 12 h of culture, the cells are observed to be adherent under a microscope, and the cells are flat. The cells are filtered through a 400 mesh sieve, the filtrate is collected, centrifuged, and the supernatant is discarded. The fetal kidney cells are cultured in a 37℃, 5% (m / v) CO2, saturated humidity incubator;

[0047] Step 2, mix the fetal kidney cells cultured in step 1 with ultrapure water to obtain a fetal kidney cell suspension, use N proteases to perform enzymatic hydrolysis treatment on N portions of the fetal kidney cell suspension respectively, extract fetal kidney cell proteins, inactivate the enzymes after the enzymatic hydrolysis is completed, and obtain corresponding N protease hydrolysis protein products; N is greater than 2;

[0048] The value of N is 4;

[0049] 4 portions of 2×10 7 cells / mL fetal kidney cells are each placed in 50 mL of ultrapure water to obtain 4 portions of fetal kidney cell suspensions; the 4 portions of fetal kidney cell suspensions are subjected to enzymatic hydrolysis using 0.24% (m / v) trypsin, 4% (m / v) alkaline protease, 1.5% (m / v) papain, and 0.43% (m / v) complex protease respectively; after the enzymatic hydrolysis is completed, the 4 portions of fetal kidney cell suspensions are placed in a 85℃ water bath for 10 minutes to inactivate the enzyme activity, and finally the corresponding enzymatic hydrolysis protein products are obtained;

[0050] Part of each enzymatic hydrolysis protein product is prepared into a freeze-dried enzymatic hydrolysis protein product, and the remainder is stored at -20℃;

[0051] Preparation of 0.24% (m / v) trypsin: take 0.12 g of trypsin dry powder, dissolve in 50 mL of PBS,

[0052] Adjust the pH to 8.0;

[0053] Preparation of 4% (m / v) alkaline protease: take 2 g of alkaline protease dry powder, dissolve in 50 mL of PBS, and adjust the pH to 8.5;

[0054] 1.5% (m / v) papain preparation: take 0.75 g of papain dry powder, dissolve in 50 mL of PBS, adjust pH = 7.0;

[0055] 0.43% (m / v) complex protease preparation: take 0.215 g of complex protease dry powder, dissolve in 50 mL of PBS, adjust pH = 7.0;

[0056] Step 3, clean and pre-wet the ultrafiltration equipment, and pass the N kinds of protein enzymatic hydrolysate products obtained in step 2 through the ultrafiltration equipment to obtain M protein components of different molecular weights of each enzymatic hydrolysate product; M is greater than 2;

[0057] The value of M is 3;

[0058] Step 3.1, load 5 mL of solvent (for cleaning and pre-wetting the ultrafiltration membrane) into the sample pool of the ultrafiltration equipment, centrifuge at 4000 rpm for 1 min, discard the filtrate and repeat this step;

[0059] Step 3.2, load one of the enzymatic hydrolysate products obtained in step 2 into the sample pool of the ultrafiltration equipment, centrifuge at 4000 rpm for 30 min to obtain the corresponding enzymatic sample; sequentially use ultrafiltration tubes with molecular weight cut-offs of 10K and 3K to perform primary separation on the enzymatic sample, thereby obtaining three protein components with molecular weights of >10K, 3K-10K and <3K for the enzymatic hydrolysate product; repeat this step for the remaining three enzymatic hydrolysate products obtained in step 2, and finally obtain three protein components with molecular weights of >10K, 3K-10K and <3K for each of the four enzymatic hydrolysate products;

[0060] The pressure of CO2 during ultrafiltration is less than 0.25 MPa,

[0061] Part of each protein component is prepared into a freeze-dried protein component;

[0062] Step 4, use the BCA (Bicinchoninic Acid) method to perform protein quantification on the multiple protein components of different molecular weights of each enzymatic hydrolysate product in step 3;

[0063] Step 4.1, configure BSA (bovine serum albumin) standard into different concentration gradient protein standards with ultrapure water;

[0064] Step 4.2, mix and configure BCA (Bicinchoninic Acid) working solution according to the volume ratio of 50:1 of A liquid and B liquid;

[0065] Step 4.3, 20 μL of each concentration of protein standard or protein sample (N protease digestion protein products and N freeze-dried protease digestion protein products obtained in step 2, M protein components and M freeze-dried protein components obtained in step 3) is taken and added to the bottom of a 96-well plate, 200 μL of BCA working solution is added to each well;

[0066] Step 4.4, incubate at 37°C for 30 min, cool the 96-well plate to room temperature, and detect the absorbance value at 562 nm on the microplate reader; plot the concentration of the protein standard against its corresponding absorbance value to obtain a standard curve; calculate the protein concentration in the protein sample according to the absorbance value of the protein sample by the standard curve;

[0067] The detection results are as follows:

[0068] The protein contents of the N protease digestion protein products obtained in step 2 were detected, and the corresponding protein contents of the trypsin, alkaline protease, protease, and papain protein digestion products were 1008.91 ± 32.38 ug, 5379.61 ± 259.85 ug, 577.86 ± 19.19 ug, and 5603.6 ± 301.26 ug, respectively.

[0069] The N freeze-dried protease digestion protein products obtained in step 2 were dissolved in 1 ml of ultrapure water, and the corresponding protein contents of the trypsin, alkaline protease, protease, and papain freeze-dried protease digestion protein products were 1261.15 ± 88.21 ug, 5699.6 ± 399.12 ug, 739.7 ± 75.23 ug, and 5699.6 ± 338.51 ug, respectively.

[0070] The M protein components obtained in step 3 were each taken 1 ml, and the M freeze-dried protein components were each added 1 ml and 50 μl of ultrapure water to dissolve, and the protein contents of the different molecular weight protein components of trypsin, alkaline protease, protease, and papain were detected. It was found that the protein content of each freeze-dried protein component after ultrafiltration was the highest, so the freeze-dried protein component was used for subsequent steps.

[0071] Step 5, after culturing and inoculating tumor cells (MCF-7 cells), the tumor cells are divided into N protease treatment groups according to the N proteases used in step 2, the i-th protease treatment group contains NT subgroups, a positive control subgroup, and M protein subgroups corresponding to the M protein components of the i-th protease digestion protein product; 1 ≤ i ≤ N; the positive control subgroup and the plurality of protein subgroups are set to have at least three concentrations; the CCK-8 (Cell Counting Kit-8) method is used to determine the cell survival rate for each protease treatment group, and the determination of each protease treatment group is repeated multiple times;

[0072] Step 5.1, MCF-7 cells were cultured in DMEM high glucose liquid containing 10% FBS, after cell passage, digestion, counting, 1x10 4 Cells were divided into alkaline protease treatment group, complex protease treatment group, trypsin treatment group and papain treatment group, each protease treatment group contained NT (No Treatment) group, positive control group and multiple protein groups with different molecular weights, and the positive control group and multiple protein groups were set with multiple concentrations, as shown in Table 1:

[0073] Table 1

[0074]

[0075]

[0076] After 48 hours of treatment, 10 μ L CCK-8 solution was added to each well, and the absorbance was measured at 450 nm using an ELISA enzyme marker, and high absorbance indicated high cell survival rate;

[0077] Each protease treatment group data was repeated 3 times, and the experimental results showed that the protein groups with molecular weight 3k-10k and molecular weight >10k in the complex protease treatment group, the protein group with molecular weight <3k in the trypsin treatment group, and the protein group with molecular weight >10k in the papain treatment group had higher tumor cell proliferation inhibition activity, and the experimental results were as follows:

[0078] Compared with the NT group, 5-fluorouracil could significantly inhibit the proliferation activity of MCF-7 cells at all concentrations, showing its anticancer effect (P<0.01);

[0079] The three protein groups with different molecular weights in the alkaline protease treatment group had no significant inhibitory effect on the proliferation activity of MCF-7 cells (all P>0.05);

[0080] The protein groups with molecular weight <3k and molecular weight >10k in the complex protease treatment group could significantly inhibit the proliferation activity of MCF-7 cells (P<0.01), and the protein group with molecular weight 3k-10k had no significant inhibitory effect on the proliferation activity of MCF-7 (P>0.05).

[0081] The protein group with molecular weight <3k in the trypsin treatment group could inhibit the proliferation activity of MCF-7 cells (P<0.05), and the protein groups with molecular weight 3k-10k and molecular weight >10k had no significant inhibitory effect on the proliferation activity of MCF-7 (P>0.05);

[0082] The protein groups with molecular weight <3k and >10k in the papain treatment group significantly inhibited the proliferation activity of MCF-7 cells (P<0.01), and the protein groups with molecular weight 3k-10k did not significantly inhibit the proliferation activity of MCF-7 cells (all P>0.05);

[0083] Step 6, the protein components obtained in step 5 are subjected to liquid chromatography mass spectrometry (LC-MS / MS) for protein identification and quantitative analysis, and a plurality of polypeptides are obtained;

[0084] According to the results of the CCK-8 experiment, the papain treatment group <3k, the complex protease treatment group <3k, and the trypsin treatment group <3k with high inhibition of MCF-7 proliferation activity are used for polypeptide identification and mass spectrometry analysis, specifically:

[0085] The corresponding freeze-dried protein components in step 3 are dissolved and separated by capillary high performance liquid chromatography, and then subjected to mass spectrometry analysis by QExactive mass spectrometer (ThermoFisher) in positive ion mode, with an analysis time of 60 min; the mass spectrometry acquisition mode is 10 fragment maps (MS2scan) after each full scan, and the mass spectrometry raw file (Raw File) is obtained;

[0086] Then the mass spectrometry raw file is processed using MaxQuant software (version 1.5.5.1), and the corresponding database is searched, and finally the protein identification and quantitative analysis results are obtained, a total of 22 polypeptides in the papain treatment group, 30 polypeptides in the trypsin treatment group, and 43 polypeptides in the complex protease treatment group are identified;

[0087] The related parameters and instructions for searching the database are shown in Table 2:

[0088] Table 2

[0089]

[0090]

[0091] Step 7, analysis of the biological activity of the peptides and synthesis of the peptides;

[0092] The polypeptides obtained in step 5 are subjected to peptide activity prediction using the website PeptideRanker (http: / / distilldeep.ucd.ie / peptideranker / ), and trained under the threshold value of 0.5 (all peptides with a score exceeding 0.5 are considered to have potential biological activity); the peptide activity prediction can also be performed using PeptideDB (http: / / www.peptidedb.org / );

[0093] According to the biological activity prediction results, polypeptides with a screening threshold value > 0.5 were synthesized by GenScript Company, three polypeptides were obtained, the amino acid sequences are shown in SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3, respectively, and the preparation of polypeptide components derived from fetal kidney tumor inhibitory activity was completed.

[0094] The three polypeptides are defined as FRCP-1, FRCP-5, and FRCP-6, corresponding to SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3.

[0095] The purity of FRCP-1, FRCP-5, and FRCP-6 prepared by the present application was determined by high performance liquid chromatography (HPLC), and the molecular weight Mw was determined by mass spectrometry (MS) analysis;

[0096] Figure 1 The chromatogram of FRCP-1 is shown in Figure 1, Figure 2 The mass spectrum of FRCP-1 is shown in Figure 2, the purity of FRCP-1 is 95.10%, and the molecular weight is 2522.06; Figure 3 The chromatogram of FRCP-5 is shown in Figure 3, Figure 4 The mass spectrum of FRCP-5 is shown in Figure 4, the purity of FRCP-5 is 95.70%, and the molecular weight is 2645.18; Figure 5 The chromatogram of FRCP-6 is shown in Figure 5, Figure 6 The mass spectrum of FRCP-6 is shown in Figure 6, the purity of FRCP-6 is 96.50%, and the molecular weight is 2347.77;

[0097] The information of FRCP-1, FRCP-5, and FRCP-6 is shown in Table 3:

[0098] Table 3

[0099]

[0100]

[0101] FRCP-1, FRCP-5, and FRCP-6 were dissolved in sterile PBS at pH 7.4 to a concentration of 10 mg / mL, filtered using a 0.22 μm microporous membrane filter, and stored in a refrigerator at -80°C.

[0102] To demonstrate the anti-tumor activity of FRCP-1, FRCP-5, and FRCP-6, the following anti-tumor activity detection was performed:

[0103] Tumor cells MCF-7, A549, HCT-116, Hela, HepG2, SGC-7901 and normal cells MCF-10A and THLE-2 were cultured in DMEM high glucose solution containing 10% FBS; after cell passage, digestion and counting, 1x10 4 cells were added to each well of a 96-well plate, and were grouped as follows:

[0104] (1) NT group used normal saline;

[0105] (2) 0.1% DMSO group, since the polypeptide needs to be dissolved in DMSO, a DMSO control group was set up to evaluate the effect of DMSO itself on cells;

[0106] (3) Positive control group: 5-fluorouracil was used, divided into 100 μM, 1 mM, 5 mM three concentrations, to verify the effectiveness of the experiment;

[0107] (4) Experimental group: cells were treated with FRCP-1, FRCP-5, FRCP-6 respectively, with concentrations of 5, 10, 15, 20, 25, 30, 35, 40, 45 μg / mL, to evaluate the strength and dose dependence of its anti-tumor activity;

[0108] After 48 hours of experiment, 10 μL CCK-8 solution was added to each well, and the absorbance at 450 nm was determined by enzyme-linked immunosorbent assay (ELISA);

[0109] Each group of data was repeated 5 times;

[0110] The inhibitory effect of polypeptides of different concentrations of three polypeptides (FRCP-1, FRCP-5, FRCP-6) on MCF-7, A549, HCT-116, Hela, HepG2, SGC-7901, MCF-10A and THLE-2 was analyzed, as follows:

[0111] (1) Normal cells: different concentrations of three polypeptides (FRCP-1, FRCP-5, FRCP-6) had no significant inhibitory effect on the proliferation of MCF-10A and THLE-2, and there was no significant difference compared with the NT group and the 0.1% DMSO group (all P>0.05), but the positive control group had a significant inhibitory effect on the proliferation of MCF-10A and THLE-2 (P<0.05);

[0112] (2) Tumor cells: Low concentrations of FRCP-1, FRCP-5, and FRCP-6 did not significantly inhibit the proliferation activity of all tumor cells at 48 h, but the inhibitory activity gradually increased with the increase of the concentration; when the concentration reached 45 μg / ml, the inhibitory activity was significantly enhanced, higher than that of the NT group and the 0.1% DMSO group (both P<0.05), and there was no significant difference in the inhibitory activity of the proliferation of all tumor cells between the low-concentration positive control group (100 μm, 1 mM) and the high-concentration positive control group (5 mM) (P>0.05), but lower than that of the high-concentration positive control group (5 mM) (P<0.05); there was no statistically significant difference compared with the NT group and the 0.1% DMSO group (P>0.05).

[0113] (3) FRCP-1, FRCP-5, and FRCP-6 showed different inhibitory activities on tumor cells MCF-7, A549, HCT-116, Hela, HepG2, and SGC-7901 within 48 hours; the inhibition rate of FRCP-1, FRCP-5, and FRCP-6 increased with the increase of the concentration, and there was no obvious inhibitory activity on normal cells MCF-10A and THLE-2 cells;

[0114] In summary, the experiments show that FRCP-1, FRCP-5, and FRCP-6 have inhibitory effects on various tumor cells and have no toxicity to normal cells. Therefore, FRCP-1, FRCP-5, and FRCP-6 can be applied in the research of anti-tumor drugs.

[0115] The experiment of the embodiment of the present application shows that:

[0116] The tumor cells MCF-7, A549, HCT-116, Hela, HepG2, and SGC-7901 and the normal cells MCF-10A and THLE-2 of the embodiment of the present application are all purchased from Shanghai Dongquan Biotechnology Co., Ltd.

[0117] The statistical analysis of the embodiment of the present application is performed by using GraphPad Prism 8 software (GraphPad Software Inc., California, USA), and Student's t test is used to compare the mean values of two groups of data to determine whether there is a significant difference between two independent samples;

[0118] One-way analysis of variance (ANOVA) is used to compare the mean values of three groups or more data;

[0119] The mean value is represented by Log rank (Mante1-Cox), and SEM is an estimate of the standard deviation, which is used to describe the reliability of the sample mean;

[0120] Differences were considered statistically significant at p < 0.05.

Claims

1. A polypeptide component with tumor-suppressing activity derived from fetal kidney, characterized in that: The amino acid sequence is shown as SEQ ID NO.

1.

2. Use of the polypeptide component with tumor inhibiting activity derived from fetal kidney in claim 1 in the preparation of an anti-tumor drug; the tumor includes MCF-7, A549, HCT-116, Hela, HepG2, SGC-7901.

3. A polypeptide component derived from fetal kidney which inhibits tumor activity, characterized by: The amino acid sequence is shown as SEQ ID NO.

2.

4. Use of the polypeptide component with tumor inhibiting activity derived from fetal kidney in claim 3 in the preparation of an anti-tumor drug; the tumor includes MCF-7, A549, HCT-116, Hela, HepG2, SGC-7901.

5. A polypeptide component derived from fetal kidney which inhibits tumor activity, characterized by: The amino acid sequence is shown as SEQ ID NO.

3.

6. Use of the polypeptide component with tumor inhibiting activity derived from fetal kidney in claim 5 in the preparation of an anti-tumor drug; the tumor includes MCF-7, A549, HCT-116, Hela, HepG2, SGC-7901.

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