Polypeptide and application thereof

Abstract

The invention discloses a polypeptide and application thereof. The polypeptide TCL6148 is a polypeptide composed of an amino acid sequence as shown in SEQ ID NO: 1. The polypeptide TCL6148 is prepared by adopting a solid-phase synthesis method, it is proved for the first time that the polypeptide TCL6148 can inhibit proliferation and migration of renal cell carcinoma cells, and a new target spot and a new medication strategy are provided for treating renal cell carcinoma.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a polypeptide and its application. Background Art

[0002] Renal cell carcinoma is the most common primary renal malignancy, accounting for approximately 2-3% of all malignant tumors worldwide. It is also one of the most common and lethal genitourinary cancers, with high morbidity and mortality. In China, there are approximately 70,000 new cases of renal cell carcinoma and 20,000 renal cell carcinoma-related deaths each year. Currently, the treatment of renal cell carcinoma mainly includes surgical treatment, tyrosine kinase inhibitor targeted therapy, immune checkpoint inhibitor immunotherapy, and cell therapy. However, the characteristics of RCC such as easy metastasis, easy drug resistance, and easy recurrence have resulted in limited effects of these treatments. Therefore, there is an urgent need to explore new treatment strategies to alleviate the malignant progression of patients with advanced renal cell carcinoma.

[0003] As an emerging biological treatment strategy, peptide drugs have high specificity and targeting, and can target cancer cell-specific receptors and molecules. The advantages of peptide drugs include relatively low toxicity, side effects and good biocompatibility. Peptides can exert anti-cancer effects through multiple mechanisms such as directly inhibiting tumor cell proliferation, promoting cell apoptosis, and regulating the tumor microenvironment. Therefore, the development of peptide drugs for renal cell carcinoma has become an important research direction. Summary of the invention

[0004] The purpose of the present invention is to provide a polypeptide and its application in view of the deficiencies in the prior art.

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

[0006] The first aspect is to provide polypeptide TCL6148, wherein the polypeptide TCL6148 is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:1.

[0007] RKKRRQRRR-MCLSFRFSFELAVTPLQFLH (SEQ ID NO: 1).

[0008] Furthermore, the polypeptide TCL6148 is prepared by solid phase synthesis.

[0009] The second aspect is to provide the use of the above-mentioned polypeptide TCL6148 in the preparation of anti-renal cell carcinoma drugs.

[0010] Furthermore, the anti-renal cell carcinoma drug inhibits the proliferation and migration of renal cell carcinoma cells; the renal cell carcinoma cells are human renal carcinoma cells 786-O.

[0011] The third aspect is to provide a pharmaceutical composition, wherein the active ingredient in the pharmaceutical composition contains the polypeptide TCL6148 as described above.

[0012] The present invention adopts the above technical solution, and has the following technical effects compared with the prior art:

[0013] The present invention adopts a solid phase synthesis method to prepare the polypeptide TCL6148, and proves for the first time that the polypeptide TCL6148 can inhibit the proliferation and migration of renal cell carcinoma cells, providing a new target and a new drug strategy for the treatment of renal cell carcinoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is the HPLC spectrum and protein spectrum detection spectrum of polypeptide TCL6148.

[0015] Figure 2 The figure shows the effect of peptide TCL6148 on cell viability after treating renal cancer cell 786-0 for 24 hours.

[0016] Figure 3 The figure shows the effect of peptide TCL6148 on cell proliferation after treating renal cancer cell 786-0 for 24 hours.

[0017] Figure 4 The figure shows the effect of peptide TCL6148 on cell migration after treating renal cancer cell 786-0 for 24 hours. DETAILED DESCRIPTION

[0018] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments, but is not intended to be limiting of the present invention. It should be noted that the embodiments and features in the embodiments of the present invention may be combined with each other without conflict.

[0019] Example 1

[0020] This example provides a method for preparing polypeptide TCL6148, and the specific steps are as follows:

[0021] 1) Take 10 g of chlororesin and place it in a peptide synthesis tube, then add 100 ml of dichloromethane (DCM) for 60 minutes of swelling treatment; after the swelling is completed, filter to remove the solvent, then wash three times with 100 ml of N,N-dimethylformamide (DMF), and finally filter and dry.

[0022] 2) Measure L-histidine (His) to ensure that the molar ratio of L-histidine to resin reaches 6:1, and dissolve it in DMF; at the same time, weigh diisopropylethylamine (DIEA) to ensure that the molar ratio of L-histidine to resin reaches 8:1; add these two substances to the peptide synthesis tube and shake the reaction overnight.

[0023] 3) After reacting overnight, the system was washed three times with 100 ml of DMF; then, methanol was added to block the active sites of the chlororesin.

[0024] 4) After the blocking step, the sample was washed again with 100 ml of DMF four times, during which Fmoc-protecting groups were added during two washings to provide protection.

[0025] 5) After removing the protecting groups, weigh leucine (Leu) so that its molar mass ratio with the resin reaches 6:1; weigh the condensation reagent so that its molar mass ratio with the resin reaches 6:1; dissolve both in DMF, add DIEA, and react in a synthesis tube for 40 minutes; then wash three times with 100 ml of DMF and filter dry.

[0026] 6) Repeat the operation process of steps 4) and 5) until the last amino acid (Met) is connected; then, remove the protecting group Fmoc, wash three times with 100 ml of DMF and then filter and dry to obtain a dry peptide resin.

[0027] 7) Add 20 ml of cleavage reagent to each gram of dry peptide resin and react in a sealed manner at room temperature for 3 hours.

[0028] 8) After the reaction is completed, the mixture is filtered, the filtrate is collected and transferred to an EP tube; then ether is added in an amount 10 times the volume of the filtrate to precipitate the polypeptide; the precipitate is obtained by centrifugation, and the supernatant is discarded; this process is repeated three times to obtain a crude polypeptide.

[0029] 9) The crude polypeptide is purified by high performance liquid chromatography (HPLC), and then freeze-dried and weighed.

[0030] The purity of the anti-renal cell carcinoma peptide TCL6148 was over 95% after HPLC analysis, and the structure was identified by electrospray ionization mass spectrometry (ESI-MS). Figure 1 shown.

[0031] Example 2

[0032] This example explores the effect of polypeptide TCL6148 on the viability of human renal cell carcinoma cell 786-O cells. The specific experimental steps and results are as follows:

[0033] First, 786-O cells in the logarithmic growth phase were digested with trypsin. After the cells separated from the bottom of the culture dish, the culture medium was immediately added to stop the digestion process. Then, the digested cell suspension was collected into a centrifuge tube and centrifuged at 1000 rpm for 3 minutes. After centrifugation, the supernatant was removed, the cell pellet was resuspended with culture medium, and the cell density was diluted to 5*10 3Then, the cell suspension was distributed into a 96-well plate at 100 μl per well and incubated at 37°C and 5% CO 2 Culture in a constant temperature incubator for 24 hours.

[0034] After the culture was completed, the old culture medium in the 96-well plate was removed and replaced with fresh culture medium containing different concentrations of peptide TCL6148, and the 786-O cells were incubated for another 24 hours.

[0035] After the incubation, 10 μl of CCK8 reagent was added to each well of the 96-well plate and incubated at 37°C for 1 hour.

[0036] The absorbance value of each well was measured at a wavelength of 450 nm using an enzyme marker, and the data was analyzed and the cell survival rate of the renal cell carcinoma cells was calculated.

[0037] The results are as follows Figure 2 As shown, with the increase of the concentration of polypeptide TCL6148, the cell survival rate showed a gradually decreasing trend, indicating that polypeptide TCL6148 has a significant inhibitory effect on the cell viability of renal cell carcinoma cells.

[0038] Example 3

[0039] This example explores the effect of polypeptide TCL6148 on the proliferation of human renal cell carcinoma cell 786-O cells. The specific experimental steps and results are as follows:

[0040] First, 786-O cells in the logarithmic growth phase were digested with trypsin. After the cells separated from the bottom of the culture dish, the culture medium was immediately added to stop the digestion process. Then, the digested cell suspension was collected into a centrifuge tube and centrifuged at 1000 rpm for 3 minutes. After centrifugation, the supernatant was removed, the cell pellet was resuspended with culture medium, and the cell density was diluted to 1*10 3 Then, the cell suspension was distributed into a 96-well plate at 100 μl per well and incubated at 37°C and 5% CO 2 Culture in a constant temperature incubator for 24 hours.

[0041] After the culture was completed, the old culture medium in the 96-well plate was removed and replaced with fresh culture medium containing different concentrations of peptide TCL6148, and the 786-O cells were incubated for another 24 hours.

[0042] After the incubation, the old medium in the 96-well plate was removed and replaced with fresh medium containing 10 μM EdU reagent, and the 786-O cells were incubated for 6 hours.

[0043] After 6 hours, the medium was removed, the cells were fixed with 4% paraformaldehyde for 15 minutes, and then washed three times with PBS solution containing 3% BSA. The cells were permeabilized with Triton X-100 for 10 minutes, and then washed three times with PBS solution containing 3% BSA.

[0044] Incubate the cells with EdU staining reagent at room temperature for 30 minutes in a light-protected environment. After 30 minutes, wash the cells three times with PBS solution containing 3% BSA.

[0045] The cells were incubated with DAPI for 5 min and then washed 3 times with PBS.

[0046] A fluorescence microscope was used to take pictures of blue light (DAPI) and green light (EdU), and the data was analyzed to calculate the cell proliferation rate of renal cell carcinoma cells.

[0047] The results are as follows Figure 3 As shown, with the increase of the concentration of peptide TCL6148, the cell proliferation rate gradually decreased, indicating that peptide TCL6148 has an inhibitory effect on the proliferation ability of renal cell carcinoma cells.

[0048] Example 4

[0049] This example explores the effect of polypeptide TCL6148 on the migration of human renal cell carcinoma cell 786-O cells. The specific experimental steps and results are as follows:

[0050] First, 786-O cells in the logarithmic growth phase were digested with trypsin. After the cells were separated from the bottom of the culture dish, the culture medium was immediately added to stop the digestion process. Then, the digested cell suspension was collected into a centrifuge tube and centrifuged at 1000 rpm for 3 minutes. After centrifugation, the supernatant was removed, the cell pellet was resuspended with culture medium, and the cell suspension was distributed into a 6-well plate and incubated at 37°C, 5% CO 2 Culture in a constant temperature incubator for 24 hours.

[0051] When the cell growth density just covers the bottom of the well, aspirate the old culture medium from the 6-well plate, use a 200μl sterilized pipette tip to draw a vertical line on the bottom of the well, then wash the detached cell fragments with PBS, add culture medium containing different concentrations of peptide TCL6148 to the 6-well plate, and use a microscope to take pictures to record the cell scratches.

[0052] After 24 hours, the old culture medium was removed from the 6-well plate, the detached cell debris was washed with PBS, and the cell scratches were recorded again using a microscope. The scratch changes of the two photos were analyzed and the migration rate of the cells was calculated.

[0053] The results are as follows Figure 4As shown in the figure, with the increase of the concentration of peptide TCL6148, the cell scratch healing area gradually decreased, indicating that peptide TCL6148 has an inhibitory effect on the migration ability of renal cell carcinoma cells.

[0054] The above description is only a preferred embodiment of the present invention, and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the contents of the specification and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. Polypeptide TCL6148, characterized in that The polypeptide TCL6148 is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:

1.

2. The polypeptide TCL6148 according to claim 1, characterized in that The polypeptide TCL6148 is prepared by solid phase synthesis.

3. Use of the polypeptide TCL6148 according to any one of claims 1 to 2 in the preparation of an anti-renal cell carcinoma drug.

4. The use according to claim 3, characterized in that: The anti-renal cell carcinoma drug inhibits the proliferation and migration of renal cell carcinoma cells.

5. The use according to claim 4, characterized in that: The renal cell carcinoma cell is human renal carcinoma cell 786-O.

6. A pharmaceutical composition, characterized in that The active ingredient in the pharmaceutical composition contains the polypeptide TCL6148 as claimed in claim 1.

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