CN113501863B - Mucin13 antagonistic polypeptide, derivative and application thereof - Google Patents

Abstract

The invention discloses a mucin13 antagonist polypeptide, a derivative and an application thereof, and particularly discloses a mucin13 antagonist polypeptide, wherein an amino acid residue sequence of the mucin13 antagonist polypeptide is shown as SEQ ID No. 1. The mucin13 antagonistic polypeptide and the derivative thereof disclosed by the invention can be specifically combined with MUC13 to inhibit a MUC13 signal channel, so that the proliferation of tumor cells is inhibited, the apoptosis of the tumor cells is promoted, the mucin13 antagonistic polypeptide can be used as a biological polypeptide medicament of a MUC13 binding site, is used for preparing medicaments for preventing and/or treating tumors, and can be widely applied to the fields of medicine and biology.

Description

Mucin13 antagonistic polypeptide, derivative and application thereof

Technical Field

The invention relates to the field of biotechnology and biomedicine, in particular to a mucin13 antagonistic polypeptide, a derivative and an application thereof.

Background

Hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC) are the two most common types of primary liver cancer, with HCC being one of the most common malignancies worldwide accounting for about 90% of primary liver cancers. Liver Cancer is one of the common malignant tumors in the world, and the incidence rate of the liver Cancer is 4 th and 3 rd after lung Cancer, breast Cancer and colorectal Cancer as reported by International Agency for Research on Cancer (IARC) of the world health organization. The new primary liver cancer of China accounts for about 55 percent of the whole world every year, and the incidence rate of the primary liver cancer is 28.71/10 ten thousand according to the incidence situation of national malignant tumors released by the Chinese tumor registration center. Risk factors for the development of hepatocellular carcinoma are: hepatitis b virus infection, hepatitis c virus infection, excessive alcohol intake, smoking, aflatoxins, dietary factors (such as high iron intake), non-alcoholic fatty liver disease, genetic predisposition, diabetes, obesity, and related metabolic syndromes. 80% of hepatocellular carcinoma in China is related to hepatitis B virus infection, while in Europe, america and other countries, the incidence of hepatocellular carcinoma is mainly related to hepatitis C virus infection. Hepatocellular carcinoma is occult in disease, symptoms are not obvious, most patients lose precious chance of surgical treatment once the patients have discovered that the patients have advanced, and radical excision cannot be performed. Meanwhile, hepatocellular carcinoma has the characteristics of high malignancy, early metastasis, easy recurrence, insensitivity to radiotherapy and chemotherapy and the like.

The survival rate of the patients with HCC in China is lower, the 5-year survival rate is lower than 15%, the operation is the first choice treatment method for HCC in early stage, but the early diagnosis rate of HCC in China is lower, about 20%, most patients are diagnosed in middle and advanced stage, and the operation chance is not available. With the development and progress of treatment means such as surgical excision, radio frequency ablation, interventional therapy and liver transplantation, the cure rate of limited and early hepatocellular carcinoma is effectively improved. With the research and development of molecular targeted therapy, new molecular targeted drugs are emerging, and compared with traditional chemotherapy drugs, the molecular targeted drugs show higher tissue specificity, better therapeutic effect and lower adverse reactions. Since the approval of sorafenib for the treatment of HCC in 2007, most clinical studies have failed over 10 years. And regorafenib, ranvatinib, cabozantinib, ramucirumab, nivolumizumab and the like are successfully obtained in clinical research, and provide new selection and direction for HCC systemic treatment. Molecular targeted drug therapy has significant efficacy in specific patients, and patients benefit from survival. However, the application of targeted drugs has limitations, on one hand, the effective targets of the existing targeted drugs are fewer, and on the other hand, before selecting appropriate targeted therapeutic drugs, corresponding molecular target detection needs to be carried out on patients. At present, aiming at HCC patients, the main targeting drugs are nivolumetriab and palivizumab taking programmed cell death receptor-1 (PD-1) as a target point and tremelimumab taking cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) as a target point.

Therefore, the search for new targeted drugs for treating liver cancer has important practical significance and application value.

Mucin13 (mucin 13, MUC 13) is a membrane-bound glycosylated protein that modulates the biological activities of a variety of cells under physiological and pathological conditions. MUC13 is up-regulated in various malignant tumor tissues, can promote the proliferation, migration and invasion of tumor cells by activating various signal pathways, and is related to tumor drug resistance. The human MUC13 gene is located on a chromosome 3q21.2, contains 12 exons, has the length of 2899bp of mRNA and codes 511 amino acids. The MUC13 protein is composed of two different subunits of alpha and beta, and comprises a plurality of domains with different functions, such as an N-terminal signal peptide Tandem Repeat (TR) domain, a SEA cucumber protein enzyme and agrin (SEA) domain, 3 Epidermal Growth Factor (EGF) like domains, a single-plex sequence Transmembrane (TM) domain and a cytoplasmic tail domain. The expression level of MUC13 mRNA and protein in liver cancer tissue was significantly higher than that of matched non-cancer tissue, and high MUC13 expression was detected in 74 (44.0%) of 168 liver cancer patients, whose expression level correlated with tumor size, TNM staging, vascular invasion, and poor prognosis. MUC13 is an important marker of HCC, and its expression level affects the prognosis of patients.

Smith et al proposed Phage Display Technology (PDT) in 1985; in 1988, the phage display peptide library was successfully constructed for the first time; from 1990 to date, phage display peptide libraries have been rapidly developed and applied. The principle of the phage display technology is that exogenous DNA is cloned to a proper phage vector through a genetic engineering technology, so that an expression product corresponding to an exogenous DNA fragment is fused on capsid protein of the phage to form fusion protein which is displayed on the surface of the phage, and displayed polypeptide or protein can keep relative spatial structure and biological activity; then washing off the phage which is not specifically combined by using the target molecule and adopting a proper elutriation method, and finally screening out the target phage which can be combined with the target molecule from the phage library; the foreign polypeptide or protein is expressed on the surface of the phage, and its encoding gene can be sequenced as part of the phage genome by phage DNA sequence. The technology has the remarkable characteristic of establishing the corresponding relation between the genotype and the phenotype. The phage display technology is suitable for preparing fully human antibody medicaments. Humira, an anti-TNF alpha, for the treatment of rheumatoid arthritis was the first fully human antibody drug produced using phage antibody library technology and approved by the U.S. food and drug administration. By 2014, 6 antibodies produced using phage antibody library technology approved by the U.S. food and drug administration, and 30 or more related drugs at the same time are in clinical trial. In addition to screening for production antibodies, phage antibody library technology can also be used to screen for the corresponding antigen. The phage display technology antibody library has the advantages of high storage capacity, high efficiency, convenience, flexible screening and the like, is widely applied to many fields of life science, and can be used as a favorable tool for screening non-small cell lung cancer surface antigen targeted antibodies particularly in the fields of tumor diagnosis, tumor antibody drug preparation and the like.

Disclosure of Invention

In order to overcome the defects and shortcomings of the prior art, the invention aims at providing the MUC13 antagonist polypeptide, which has high specificity affinity with the receptor MUC13, can block the signal path of MUC13 by combining with MUC13, plays an important role in the aspects of targeted inhibition of liver cancer cell proliferation, promotion of liver cancer cell apoptosis and the like, and has great application value in the aspect of targeted treatment of liver cancer.

Another object of the present invention is to provide derivatives of the aforementioned MUC13 antagonist polypeptide, which have a high specific affinity for the receptor MUC13 and specifically bind to MUC 13.

Still another object of the present invention is to provide the use of the MUC13 antagonist polypeptide and derivatives thereof.

In order to realize the task, the specific technical scheme is as follows:

the invention provides a mucin13 antagonistic polypeptide, the amino acid residue sequence of which is Leu-Leu-Leu-Leu-Phe-Ala-Tyr-Phe-Ser-Val-Cys-Thr, and the amino acid residue sequence is shown in SEQ ID No. 1.

The invention provides a mucin13 antagonistic polypeptide derivative, which is a product obtained by carrying out conventional modification on a side chain group, an amino terminal or a carboxyl terminal of an amino acid of the mucin13 antagonistic polypeptide; or is the product obtained by connecting the mucin13 antagonist polypeptide with a label;

the conventional modification is more than one of amination, amidation, hydroxylation, carboxylation, carbonylation, alkylation, acetylation, phosphorylation, esterification, glycosylation, cyclization, biotinylation, fluorescent group modification, polyethylene glycol modification and immobilization modification; preferably, the mucin13 antagonist polypeptide is terminally amidated;

the tag is used for polypeptide or protein detection or purification, and is preferably His ₆ More than one of a tag, GST tag, EGFP tag, MBP tag, nus tag, HA tag, igG tag, FLAG tag, c-Myc tag and ProfinityXact tag.

The mucin13 antagonist polypeptide is a hydrophobic polypeptide that is soluble in an aqueous solution containing 20wt% acetonitrile as determined by hydrophilicity analysis.

The mucin13 antagonist polypeptide and derivatives thereof can be derived from mammals or birds, such as primates (humans); rodents, including mice, rats, hamsters, rabbits, horses, cattle, dogs, cats, and the like.

The mucin13 antagonistic polypeptide and the derivative thereof are obtained by adopting a known method in the prior art and chemically synthesized by using an automatic polypeptide synthesizer; or deducing a nucleotide sequence from the short peptide sequence, and cloning the nucleotide sequence into a vector for biosynthesis; or by extensive extraction and purification from existing organisms.

The present invention also provides a polynucleotide encoding said mucin13 antagonist polypeptide or a derivative of said mucin13 antagonist polypeptide.

The invention also provides a vector containing the polynucleotide, wherein the vector contains the nucleotide, and can be connected with a promoter sequence by gene technical means.

The invention also provides a host cell or host bacterium containing the vector.

In another aspect, the invention provides a pharmaceutical composition comprising said mucin13 antagonist polypeptide and/or a derivative of said mucin13 antagonist polypeptide;

preferably, the pharmaceutical composition further comprises a therapeutic agent; the therapeutic agent is any substance with anti-tumor activity;

preferably, the pharmaceutical composition contains more than one pharmaceutically acceptable carrier; preferably, the pharmaceutically acceptable carrier is preferably a diluent, excipient, filler, binder, wetting agent, disintegrant, absorption enhancer, adsorption carrier, surfactant, lubricant, or the like.

The pharmaceutical composition can be further prepared into various forms such as tablets, granules, capsules, oral liquid or injection, and the like, and the medicines of various formulations can be prepared according to the conventional method in the pharmaceutical field.

The invention also provides the application of the mucin13 antagonist polypeptide and/or the mucin13 antagonist polypeptide derivative in the preparation of medicaments for inhibiting the proliferation of tumor cells with high expression of mucin13 or promoting the apoptosis of tumor cells with high expression of mucin 13;

preferably, the tumor cell highly expressing mucin13 is a liver cancer cell.

According to another aspect of the invention, there is provided the use of said mucin13 antagonist polypeptide and/or said mucin13 antagonist polypeptide derivative for the manufacture of a medicament for the treatment of a neoplastic disease state that is highly expressing mucin 13;

preferably, the tumor disease highly expressing mucin13 is selected from liver cancer, rectal cancer, colon cancer, cholangiocarcinoma, lung cancer and breast cancer; more preferably, the neoplastic disease is liver cancer.

The present invention also provides a detection reagent comprising the mucin13 antagonist polypeptide or the mucin13 antagonist polypeptide derivative.

The invention also provides an antibody to said mucin13 antagonist polypeptide or to a derivative of said mucin13 antagonist polypeptide.

The invention has the beneficial effects that:

(1) The MUC13 antagonistic polypeptide and the derivative thereof provided by the invention can be specifically combined with MUC13, and inhibit a MUC13 signal channel, thereby inhibiting the proliferation of tumor cells and promoting the apoptosis of the tumor cells.

(2) The MUC13 antagonistic polypeptide and the derivative thereof provided by the invention can be used as a biological polypeptide medicament of a MUC13 binding site, are used for preparing medicaments for preventing and/or treating tumors, solve the problems that the effective target of the current targeted medicament is less, and the corresponding molecular target detection needs to be carried out on a patient before the proper targeted treatment medicament is selected, and have great social and economic benefits.

Drawings

Figure 1 is a High Performance Liquid Chromatography (HPLC) assay of the MUC13 antagonist polypeptide of example 1;

FIG. 2 is a Mass Spectrometry (MS) analysis of the MUC13 antagonist polypeptide of example 1;

FIG. 3 is a graph showing the results of measuring the expression level of MUC13 in hepatoma cells in Experimental example 1; wherein, A: western blotting; b: protein expression amount statistics results;

FIG. 4 is a result of examining the effect of the MUC13 antagonist polypeptide on the proliferation potency of HCCLM3 cells of hepatoma carcinoma cells in Experimental example 1;

FIG. 5 shows the condition of MUC13 antagonist polypeptide promoting the apoptosis of HCCLM3 of hepatoma cells detected by Western Blotting method in Experimental example 2; wherein, A: detecting the change of apoptosis-related signal protein after HL12-MP1 action by Western Blotting; B. c and D are the expression level statistics of Bcl-2, bax and cleared Caspase-3 protein respectively;

FIG. 6 shows the results of the clone formation assay in Experimental example 3, in which MUC13 antagonist polypeptide inhibits the in vitro clone formation of HCCLM3 of hepatocarcinoma cells; wherein, A: detecting the cell clone forming ability by a clone experiment; b: and (5) counting the number of clones.

Detailed Description

In order that the invention may be more clearly understood, it will now be further described with reference to the following examples and the accompanying drawings. The examples are for illustration only and do not limit the invention in any way. In the examples, each raw reagent material is commercially available, and the experimental method not specifying the specific conditions is a conventional method and a conventional condition well known in the art, or a condition recommended by an instrument manufacturer.

Example 1: and (3) panning, amplifying, purifying, sequencing and synthesizing the MUC13 antagonist polypeptide HL12-MP1.

This example mainly aims to obtain a positive phage specifically bound to MUC13 by screening, amplify and purify the positive phage, extract phage single-stranded DNA (ssDNA) for sequencing, analyze and compare the obtained sequences, and finally synthesize a high-purity antagonist polypeptide, which is named HL12-MP1.

The method comprises the following specific steps:

1. establishing 293T cell line permanently expressing MUC 13: 293T-MUC13

(1) Vigorous growing human 293T cells were selected at 5X 10 days before transfection ⁵ One/well, inoculating in 6-well plate, culturing until the cell fusion degree is 60% after the second day;

(2) the transfection was performed on the next day by diluting 3. Mu.g of plasmid with 200. Mu.L of opti-MEM medium and 6. Mu.L of Lipofectamine2000 with 200. Mu.L of opti-MEM medium in units of one culture well of a 6-well plate, gently mixing the diluted solutions, and then leaving the mixture at room temperature for 5 minutes;

(3) gently mixing the two tube dilutions, standing for 20 minutes at room temperature, and gently adding 600. Mu.L of opti-MEM medium to the mixed dilution;

(4) gently rinsing the cells to be transfected once with PBS, then gently adding the mixed diluent into the culture hole, and placing the culture hole in a carbon dioxide incubator for culture;

(5) after culturing for 4-6 hours, abandoning the culture medium used for transfection, and adding 3mL of complete culture medium into the hole;

(6) after 48 hours, a culture medium containing 1 mu g/mL puromycin (puromycin) is selected for screening; obtaining the 293T cell line which stably expresses the MUC13 after the cell is not dead any more.

(7) Total RNA was extracted with TRIzol, 2. Mu.g of RNA was quantified for reverse transcription (reverse transcription kit, purchased from Promega) and qPCR was performed with specific primer sequences.

The sequence of the specific primer is Hu-MUC13 primer sequence:

fw 5 'ACAATGGTTCCTGTAAC-3' as shown in SEQ ID No: 2;

rv 5 'ACCCTTCTAAACACAGAGGCAA-3', as shown in SEQ ID No: 3.

(8) In comparison with the transfected pSM2c-Hu-scramble RNA, the high expression level of MUC13 was detected and designated: 293T-MUC13, i.e., can be used for positive phage selection.

2. Performing panning, amplification, purification, sequencing and synthesis of MUC13 antagonist polypeptide

(1) Preparation of ER2738 host bacterial liquid: performing aseptic technique operation, namely taking 200 mu L of LB-Tet liquid culture medium in a 1.5mL sterile centrifuge tube, taking 0.2 mu L of bacterial liquid from the glycerol frozen product of E.coli ER2738, fully and uniformly mixing the bacterial liquid with the glycerol frozen product, completely absorbing and coating the bacterial liquid on an LB-Tet plate, marking the plate, standing the plate at room temperature for 3min, and then placing the plate in a 37 ℃ constant temperature incubator for inversion overnight culture. Observing the next day, sealing with sealing film after the clone grows out, and storing at 4 deg.C in dark for use. Single colonies were picked aseptically with a sterile pipette tip and placed into 10mL sterile centrifuge tubes pre-filled with 3mL LB-Tet broth, labeled and shake-cultured overnight on a constant temperature shaker at 37 ℃ and 300 rpm/min. The next day, the bacterial amplification solution was stored at 4 ℃ until use. Taking 10mL of a sterilized centrifuge tube, adding 3mL of LB-Tet liquid culture medium in an aseptic operation, inoculating 30 mu L of overnight-cultured bacteria, carrying out shake culture at constant temperature of 37 ℃ and 300rpm/min for 2-3 h, wherein the bacteria are in an exponential growth phase and are in a mist shape (OD) by visual observation ₆₀₀ ～0.5)。

(2) Panning of MUC13 antagonist peptide: high expression MUC13 cells were treated as follows 10 ⁵ The culture dish is inoculated on 60X 15mm which is coated with polylysine in advance ² In a culture dish, when the cells are cultured to 80-90% in a conventional way, 1 mu L of eluent is firstly taken for elutriation (simultaneously, a cell line which does not express MUC13 is used as a blank control), and the rest eluent is added into 20mL of LB culture solution for amplificationThe amplification product is then purified and finally re-titrated, stored at 4 ℃ for a short period and an equal number of levels for the next round of panning, and the remaining amplification product is stored at-20 ℃ in 50% glycerol.

(3) Determination of phage titer: 4 sterilized 10mL centrifuge tubes were prepared for each phage dilution, and Top agar (agar Top) was melted in a microwave oven, 3mL Top agar was added to each tube, and a water bath was run at 45 ℃ until needed. For each dilution of phage, 1 LB/IPTG/Xgal plate was prepared and pre-warmed in a 37 ℃ incubator for use. Will OD ₆₀₀ Coli ER2738 E.coli 0.5 was aliquoted at phage dilution 200. Mu.L/tube and stored at 4 ℃ for future use. Taking 4 sterilized 1.5mL centrifuge tubes, respectively containing 100 μ L, 90 μ L LB-Tet culture medium, sucking 1 μ L of bacteriophage to be tested into 100 μ L LB-Tet culture medium, diluting according to 10 times gradient, respectively marking as 10 ^-1 、10 ^-2 、10 ^-3 、10 ^-4 Each dilution was gently shaken and mixed well and centrifuged instantaneously. Mix 10 μ L of each dilution of phage to be titrated with 200 μ L of e.coli ER2738, mix by gentle shaking, centrifuge instantaneously, incubate for 5min at room temperature. Quickly adding the mixed bacterial liquid into top agar, quickly shaking and uniformly mixing, immediately pouring into a preheated LB/IPTG/Xgal flat plate, uniformly flattening, cooling for 5min at room temperature, culturing in a constant-temperature incubator at 37 ℃, and inverting the flat plate for overnight culture.

(4) Amplification and purification of eluted phages: taking a 250mL conical flask, adding the overnight cultured ER2738 host bacterial liquid into 20mL of LB liquid culture medium according to the proportion of 1; then adding the phage liquid to be amplified into an erlenmeyer flask, and carrying out vigorous shaking culture at 37 ℃ and 250rpm for 4.5h; the culture was transferred to a 50mL centrifuge tube and centrifuged at 10,000rpm for 10min at 4 ℃. Transferring the supernatant into another clean centrifugal tube, and centrifuging again at 10,000rpm at 4 ℃ for 10min; transferring 80% of the supernatant into another clean centrifugal tube, adding 1/4 volume of PEG/NaCl, reversing, mixing uniformly, and precipitating at 4 ℃ overnight; the next day, the pellet was centrifuged at 12000rpm for 20min at 4 ℃. Carefully sucking the supernatant with a clean gun head, centrifuging at 12,000rpm at 4 deg.C for 1min, and removing the residual supernatant; the pellet was then resuspended in 1mL TBS and gently pipetted 100 times. Then transferring the suspension into a 2mL centrifuge tube, and centrifuging at 4 ℃ and 10000rpm for 5min to remove residual cells; adding 1/4 volume of PEG/NaCl into the supernatant, and incubating on ice for 60min for re-precipitation; taking out the centrifuge tube, centrifuging at 12000rpm at 4 deg.C for 20min, and removing supernatant; the pellet was resuspended in 200. Mu.L TBS and centrifuged at 10,000rpm for 1min at 4 ℃. The supernatant was transferred to another centrifuge tube. Short-term storage at 4 deg.C, or long-term storage at-20 deg.C with 50% glycerol. The amplification of the monoclonal phage comprises the steps of adding overnight cultured ER2738 host bacterial liquid into 2mL of LB liquid culture medium according to the proportion of 1 to 100, and carrying out vigorous shaking culture at 37 ℃ and 250rpm for 2h; selecting a plate with less than 100 plaques from the fourth round of titer plates by using a sterilizing toothpick, picking well-separated blue plaques, adding the blue plaques into a culture tube, and carrying out violent shake culture at 37 ℃ at 250r/min for 4.5 hours; the culture was then transferred to fresh centrifuge tubes and centrifuged at 10,000rpm for 30sec at 4 ℃. Transferring the supernatant into a fresh tube, and centrifuging once again; 80% of the supernatant was transferred to fresh centrifuge tubes and stored at 4 ℃ or stored with 50% glycerol for a long period at-20 ℃.

(5) Identification of M13 phage ssDNA by agarose gel electrophoresis: horizontally placing a gel forming mold, placing the selected comb, and reserving a space of 1mm between the bottom of the comb and the mold; weighing 1g of agarose for DNA electrophoresis, putting the agarose into a 250mL triangular flask, adding 100mL 1 XTAE buffer solution, uniformly mixing, putting the flask into a microwave oven, heating and boiling until the agarose is completely dissolved; and closing the induction cooker, taking out the triangular flask, cooling the triangular flask to room temperature (the flask can be held by hand for tolerance), adding 5 mu L of ethidium bromide, and pouring the gel solution into a rubber plate for paving after uniform mixing. The rubber plate used in the experiment needs about 100mL of rubber solution; after the gel is completely solidified at room temperature and takes about 30 minutes, pulling out the comb teeth, and putting the rubber plate into an electrophoresis tank; adding 1 XTAE buffer solution into the electrophoresis tank, wherein the buffer solution is preferably 2mm higher than the surface of the gel; diluting a sample by using a Loading buffer, adding the diluted sample into a rubber plate, paying attention to the fact that a suction head of a sample injector is just placed in a gel point sample hole, the gel cannot be punctured, and preventing the sample from overflowing out of the hole; switching on a power supply, adjusting the voltage to 50V, performing electrophoresis for 90min, taking out the gel plate, and observing the result under an ultraviolet lamp.

(6) ssDNA sequencing and sequence analysis: the extracted M13 phage ssDNA was sent to Shanghai Yiciji Biotechnology Ltd for DNA sequencing. Sequencing was followed by sequence analysis using Bioedit software. According to the analysis result, the sequence of the sample is Leu-Leu-Leu-Leu-Phe-Ala-Tyr-Phe-Ser-Val-Cys-Thr, expressed as HL12-MP1, and finally the short peptide is synthesized by Fei peptide Biotechnology Limited.

Synthesis and characterization of MUC13 antagonist Polypeptides

The peptide was synthesized by Fmoc solid phase synthesis (from Hippocampus Kogyo) using a CS936 peptide synthesizer (CSBio, USA) by the following steps:

(1) Deprotection: removing the protecting group of the amino group by using piperidine (piperidine, shanghai purple reagent factory);

(2) Activation and crosslinking: the carboxyl of the next amino acid is activated and dissolved by activator HBTU (HCTU/HITU) + NMM, and the activated monomer reacts with free amino to form peptide bond;

(3) And (3) circulation: (1) And (2) the two steps of reaction are repeatedly circulated until the synthesis of the whole peptide chain is finished;

(4) Elution and deprotection: eluting the column with different resin-removing solvents according to the residues contained in the peptide chain, wherein the protecting groups are eluted and deprotected by a deprotection agent (TFA);

(5) The synthesized short peptide is purified by a Varian Prostar210 purification column (VARIAN, USA), and a UV-Vis-detector which is Varian Prostar345 (VARIAN, USA) is adopted in the purification process;

(6) The purity is verified to reach more than 99% by adopting System Gold HPLC (Beckman company in America);

(7) The molecular weight of the synthesized short peptide was measured by Thermo Finnigan LCQ deca XPplus (Thermo Co., USA).

Figure 1 is a High Performance Liquid Chromatography (HPLC) assay of a MUC13 antagonist polypeptide showing that the purity of the synthesized MUC13 antagonist polypeptide is as high as 98.25%.

Figure 2 is a Mass Spectrometry (MS) analysis of the MUC13 antagonist polypeptide showing that the size of the synthesized MUC13 antagonist polypeptide is 1487.2Da, which is consistent with predicted values.

Experimental example 1: HL12-MP1 can obviously inhibit the proliferation of HCCLM3 of hepatoma cells

(1) Mixing HCCLM3 of hepatocarcinoma cell at 5 × 10 ³ Inoculating each well into a 96-well cell culture plate, culturing for 24h with the culture medium volume of 200 mu L per well, and then starving overnight;

(2) adding HL12-MP1 polypeptides with different concentration gradients (100. Mu.M, 50. Mu.M, 25. Mu.M, 12.5. Mu.M, 6.5. Mu.M, 3.25. Mu.M) to culture for 24 hours, 48 hours, 72 hours, respectively;

(3) adding 20 mu L of MTT (thiazole blue) working solution into each hole, and continuously putting the mixture into a carbon dioxide incubator to culture for 4 hours;

(4) the supernatant in the culture plate is discarded, 150 mu L DMSO (dimethyl sulfoxide) is added, the mixture is shaken for 10 minutes, the 490nm wavelength is selected on a microplate reader for detection, and the growth curve of the cells is drawn.

FIG. 3 shows the detection result of MUC13 expression level in liver cancer cells, and the results of western blot experiments show that liver cancer cells HepG2, huh7 and HCCLM3 all highly express MUC13, and the MUC13 expression level in HCCLM3 is the highest, and the liver cancer cells HCCLM3 are taken as an example to detect the effect of antagonistic polypeptide HL12-MP1. Fig. 4 is a detection result of the influence of HL12-MP1 short peptide on the proliferation ability of HCCLM3, and the results show that HL12-MP1 short peptide with different concentrations can significantly inhibit the proliferation of HCCLM3, and the effect of the short peptide on inhibiting the proliferation of HCCLM3 is more significant as the action time increases.

Experimental example 2: HL12-MP1 can promote apoptosis of HCCLM3 of liver cancer cell

(1) Mixing HCCLM3 of hepatocarcinoma cell at a ratio of 1.2 × 10 ⁶ Inoculating the cells/well into a 6-well cell culture plate, culturing for 24h with the volume of culture medium per well being 2mL, and then starving overnight;

(2) adding HL12-MP1 polypeptides with different concentration gradients (0 mu M and 12.5 mu M), respectively culturing for 48 hours, collecting protein samples, and performing western blotting detection.

FIG. 5 shows that the expression level of Bax and cleared Caspase-3 protein in HCCLM3 cells acted by MUC13 antagonist polypeptide HL12-MP1 is obviously increased, the expression level of Bcl-2 protein is obviously reduced, and MUC13 antagonist polypeptide HL12-MP1 can activate Caspase-3 mediated apoptosis pathway to promote apoptosis of liver cancer cells by promoting the expression of Bax and cleared Caspase-3 protein and inhibiting the expression of Bcl-2 protein through the Western Blotting method to detect the condition that HL12-MP1 short peptide promotes the apoptosis of liver cancer cells.

Experimental example 3: HL12-RP1 can obviously inhibit the in vitro clone formation capability of hepatoma cell HCCLM3

(1) Inoculating the HCCLM3 of the liver cancer cells into a 6-well cell culture plate at 1000/well, wherein the volume of a culture medium in each well is 1mL, culturing for 24h, and then starving overnight;

(2) adding HL12-MP1 polypeptide with different concentration gradients (12.5 mu M,25 mu M and 50 mu M) for culturing for 10 days;

(3) washing with PBS for 3 times, and fixing with 4% paraformaldehyde for 30min;

(4) washing with PBS for 3 times, adding 0.5% crystal violet, and dyeing for 1h;

(5) washed 3 times with PBS and photographed microscopically.

FIG. 6 shows the experimental results of HL12-MP1 short peptide inhibiting the in vitro cloning formation of liver cancer cell HCCLM3, and the results show that HL12-MP1 short peptide can obviously inhibit the cloning formation of liver cancer cell HCCLM3 along with the increase of the action concentration.

It will be appreciated by those skilled in the art that the use of the present invention is not limited to the specific applications described above. The invention is also not limited to the preferred embodiments thereof with respect to the specific elements and/or features described or depicted herein. It should be understood that the invention is not limited to the disclosed embodiment or embodiments, but is capable of numerous rearrangements, modifications and substitutions without departing from the scope of the invention as set forth and defined by the following claims.

SEQUENCE LISTING

<110> Shenzhen International institute for graduate of Qinghua university

<120> mucin13 antagonistic polypeptide, derivative and application thereof

<130> 2021

<160> 3

<170> PatentIn version 3.3

<210> 1

<211> 12

<212> PRT

<213> Artificial sequence

<400> 1

Leu Leu Leu Leu Phe Ala Tyr Phe Ser Val Cys Thr

1 5 10

<210> 2

<211> 20

<212> DNA

<213> Artificial sequence

<400> 2

acaatggttc cttctgaaac 20

<210> 3

<211> 20

<212> DNA

<213> Artificial sequence

<400> 3

acccttctaa acacaggcaa 20

Claims (14)

1. A mucin13 antagonist polypeptide, wherein the amino acid residue sequence of said mucin13 antagonist polypeptide is represented by SEQ ID No. 1.

2. A polynucleotide encoding the mucin13 antagonist polypeptide of claim 1.

3. A vector comprising the polynucleotide of claim 2.

4. A host cell or host bacterium comprising the vector of claim 3.

5. A pharmaceutical composition comprising the mucin13 antagonist polypeptide of claim 1.

6. The pharmaceutical composition of claim 5, further comprising a therapeutic agent; the therapeutic agent is any substance having anti-tumor activity.

7. The pharmaceutical composition of claim 5, wherein the pharmaceutical composition comprises one or more pharmaceutically acceptable carriers.

8. The pharmaceutical composition of claim 7, wherein the pharmaceutically acceptable carrier is a diluent, excipient, filler, binder, wetting agent, disintegrant, absorption enhancer, adsorptive carrier, surfactant, or lubricant.

9. The use of the mucin13 antagonist polypeptide of claim 1 in the manufacture of a medicament for inhibiting proliferation or promoting apoptosis of tumor cells that express mucin 13.

10. The use of claim 9, wherein said tumor cells highly expressing mucin13 are hepatoma cells.

11. Use of the mucin13 antagonist polypeptide of claim 1 in the manufacture of a medicament for the treatment of a tumor disease in which mucin13 is highly expressed.

12. The use according to claim 11, wherein the tumor disease highly expressing mucin13 is liver cancer, rectal cancer, colon cancer, cholangiocarcinoma, lung cancer or breast cancer.

13. The use according to claim 12, wherein the tumor disease highly expressing mucin13 is liver cancer.

14. A detection reagent comprising the mucin13 antagonist polypeptide of claim 1.

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