CN113461783B - Podophyllin antagonistic polypeptide and derivative and application thereof - Google Patents

Abstract

The invention belongs to the field of biological medicines, and particularly relates to podophyllin antagonistic polypeptide, a derivative and an application thereof. The invention discloses a podophyllum antagonist polypeptide, the amino acid residue sequence of which is shown in SEQ ID No. 1. The podophyllum antagonist polypeptide derivative disclosed by the invention is a product obtained by conventionally modifying a side chain group, an amino end or a carboxyl end of amino acid of the podophyllum antagonist polypeptide, or a product obtained by connecting a label with the podophyllum antagonist polypeptide. The podophyllin antagonist polypeptide and the derivative thereof disclosed by the invention can be specifically combined with PDPN to block a downstream signal channel, so that the proliferation of tumor cells is inhibited, the apoptosis of the tumor cells is promoted, effective treatment micromolecule medicines are provided for tumors and the like, and the podophyllin antagonist polypeptide and the derivative thereof can be widely applied to the fields of medicine and biology.

Description

Podophyllin antagonistic polypeptide and derivative and application thereof

Technical Field

The invention relates to the field of biotechnology and biomedicine, in particular to podophyllin antagonist polypeptide and derivatives and application thereof.

Background

The incidence of malignant tumors (tumors) is in priority in disease development worldwide, seriously threatening human health. Melanoma, a highly malignant cancer type, varies widely in incidence and mortality worldwide. The data from the investigation of melanoma in 2012 showed that the incidence of melanoma varies from 0.2/10 million years in southeast asia to 7.7/10 million years in america.

Melanoma is a cancer that originates from melanocytes, a cell type in the skin that normally produces pigment, which originate in the neural crest during development. Melanocytes differentiation, survival and migration from neural crest precursors are largely dependent on the canonical Wnt signaling pathway (via β -catenin), c-kit receptor tyrosine kinase, and downstream transcription factors, such as MITF. Malignant transformation of melanocytes into melanoma is a result of the need for exogenous and endogenous triggers as well as the interaction of factors intrinsic to the tumor and immune-related factors. A prerequisite for the development of melanoma is the development of malignant melanoma after the hyperproliferation and abnormal differentiation of melanocytes under the influence of internal and external factors (race and inheritance, gene mutation, external wound stimulation, ultraviolet rays, etc.). Most cancers acquire the capacity for this malignant proliferation by modulating the signaling pathway of this process and continuing activation.

For melanoma patients, the mutation frequency of the BRAF gene in the melanocytes is 700 times higher than that of normal people. BRAF mutations are detected in approximately 50% of cases of melanoma, which is the most common genetic change in melanoma. The high frequency of such mutations and the "addiction" of melanoma cells to BRAF make it an excellent therapeutic target. To date, two drugs directed against BRAF, vitamin Mo Feini (vemurafenib) and dabrafenib (dabrafenib), have been accepted by the united states Food and Drug Administration (FDA) and are standard treatment regimens for advanced melanoma patients with BRAF mutations. However, patients receiving targeted therapy have an average Overall Survival (OS) of 14 months, while the first-line chemotherapeutic for melanoma is dacarbazine for 9 months. The rapid recurrence of tumors after treatment is due to the intrinsic or acquired resistance of melanoma to BRAF inhibitors. To overcome the resistance of targeted drugs, another MAPK pathway inhibitor, trametinib (MEK inhibitor), was approved in 2013. The combination of BRAF and MEK inhibitors (dabrafenib and trametinib or vilrafenib and cobinib) showed a longer survival period compared to BRAF inhibitor monotherapy.

In addition to genetic mutations, immune escape is another hallmark of the ongoing development of malignant cells, a strategy for cancer cells to evade the host immune response. Lymphocyte T recruitment is impaired due to failure of specific chemokine production, and activation of these cells is inhibited due to T cell anergy, inhibited by regulatory T cells or linked to negative regulatory receptors. The latter mechanism involves the expression of cytotoxic T lymphocyte-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1) proteins, and programmed death ligands-1, -2 (PD-L1, -2) on melanoma cells on T cells. Two drugs directed against the PD-1 molecule on T cells have been approved by the FDA for the treatment of advanced melanoma: ni Gu Lu monoclonal antibody and pembrolizumab. Because of their superior performance in clinical trials to ipilimumab, they have become the first choice for immunotherapy of melanoma. Both Overall Response Rates (ORR) and efficacy were similar. Almost 60% of patients treated with the nim Gu Lu mab survived for 2 years. Another immunotherapy approved by the FDA for the treatment of melanoma is talimogene laherparepvec (T-VEC). It is an engineered oncolytic herpes simplex type 1 virus in which the neurotoxic factor is stimulated by granulocyte-macrophage colony stimulating factor (GM-CSF). T-VECs are injected directly into melanoma tumors (regional or cutaneous metastases), inducing melanoma cell death, and are produced by local production of GM-CSF, which recruits antigen presenting cells into the tumor microenvironment, enhances dendritic cell function, and promotes cytotoxic T cell responses to tumor-associated antigens. In clinical trials, the objective response rate of T-VEC was 25%, with a lasting response in 16% of patients.

Podoplanin (PDPN), also known as platyhod protein, is a transmembrane glycoprotein expressed primarily in lymphatic endothelial cells and widely used as a marker of lymphatic endothelia and lymphangiogenesis. The structure of PDPN is composed of three parts, the extracellular region is composed of 162 amino acids and contains a large number of serine and threonine residues, wherein the PLAG domain is positioned between 29 and 54 amino acids of the amino acid sequence, the sequence is highly conserved among species, and the PLAG region is composed of three repeated sequences, wherein the PLAG3 mainly plays a role. PDPN, the main ligand of which is expressed on the surface of platelets, PDPN expressed by tumor cells can be combined with C-type lectin receptor (CLEC-2) indicated by the platelets to induce platelet activation and aggregation, and the aggregated platelets can wrap around the tumor cells to form a barrier between the tumor cells and immune cells, so that the tumor cells are protected from being attacked by the immune cells. In addition, after platelets bind to tumor cells, activated platelets may also secrete a number of cytokines that affect the activity of tumor cells and immune cells in the tumor microenvironment. In recent years, the PDPN protein is also expressed in some malignant tumor tissues including melanoma, squamous cell lung carcinoma, breast cancer, brain glioma and the like, the PDPN is up-regulated on the surfaces of various tumor cells and has close relation with the occurrence, development, invasion and metastasis of tumors, and the expression difference of the PDPN protein in different malignant tumors has good clinical guiding significance for the diagnosis, treatment and prognosis of the malignant tumors. At present, few biological polypeptide drugs for preventing and/or treating malignant tumors are reported by taking PDPN protein as a binding site.

Phage Display Technology (PDT) was proposed by Smith et al 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 obvious 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 targeted antibody tool for screening tumor surface antigens 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 primary object of the present invention is to provide a PDPN antagonist polypeptide obtained by screening a phage display library, wherein the antagonist polypeptide has high specificity affinity with a receptor PDPN, can block a signal pathway of PDPN by binding with PDPN, plays an important role in the aspects of targeted inhibition of proliferation, migration and invasion of melanoma cells, promotion of apoptosis of melanoma cells, and the like, and has great application value in the aspect of targeted therapy of melanoma.

Another object of the present invention is to provide a derivative of the PDPN antagonist polypeptide, which has a high specific affinity for PDPN and binds to PDPN.

The invention also aims to provide application of the PDPN antagonistic polypeptide and derivatives thereof.

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

the invention provides a podophyllotoxin antagonist polypeptide, wherein the amino acid residue sequence of the podophyllotoxin antagonist polypeptide is His-Met-Asp-Asn-Asn-Ala-Phe-Ser-Pro-Leu-Gly-Arg, and is shown in SEQ ID No. 1.

The invention provides a podophyllum antagonist polypeptide derivative, which is a product obtained by conventionally modifying a side chain group, an amino end or a carboxyl end of amino acid of the podophyllum antagonist polypeptide; or is a product obtained by connecting the podophyllum protein antagonistic 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 podophyllin 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 label, a GST label, an EGFP label, an MBP label, a Nus label, an HA label, an IgG label, a FLAG label, a c-Myc label and a ProfinityXact label.

The hydropathic analysis shows that the podophyllin antagonist polypeptide is a hydrophilic polypeptide.

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

The podophyllum protein 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 the nucleotide sequence from the short peptide sequence, and then cloning the short peptide sequence into a vector for biosynthesis; or by extensive extraction and purification from existing organisms.

The invention also provides a polynucleotide encoding the podophyllin antagonist polypeptide or the podophyllin antagonist polypeptide derivative.

The invention also provides a vector containing the polynucleotide.

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

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

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 application of the podophyllum protein antagonistic polypeptide and/or the podophyllum protein antagonistic polypeptide derivative in preparing a medicament for inhibiting proliferation of tumor cells with high expression of the podophyllum protein or promoting apoptosis of the tumor cells with high expression of the podophyllum protein;

preferably, the tumor cell highly expressing the pedigree protein is a melanoma cell.

The invention also provides the application of the podophyllum protein antagonistic polypeptide and/or the podophyllum protein antagonistic polypeptide derivative in preparing a medicament for treating tumor diseases with high expression of podophyllum protein;

preferably, the tumor disease with high expression of the podophyllin is melanoma, brain glioma, squamous cell lung cancer, skin cancer, breast cancer or oral squamous cell carcinoma; more preferably, the neoplastic disease is melanoma.

The invention also provides a detection reagent which comprises the podophyllin antagonist polypeptide or the podophyllin antagonist polypeptide derivative.

The invention also provides an antibody of the podophyllin antagonist polypeptide or the podophyllin antagonist polypeptide derivative.

The invention has the beneficial effects that:

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

(2) The PDPN antagonistic polypeptide and the derivative thereof obtained by screening can be used as a biological polypeptide medicament of a PDPN binding site by blocking a signal path of PDPN, are used for preparing medicaments for preventing and/or treating tumors, can be widely applied in the fields of medicine and biology, and generate huge social and economic benefits.

Drawings

FIG. 1 is a High Performance Liquid Chromatography (HPLC) assay of the PDPN antagonist polypeptide of example 1;

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

FIG. 3 shows the results of detecting the expression levels of PDPN mRNA and protein in melanoma cells A375 and A875 in Experimental example 1, wherein A is the mRNA level detected by RT-PCR, B is western blotting, and C is the statistical result of protein expression;

FIG. 4 is a graph showing the results of examining the effect of PDPN antagonist polypeptide on the proliferation ability of melanoma cells A375 and A875 in Experimental example 2;

FIG. 5 is a graph showing the results of examining the migration inhibitory activity of the PDPN antagonist polypeptide against melanoma cells A375 in Experimental example 3;

FIG. 6 shows the results of examining the ability of PDPN antagonist polypeptide of Experimental example 4 to inhibit the invasion of melanoma cell A375;

FIG. 7 is a graph showing the results of examining the ability of PDPN antagonist polypeptide of Experimental example 5 to inhibit the tumorigenicity of melanoma cells A375 in nude mice.

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) carrying out panning, amplification, purification, sequencing and synthesis on the PDPN antagonistic polypeptide CY12-RP1.

This example is mainly to obtain a positive phage specifically bound to PDPN by screening, amplify and purify the positive phage, extract phage single-stranded DNA (ssDNA) for sequencing, compare the obtained sequences, and finally synthesize a high-purity antagonist polypeptide CY12-RP1.

The method comprises the following specific steps:

1. establishing a 293T cell line with permanent high expression of PDPN: 293T-PDPN ^+/+ :

(1) The well-grown human 293T cells which can emit light are selected and transfected 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 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 one well of a 6-well plate, gently mixing them, and then leaving them at room temperature for 5 minutes;

(3) gently mixing the two tube dilutions, standing at room temperature for 20 minutes, 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 for stably expressing PDPN 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 corporation), and RT-PCR was performed with specific primer sequences.

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

forward 5'-TGACTCCAGGAACCAGCGAAG-3' as shown in SEQ ID No. 2;

reverse 5'-GCGAATGCCTGTTACACTGTTGA-3' as shown in SEQ ID No. 3.

(8) High expression levels of PDPN were detected in comparison to pSM2c-Hu-scramble RNA transfected and designated: 293T-PDPN ^+/+ And the method can be used for screening positive phage.

2. Performing panning, amplification, purification, sequencing and synthesis of PDPN antagonistic polypeptide

(1) Preparation of ER2738 host bacterial liquid: performing aseptic technique operation, namely taking 200 mul of LB-Tet liquid culture medium in a 1.5ml sterilized centrifugal tube, taking 0.2 mul 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 for 3min at room temperature, and then placing the plate in a constant temperature incubator at 37 ℃ for inversion overnight culture. Observing the next day, sealing with sealing film after clone grows out, and storing at 4 deg.C in dark for use. Single colonies were picked by aseptic technique using a sterilized tip, placed in 10ml sterilized centrifuge tubes previously supplemented with 3ml LB-Tet broth, labeled and cultured overnight on a constant temperature shaker at 37 ℃ under shaking at 300 rpm/min. The next day, the bacterial amplification solution was stored at 4 ℃ for future 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 PDPN antagonist peptides: high expression PDPN cells are expressed according to 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 taken for elutriation (meanwhile, a cell line which does not express PDPN is used as a blank control) in each round to measure the titer, the rest eluent is added into 20ml of LB culture solution for amplification, then purification and the titer after amplification are measured, an amplified product is stored at 4 ℃ for a short time, the same magnitude is taken for next round of elutriation, and the rest amplified product is stored at-20 ℃ by using 50% of glycerol.

(3) The titer of the phage was determined by taking 4 sterilized 10ml centrifuge tubes, preparing 1 sterilized centrifuge tube for each phage dilution, melting Top agar (agar Top) in a microwave oven, adding 3ml Top agar to each tube, and using a water bath at 45 ℃. Prepare 1 block LB/IPTG @ for each phage dilutionXgal plate, 37 ℃ incubator preheating standby. Will OD ₆₀₀ Coli ER2738 E.coli 0.5 was aliquoted at 200. Mu.l phage dilution per tube and stored at 4 ℃ until use. Taking 4 sterilized 1.5ml centrifuge tubes, respectively containing 100. Mu.l, 90. Mu.l LB-Tet culture medium, sucking 1. Mu.l of bacteriophage to be tested into 100. Mu.l LB-Tet culture medium, diluting according to 10-fold 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 12,000rpm for 20min at 4 ℃. Carefully sucking the supernatant with a clean gun head, centrifuging at 12,000rpm at 4 ℃ 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 10,000rpm at 4 ℃ for 5min to remove residual cells; adding 1/4 volume of PEG/NaCl into the supernatant, and incubating on ice for 60min for reprecipitation; taking out the centrifuge tube, centrifuging at 12,000rpm 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 of 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, preferably 2mm higher than the surface of the gel; diluting the sample with a Loading buffer, adding the diluted sample into a gel plate, and paying attention to that a suction head of a sample injector is just placed in a gel point sample hole, the gel cannot be punctured, and the sample is prevented 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. As a result of analysis, the sequence of the sample was His-Met-Asp-Asn-Asn-Ala-Phe-Ser-Pro-Leu-Gly-Arg (abbreviated as HMDNNAFSPLGR), and CY12-RP1, and finally, a short peptide was synthesized by Qiaozhou Biotech Co., ltd.

Synthesis and characterization of PDPN antagonist polypeptide

The polypeptide was synthesized by Fmoc solid phase synthesis (synthesized by Shanghai Qiaozao Bio Inc.) using a CS936 polypeptide synthesizer (CSBio Inc., USA), and the synthesis process included 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 an activator HBTU (HCTU/HITU) + NMM, and the activated monomer reacts with free amino to form peptide bonds;

(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 be more than 99% by adopting System Gold HPLC (Beckmann company, USA);

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

FIG. 1 is a High Performance Liquid Chromatography (HPLC) assay of PDPN antagonist polypeptides, the HPLC chromatographic conditions are as follows:

HPLC results show that the purity of the synthesized PDPN antagonistic polypeptide reaches 98.63%.

FIG. 2 is a Mass Spectrometric (MS) detection of a PDPN antagonist polypeptide under the following MS mass spectrometric conditions:

Ion Source：ESI Nebulizer Gas(NEB)：12.00

Curtain Gas(CUR)：6.00 lonspray Voltage(IS)：±4500

Temperature(TEM)：0.00 Run Time：0.5-1min

MS results show that the size of the synthesized PDPN antagonistic polypeptide is 1358.51Da, and the molecular weight is consistent with a predicted value.

Experimental example 1: expression levels of PDPN mRNA and protein in melanoma cells

(1) Two melanoma cell lines A375 and A875 were selected, total RNA was extracted using TRIzol, 2. Mu.g of RNA was quantitated for reverse transcription (reverse transcription kit, available from Promega corporation), and RT-PCR was performed using specific primer sequences.

The sequence of the PDPN specific primer is as follows:

forward:5'-TGACTCCAGGAACCAGCGAAG-3', as shown in SEQ ID No:2 is shown in the specification;

reverse:5'-GCGAATGCCTGTTACACTGTTGA-3', as shown in SEQ ID No:3, respectively.

(2) Collecting melanoma cells A375 and A875 cells, adding precooled PBS, washing for three times, adding cell lysate (RIPA: PMSF = 100: 1), incubating at 4 ℃ for 40min, collecting protein, placing in a 4 ℃ refrigerated centrifuge, centrifuging for 5min (12000 g/min), adding 5 x Buffer, and preparing into protein sample. The protein sample was subjected to electrophoresis and membrane transfer to obtain a PVDF membrane containing the protein, which was blocked at room temperature for 1 hour with TBST containing 5% by mass of skim milk powder, and then the PDPN antibody (Biolegend, 916605) and the β -actin antibody (ABClonal, AC 026) were diluted with TBST containing 5% by mass of BSA, and the above primary antibody was added thereto, followed by incubation at 4 ℃ overnight. The next day, after addition of goat anti-mouse secondary antibody (1.

The results are shown in fig. 3, wherein a is mRNA level detected by RT-PCR, B is Western blotting Western blot, and C is statistical result of protein expression, and it can be seen that PDPN is highly expressed in mRNA and protein levels in melanoma cells a375 and a 875.

Experimental example 2: CY12-RP1 can remarkably inhibit the proliferation of melanoma cells A375

(1) Melanoma cells A375 x 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 CY12-RP1 polypeptide with different concentration gradients (100. Mu.M, 50. Mu.M, 25. Mu.M, 10. Mu.M, 5. Mu.M, 1. Mu.M) for culturing for 48h;

(3) adding 10 mu L of CCK8 working solution into each hole, and continuously putting into a carbon dioxide incubator to culture for 1-2h;

(4) and (4) taking out the 96-well plate, slightly shaking the 96-well plate on a shaking table for 2-3min, selecting a wavelength of 450nm on a microplate reader for detection, and drawing a growth curve of the cells.

Fig. 4 is a result of examining the effect of PDPN antagonist polypeptide on the proliferation ability of melanoma cells a375 and a875, and the result shows that different concentrations of CY12-RP1 short peptide can significantly inhibit the proliferation of melanoma cells a375 and a 875.

Experimental example 3: CY12-RP1 can remarkably inhibit the migration of melanoma cells A375

(1) Melanoma cell line A375 was inoculated into the upper chamber of Transwell according to 5 x 10 ⁴ Adding CY12-RP1 polypeptide with different concentration gradients (0. Mu.M, 5. Mu.M and 10. Mu.M) into each cell/well, and culturing for 48h;

(2) the lower chamber was filled with 800ul of medium containing 10% FBS;

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

(4) washing with PBS for 3 times, and staining with 0.5% crystal violet for 1 hr;

(5) carefully scraping the upper layer of cells by using a cotton swab, and taking a picture under a microscope;

FIG. 5 shows the result of testing the migration ability of PDPN antagonist polypeptide to inhibit melanoma cells A375, and it was found by Transwell experiment that CY12-RP1 can significantly inhibit the migration ability of melanoma cells A375 with the increase of action concentration.

Experimental example 4: CY12-RP1 can obviously inhibit the invasive capability of a melanoma cell line A375

(1) Transwell upper chamber as per 1:8 proportion of Matrigel was previously laid out and the next day 5 x 10 inoculation was carried out in the Transwell upper chamber ⁴ Melanoma cells A375, were cultured by adding different concentrations of A375 (0. Mu.M, 5. Mu.M, 10. Mu.M) CY12-RP1 polypeptide to 2% FBS-containing medium for 48h;

(2) the lower chamber was filled with 800ul of medium containing 10% FBS;

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

(4) washing with PBS for 3 times, and staining with 0.1% crystal violet for 1h;

(5) the upper cells were carefully scraped off with a cotton swab and photographed under a microscope.

FIG. 6 shows the result of testing the ability of PDPN antagonist polypeptide to inhibit the invasion of melanoma cell line A375, and it was found through Transwell experiments that CY12-RP1 can significantly inhibit the invasion of melanoma cell line A375 with the increase of action concentration.

Experimental example 5: CY12-RP1 can remarkably inhibit the tumorigenic capacity of A375

(1) Will be 3 x 10 ⁶ A375 melanoma cells were inoculated subcutaneously into about 18g of nude mice at 6 weeks, 5 per group;

(2) on the third day, tail vein administration treatment is carried out on tumor-bearing mice, namely a buffer group, a CY12-RP1 (50 mg/kg) group and a CY12-RP1 (100 mg/kg) group, wherein the administration frequency is 2 days/1 time;

(3) body weight and tumor size of tumor-bearing mice were measured 2 days/1 time.

FIG. 7 shows the result of testing the ability of PDPN antagonist polypeptide to inhibit the tumor formation of melanoma cells A375 in nude mice, and it can be seen from the tumor formation experiment in nude mice that CY12-RP1 can significantly inhibit the tumor formation of A375, and with the increase of concentration, CY12-RP1 has stronger tumor inhibition ability.

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 institute in Qinghua university

<120> podophyllin antagonistic polypeptide, and derivative and application thereof

<130> CP121010575C

<160> 3

<170> PatentIn version 3.3

<210> 1

<211> 12

<212> PRT

<213> Artificial sequence

<400> 1

His Met Asp Asn Asn Ala Phe Ser Pro Leu Gly Arg

1 5 10

<210> 2

<211> 21

<212> DNA

<213> Artificial sequence

<400> 2

tgactccagg aaccagcgaa g 21

<210> 3

<211> 23

<212> DNA

<213> Artificial sequence

<400> 3

gcgaatgcct gttacactgt tga 23

Claims (14)

1. A podophyllum antagonist polypeptide is characterized in that the amino acid residue sequence of the podophyllum antagonist polypeptide is shown as SEQ ID No. 1.

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

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

7. The pharmaceutical composition of claim 6, wherein the pharmaceutical composition comprises more than one pharmaceutically acceptable carrier.

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 podophyllin antagonist polypeptide of claim 1 in the preparation of a medicament for inhibiting proliferation of tumor cells highly expressing podophyllin or promoting apoptosis of tumor cells highly expressing podophyllin.

10. The use according to claim 9, wherein the tumor cells highly expressing the pediocin are melanoma cells.

11. Use of the podophyllin antagonist polypeptide of claim 1 in the preparation of a medicament for the treatment of neoplastic disease in which the podophyllin is highly expressed.

12. The use according to claim 11, wherein the tumor disease highly expressing podophyllin is melanoma, brain glioma, squamous cell lung carcinoma, skin cancer, breast cancer or oral squamous cell carcinoma.

13. The use according to claim 12, wherein the tumor disease highly expressing pediocin is melanoma.

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

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