CN113214360B - RHAMM antagonistic polypeptide, derivative and application thereof - Google Patents
RHAMM antagonistic polypeptide, derivative and application thereof Download PDFInfo
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- CN113214360B CN113214360B CN202110459921.4A CN202110459921A CN113214360B CN 113214360 B CN113214360 B CN 113214360B CN 202110459921 A CN202110459921 A CN 202110459921A CN 113214360 B CN113214360 B CN 113214360B
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Abstract
The invention relates to a RHAMM antagonistic polypeptide, a derivative and an application thereof, and particularly discloses the RHAMM antagonistic polypeptide: the amino acid residue sequence of the RHAMM antagonist polypeptide HL12-RP1 is as follows: Leu-Ile-Leu-Arg-Cys-Arg-Arg-Ser-Phe-Ile-Gly-Tyr SEQ ID NO. 1. Also discloses the application of the RHAMM antagonistic polypeptide and the derivative thereof in resisting tumors. The invention provides a RHAMM antagonist polypeptide HL12-RP1 and derivatives thereof, wherein the antagonist polypeptide and the derivatives thereof can be specifically combined with RHAMM and specifically combined with RHAMM to inhibit a RHAMM signal path; the cell proliferation of the colorectal cancer is inhibited by blocking a RHAMM signal path, and the apoptosis of the colorectal cancer cell is promoted.
Description
Technical Field
The invention relates to the field of biotechnology and biomedicine, in particular to colorectal cancer target RHAMM receptor antagonist polypeptide HL12-RP1 and derivatives and application thereof.
Background
1.1 colorectal cancer
Colorectal cancer (CRC) is one of the most common malignancies in the world. The International Agency for Research on Cancer (IARC) report of the world health organization indicates that the incidence of colorectal Cancer is located at the 3 rd position of malignant tumor after lung Cancer and breast Cancer, and the mortality is located at the 4 th position of malignant tumor after lung Cancer, liver Cancer and stomach Cancer. Colorectal cancer incidence region distribution has obvious difference in the world, and the incidence rate in European and American regions is higher than that in Asian regions. The CRC incidence rate of China is 29/10 ten thousand, and the death rate is 14/10 ten thousand. However, with the continuous improvement of the living standard of the substance, the change of the living style, especially the change of the eating habit, the incidence rate of colorectal cancer in China shows a trend of gradually rising. In contrast, the incidence and fatality rate of colorectal cancer has been significantly reduced in the european and american countries over the last 20 years due to the early development of colorectal cancer screening. Compared with European and American countries, the colorectal cancer of Chinese presents the characteristics of three highs and one lowness: 1. the incidence rate of colon cancer is slightly lower than that of rectal cancer, but the incidence rate of colon cancer is in an increasing trend in recent years; 2. the low rectal cancer accounts for a higher proportion; 3. the proportion of young people is about 10-15%; 4. the early screening and diagnosis rate is low.
At present, the cause of colorectal cancer is not completely clear, but with the continuous development and research of medicine and molecular biology, the development and development of colorectal cancer are gradually realized as the evolution process of 'inflammation-hyperplasia-canceration' caused by the joint action of multiple genes and the participation of multiple signal pathways. Domestic and foreign researches find that gene mutation of K-ras, methylation of DNA molecules and insulin-like growth factors are important reasons for promoting CRC generation and development. With the continuous research on CRC etiology, high-risk factors related to the CRC pathogenesis are gradually recognized, such as long-term high fat intake, nitrite compound food intake, vitamin deficiency, long-term alcoholism and other dietary factors; ulcerative colitis, colon adenoma, Familial Adenomatous Polyposis (FAP), and the like are diseases considered as precancerous lesions; factors such as age increase (> 60 years) and family inheritance are high risk factors for CRC.
At present, the treatment means of the colorectal cancer patient mainly comprises surgical treatment, chemical drug treatment and radioactive ray radiation treatment. Among them, surgery is currently the most important and effective means for colorectal cancer treatment, and radiotherapy and chemotherapy have become important components of adjuvant therapy for colorectal cancer. Meanwhile, therapeutic means such as targeted therapy, gene therapy, neoadjuvant chemotherapy, immunotherapy, etc. are also increasingly applied. Molecular targeted drug therapy has significant efficacy in specific patients, and patients benefit from survival. However, the application of targeted drugs is limited mainly because the effective targets of the existing targeted drugs are few, and the corresponding molecular target detection needs to be carried out on patients before the appropriate targeted therapeutic drugs are selected. Currently, for CRC patients, the main two targeted drugs are bevacizumab targeting Vascular Endothelial Growth Factor (VEGF) and cetuximab targeting Epidermal Growth Factor Receptor (EGFR). The study of domestic scholars suggests that patients with advanced and late colorectal cancer benefit from using cetuximab and bevacizumab for survival.
1.2 cancer-promoting factor RHAMM and tumors
The hyaluronic acid-mediated movement Receptor (RHAMM), also called CD168, was originally found to be a soluble protein secreted by migrating cells and capable of promoting cell movement and infiltration by interacting with Hyaluronic Acid (HA). RHAMM is an acidic coiled-coil protein that was first discovered in vertebrates and is not expressed in lower organisms and insects. RHAMM has no transmembrane domain nor signal peptide sequence. In general, RHAMM is localized primarily within cells, but can be secreted extracellularly under certain stimuli. The RHAMM has a full length of about 85kDa and consists of an N-terminal overbased HEAD region, a C-terminal TAIL region, and an a-helical region of more than about 600 amino acids in between. The RHAMM on the cell surface is used as a co-receptor of transmembrane membrane proteins to interact with HA, so that a signal cascade system in a cell is activated, and the RHAMM participates in intercellular adhesion and cell migration of normal cells and tumor cells. RHAMM was identified as a novel Microtubule Associated Protein (MAP) whose N-terminal basic sequence of amino acids 1-69 and the amino acid sequence encoded by exon 4 are binding sites for microtubules. If the sequence encoded by the N-terminal domain of RHAMM and the adjacent exon 4 is knocked out simultaneously, the interaction with microtubules is completely destroyed. The expression of intracellular RHAM is regulated by the cell cycle, and its expression curve is very similar to that of Cyclin B. RHAMM is expressed in high amount in G2/M phase and mitosis prophase, and changes periodically with cell cycle.
In recent years, the molecular mechanism of invasion and metastasis of malignant tumors has become a hot point of research. Cell-cell or cell-extracellular matrix interactions mediated by cell surface adhesion molecules are the biological basis for cancer cell invasion and thus metastasis. The extracellular matrix is an important component of HA, and promotes the migration of normal cells and the invasion of tumor cells by binding to corresponding receptors on the cell surface, so that the interaction between HA in the extracellular matrix and corresponding receptors on the surface of cancer cells is of great interest among many factors affecting the invasion and metastasis of cancer cells. Although RHAMM mRNA is difficult to detect in most homeostatic tissues, RHAMM is often consistently highly expressed in a variety of tumors. In some malignancies, including leukemia, breast cancer, melanoma, prostate cancer and ovarian cancer, RHAMM on the cell surface has been detected as a tumor antigen. Intracellular RHAMM expression levels are also elevated in brain tumors, gastric cancer, colorectal cancer, multiple myeloma, oral squamous cell carcinoma, endometrioma, and bladder cancer compared to normal tissues. RHAMM is usually highly expressed in advanced tumor stages, and its expression level is positively correlated with the stage of tumor and with the poor prognosis of some tumors. RHAMM was originally discovered as a secreted HA-binding protein, and extracellular RHAMM was able to activate cell surface transmembrane adhesion molecules and the pro-cell migration and infiltration functions of the HA receptor CD 44. This RHAMM-regulated activation process leads to an increase in cell surface CD44 expression and CD 44-mediated activation of ERK 1/2. In breast cancer tumorigenesis, extracellular HA binding mediated activation of CD44-RHAMM confers the potential for tumor malignancy. The strong association between the RHAMM-CD44 and RAS/ERK1/2 signaling pathways suggests that these HA receptor complexes can control signaling pathways that are overactivated in many tumors. RHAMM on the cell surface is capable of forming a complex with CD44 to function as a motor receptor during epithelial-mesenchymal transition (EMT) in invasive breast cancer cell lines. In addition, RHAMM on the cell surface can interact with RON, a tyrosine receptor kinase of the c-Met family. Thus, extracellular RHAMM acts with one or more adhesion/growth factor receptors on the cell surface to activate key signaling pathways, promoting tumor metastasis.
1.3 phage display technology
Phage Display Technologies (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 external 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, the fusion protein 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. Therefore, the antibody library of the phage display technology is widely applied in many fields of life science due to the advantages of high storage capacity, high efficiency, convenience, flexible screening and the like, and particularly receives more and more attention in the fields of tumor diagnosis, tumor antibody drug preparation and the like, and can be used as a targeted antibody tool for screening tumor surface antigens.
Disclosure of Invention
In order to overcome the defects and shortcomings of the prior art, the invention aims at providing the RHAMM antagonistic polypeptide obtained by screening the phage display library, wherein the antagonistic polypeptide has high specificity affinity with a receptor RHAMM, and can block a signal path of the RHAMM by combining with the RHAMM, so that the polypeptide plays an important role in the aspects of targeted inhibition of colorectal cancer cell proliferation, promotion of colorectal cancer cell apoptosis and the like, and has great application value in the aspect of targeted therapy of colorectal cancer.
Another object of the present invention is to provide derivatives of the RHAMM receptor antagonist polypeptide as defined above, which derivatives are also capable of having a high specific affinity for the RHAMM receptor and specifically binding to RHAMM.
The invention also aims to provide application of the RHAMM antagonistic polypeptide and derivatives thereof.
In order to realize the task, the invention adopts the following technical solution:
in one aspect of the invention, the RHAMM specific antagonistic polypeptide is obtained by screening a phage display random peptide library to obtain 1 polypeptide fragment, wherein the amino acid sequence HL12-RP1 is Leu-Ile-Leu-Arg-Cys-Arg-Ser-Phe-Ile-Gly-Tyr SEQ ID NO.1 (abbreviated as LILRCRRSFIGY).
The screening method of the RHAMM antagonistic polypeptide utilizes a phage random peptide library, firstly adopts RHAMM plasmid to transfect 293T cells to obtain a stable cell line permanently expressing RHAMM at high level, takes wild 293T cells as control adsorption cells, carries out 5 rounds of whole cell reduction screening, randomly picks 50 positive phages to amplify, extracts a clone single-stranded DNA and sequences. The basic characteristics of the amino acid sequence of the polypeptide are analyzed, polypeptide homology comparison is carried out, and a polypeptide motif with high occurrence frequency is searched.
Hydrophilicity analysis shows that HL12-RP1 is weak hydrophobic polypeptide;
in another aspect, the invention provides a derivative of RHAMM antagonist polypeptide, wherein the derivative of RHAMM antagonist polypeptide is a product obtained by performing conventional modification on the amino acid side chain group, amino terminal or carboxyl terminal of antagonist polypeptide, or a product obtained by connecting a tag for polypeptide or protein detection or purification to RHAMM antagonist polypeptide;
the conventional modification is amination, amidation, hydroxylation, carboxylation, carbonylation, alkylation, acetylation, phosphorylation, esterification, glycosylation, cyclization, biotinylation, fluorescent group modification, polyethylene glycol (PEG) modification or immobilization modification;
the label is His6GST, EGFP, MBP, Nus, HA, IgG, FLAG, c-Myc or ProfinityXact.
The RHAMM antagonist polypeptide and derivatives thereof may be derived from mammals or birds, such as primates (humans); rodents, including mice, rats, hamsters, rabbits, horses, cattle, canines, cats, and the like.
The derivative of the RHAMM antagonistic polypeptide is preferably: the end of the RHAMM antagonistic polypeptide is amidated and modified.
The RHAMM antagonistic polypeptide and the derivatives thereof are obtained by a known method in the prior art, and can be chemically synthesized by an automatic polypeptide synthesizer; deducing a nucleotide sequence from the short peptide sequence, and cloning the nucleotide sequence into a vector for biosynthesis; it can also be extracted and purified in large quantities from existing organisms.
In a further aspect, the invention provides a polynucleotide comprising a RHAMM antagonist polypeptide as defined above or a derivative thereof.
In yet another aspect, the invention provides a vector comprising a polynucleotide as described above.
In a further aspect the invention provides a host cell transfected with a vector as hereinbefore described.
In a further aspect the invention provides a medicament comprising a RHAMM antagonist polypeptide as hereinbefore described or a derivative as hereinbefore described.
The medicament can contain one or more pharmaceutically acceptable carriers;
the carrier is preferably a diluent, an excipient, a filler, an adhesive, a wetting agent, a disintegrating agent, an absorption enhancer, an adsorption carrier, a surfactant or a lubricant and the like;
the medicine can be further prepared into various forms such as tablets, granules, capsules, oral liquid or injection, and the medicines of various formulations can be prepared according to the conventional method in the pharmaceutical field.
Preferably, the pharmaceutically acceptable carrier is a diluent, excipient, filler, binder, wetting agent, disintegrant, absorption enhancer, adsorption carrier, surfactant or lubricant.
In a further aspect, the invention provides a test reagent comprising a RHAMM antagonist polypeptide as defined above or a derivative as defined in claim 2.
In a further aspect of the invention there is provided an antibody to a RHAMM antagonist polypeptide, such as the RHAMM antagonist polypeptide shown above, or a derivative of the RHAMM antagonist polypeptide, such as the derivative of claim 2.
In a further aspect, the invention provides the use of the RHAMM antagonist polypeptide as described above, or of a derivative of the RHAMM antagonist polypeptide as described above, in the preparation of a medicament for inhibiting proliferation of tumor cells highly expressing RHAMM, promoting apoptosis of tumor cells highly expressing RHAMM, inhibiting cell migration or inhibiting angiogenesis of tumor;
preferably, the tumor cells highly expressing RHAMM are selected from the group consisting of rectal cancer cells, colon cancer cells, brain glioma cells, gastric cancer cells, multiple myeloma cells, oral squamous cell carcinoma cells, endometrioma cells and bladder cancer cells.
In a further aspect, the invention provides the use of the RHAMM antagonist polypeptide as defined above, or of a derivative of the RHAMM antagonist polypeptide as defined above, for the preparation of a medicament for the treatment of a tumor disease in which RHAMM is highly expressed;
preferably, the tumor diseases with high expression of RHAMM are selected from rectal cancer, colon cancer, brain glioma, gastric cancer, multiple myeloma, oral squamous cell carcinoma, endometrioma and bladder cancer.
In the specific experiment of the invention, the cell 293T RHAMM which can specifically and highly express RHAMM can be prepared by using the RHAMM antagonist polypeptide HL12-RP1+/+And (4) combining.
In further experiments, the function of the RHAMM antagonist polypeptide HL12-RP1 is verified by using the colorectal cancer cell HCT-116 with high RHAMM expression as a cell model, and compared with a control group, HL12-RP1 can obviously inhibit the proliferation of the colorectal cancer cell HCT-116.
In another specific experiment of the invention, the RHAMM antagonist polypeptide HL12-RP1 is detected to promote the apoptosis of HCT-116 by adopting a flow cytometry technology.
Meanwhile, the detection in Western Blotting experiment shows that RHAMM antagonist polypeptide HL12-RP1 activates Caspase-3 mediated apoptosis pathway by promoting the expression of Bax and cleared Caspase-3 protein and inhibiting the expression of Bcl-2 protein.
Compared with the prior art, the invention has the following advantages and effects:
(1) the invention provides a RHAMM antagonist polypeptide HL12-RP1 and derivatives thereof, wherein the antagonist polypeptide and the derivatives thereof can be specifically combined with RHAMM and specifically combined with RHAMM to inhibit a RHAMM signal path.
(2) The RHAMM antagonistic polypeptide and the derivative thereof obtained by screening can inhibit the proliferation of colorectal cancer cells by blocking a RHAMM signal channel and promote the apoptosis of the colorectal cancer cells, can be used as biological polypeptide drugs of RHAMM binding sites, and can be used for preparing drugs for preventing and/or treating colorectal cancer. Can be widely applied in the medical and biological fields and can generate huge social and economic benefits.
Drawings
FIG. 1 shows Western blotting to detect the expression level of RHAMM in colorectal cancer cells. A: western blotting; b: and (5) counting the protein expression amount.
Figure 2 shows the purity of the RHAMM antagonist polypeptide as measured by HPLC.
Figure 3 shows MS mass spectrometry identification of the size of RHAMM antagonist polypeptides.
FIG. 4 shows the effect of the RHAMM antagonist polypeptide on the proliferative capacity of colorectal cancer cells HCT-116 measured by the MTT method.
FIG. 5 shows the detection of RHAMM antagonist polypeptide promoting the apoptosis of HCT-116 cells of colorectal cancer by cell flow technique. A: detecting apoptosis by a flow cytometer; b: western Blotting detects the change of apoptosis-related signal protein after HL12-RP1 action; c: counting apoptosis data; d, E and F are statistics of the expression level of Bcl-2, Bax and cleared Caspase-3 proteins respectively.
FIG. 6 shows the inhibition of migration of HCT-116 from colorectal cancer cells by rhaMM antagonist polypeptides as detected by the scratch test. A: detecting the migration capacity of the cells by a Transwell experiment; b: and counting the number of the migrated cells.
FIG. 7 shows the clonogenic assay to detect inhibition of the RHAMM antagonist polypeptide by colorectal cancer cell HCT-116 in vitro clonogenic assays. A: detecting the cell clone forming ability by a clone experiment; b: and (5) counting the number of clones.
FIG. 8 shows the ability of RHAMM antagonist polypeptides to inhibit HUVEC angiogenesis as measured by the HUVEC angiogenesis assay. A: HUVEC angiogenesis test to detect the angiogenesis ability; b: is the average length of blood vessels formed by HUVECs after administration; c, is the ratio of the area of HUVEC vascularized after administration; d, is the branching density of the HUVECs vascularized after administration.
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: elutriating, amplifying, purifying, sequencing and synthesizing RHAMM antagonist polypeptide HL12-RP 1.
The embodiment mainly aims to obtain the positive phage specifically combined with RHAMM through screening, amplifying and purifying the positive phage, extracting phage single-stranded DNA (ssDNA) for sequencing, analyzing and comparing the obtained sequences, and finally synthesizing the antagonist polypeptide HL12-RP1 with high purity.
The method comprises the following specific steps:
example 1 Western blotting to detect the expression level of RHAMM protein in different cancer cells
293T, colorectal cancer cells Caco2, HCT-116 and LOVO cells were taken. Washing with precooled PBS for three times, adding cell lysate (RIPA: PMSF ═ 100:1), incubating at 4 deg.C for 40min, collecting protein, centrifuging at 4 deg.C in a refrigerated centrifuge for 5min (12000g/min), and adding 5 × Buffer to obtain protein sample. After the protein sample was subjected to electrophoresis and membrane transfer to obtain a PVDF membrane containing proteins, the PVDF membrane was blocked with TBST containing 5% by mass of skimmed milk powder at room temperature for 1 hour, and then RHAMM antibody (ABClonal, A12445) and β -actin antibody (ABClonal, AC026) 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-rabbit secondary antibody (1:2000), the color was developed with ECL kit, and the results were analyzed with Quantity-One software.
As shown in FIG. 1, it can be seen that the RHAMM receptor is underexpressed in 293T, and is highly expressed in colorectal cancer cells Caco2, HCT-116 and LOVO.
Example 2 screening, Synthesis and characterization of RHAMM antagonistic Polypeptides
1. Screening of RHAMM antagonistic Polypeptides
(1) Establishing a 293T cell line with permanently high expression of RHAM: 293T-RHAMM+/+:
Selecting luminous human 293T cell with vigorous growth, and culturing at 5X 10/day before transfection5One/well, inoculating in 6-well plate, culturing until the cell fusion degree is 60% after the second day;
② the second day, with 6 hole plate culture hole as a unit, using 200 u L of opti-MEM medium dilution 3 u g plasmid, another 200 u L opti-MEM medium dilution 6 u L liposome Lipofectamine2000, after gently mixing, placed at room temperature for 5 minutes;
③ mixing the two tube dilutions gently, standing for 20 minutes at room temperature, and then adding 600 μ L of opti-MEM culture medium gently into the mixed dilutions;
rinsing the cells to be transfected with PBS slightly once, then adding the mixed diluent into the culture holes slightly, and culturing in a carbon dioxide incubator;
fifthly, after culturing for 4-6 hours, abandoning the culture medium used for transfection, and adding 3mL of complete culture medium into the hole;
sixthly, selecting a culture medium containing 1 mu g/mL puromycin (puromycin) for screening after 48 hours; obtaining the 293T cell line for stably expressing the RHAMM after the cells are not dead any more.
Seventhly, extracting total RNA by using TRIzol, quantifying 2 mug of RNA for reverse transcription (a reverse transcription kit, purchased from Promega corporation), and performing qPCR by using a specific primer sequence.
The specific primer sequence is Hu-RHAMM primer sequence:
Fw 5′-AGAACCAACTCAAGCAACAGG-3′SEQ ID NO.2
Rv 5′-AGGAGACGCCACTTGTTAATTTC-3′SEQ ID NO.3
(viii) comparing with the transfected pSM2c-Hu-scramble RNA, detecting the high expression level of RHAMM, and naming as: 293T-RHAMM+/+And the method can be used for screening positive phage.
(2) Performing panning, amplification, purification, sequencing and synthesis of RHAMM antagonistic polypeptide
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 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 ℃ for future use. Taking 10mL of a sterilized centrifuge tube, adding 3mL of LB-Tet liquid culture medium in a sterile operation manner, 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 fog shape (OD) by visual observation600～0.5)。
Elutriation of RHAMM antagonistic peptides: highly expressing RHAMM cells are treated as 105The culture dish is inoculated on 60X 15mm which is coated with polylysine in advance2In a culture dish, the cells are routinely cultured until the cell density is 80-90%, and the cells are used for elutriation (simultaneously)Using cell line not expressing RHAMM as blank control), collecting 1 μ L of eluate from each round, adding the rest into 20mL of LB culture solution, amplifying, purifying, measuring the amplified titer, storing the amplified product at 4 deg.C for a short period, collecting the same amount for the next round, and storing the rest in 50% glycerol at-20 deg.C.
And thirdly, measuring the titer of the phage, namely taking 4 sterilized 10mL centrifuge tubes, preparing 1 sterilized centrifuge tube for each phage dilution, melting Top agar (agar Top) by a microwave oven, adding 3mL Top agar into each tube, and carrying out water bath at 45 ℃ for later use. For each dilution of phage, 1 LB/IPTG/Xgal plate was prepared and pre-warmed in a 37 ℃ incubator for use. Will OD600Coli 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、10-4And each dilution is mixed evenly by gentle oscillation and then is centrifuged instantly. 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 plate, uniformly flattening, cooling at room temperature for 5min, and inversely culturing the plate in a constant-temperature incubator at 37 ℃ overnight.
Amplification and purification of eluted phage: taking a 250mL conical flask, adding the overnight cultured ER2738 host bacterial liquid into 20mL LB liquid culture medium according to the proportion of 1:100, and carrying out vigorous shaking culture at 37 ℃ and 250rpm for 2 h; then adding the phage liquid to be amplified into an erlenmeyer flask, and carrying out vigorous shaking culture at 37 ℃ and 250rpm for 4.5 h; the culture was transferred to a 50mL centrifuge tube and centrifuged at 10,000rpm at 4 ℃ for 10 min. Transferring the supernatant into another clean centrifugal tube, and centrifuging again at 10,000rpm at 4 ℃ for 10 min; transferring 80% of the supernatant into another clean centrifuge 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 4 deg.C and 12,000rpm 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 10,000rpm for 5min to remove residual cells; adding 1/4 volume of PEG/NaCl to the supernatant, and incubating on ice for 60min for reprecipitation; taking out the centrifuge tube, centrifuging at 4 deg.C and 12,000rpm 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 1:100, and carrying out vigorous shaking culture at 37 ℃ and 250rpm for 2 h; 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 ℃ and 250r/min for 4.5 h; the culture was then transferred to a fresh centrifuge tube 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 ℃.
Identifying M13 bacteriophage 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 Erlenmeyer 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 (3) closing the induction cooker, taking out the triangular flask, cooling the triangular flask to room temperature (which can be tolerated by holding the flask by hand), adding 5 mu L of ethidium bromide, and pouring the gel solution into a rubber plate paving plate after uniformly 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.
Sequencing and sequence analysis of ssDNA: the extracted M13 phage ssDNA was sent to Shanghai Yingji 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 Leu-Ile-Leu-Arg-Cys-Arg-Arg-Ser-Phe-Ile-Gly-Tyr SEQ ID NO.1 (abbreviated as: LILRCRRSFIGY), represented by HL12-RP1, and finally, the short peptide was synthesized by Qianzhou Biotech Co., Ltd.
(3) Synthesis and identification of RHAMM antagonist polypeptides
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 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 decap XPplus (Thermo Co., USA).
Fig. 2 shows the purity of the synthesized RHAMM antagonist polypeptide detected by HPLC, and the results show that the purity of the synthesized RHAMM antagonist polypeptide is as high as 98.22%.
FIG. 3 shows MS mass spectrometry of RHAMM antagonist polypeptide size, and the results show that the synthesized RHAMM antagonist polypeptide size is 1496.84Da
Example 3 HL12-RP1 can significantly inhibit the proliferation of colorectal cancer cell HCT-116
(ii) colorectal cancer cell HCT-116 was reduced by 5 x 103Inoculating 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;
adding HL12-RP1 polypeptide with different concentration gradients (100 mu M, 10 mu M, 1 mu M, 0.1 mu M, 0.01 mu M and 0.001 mu M) to culture for 24 hours, 48 hours and 72 hours respectively;
③ adding 20 mu L of MTT working solution into each hole, and continuously putting into a carbon dioxide incubator to culture for 4 hours;
and fourthly, abandoning the supernatant in the culture plate, adding 150 mu L DMSO (dimethyl sulfoxide), shaking for 10 minutes, selecting 490nm wavelength on a microplate reader for detection, and drawing a growth curve of the cells.
FIG. 4 shows that different concentrations of HL12-RP1 short peptide can obviously inhibit the proliferation of the colorectal cancer cell HCT-116, and the effect of the short peptide on inhibiting the proliferation of the colorectal cancer cell HCT-116 is more obvious along with the increase of the action time.
Example 4 HL12-RP1 can promote apoptosis of colorectal cancer cell HCT-116
(ii) colorectal cancer cell HCT-116 was reduced by 5 x 105Inoculating each cell/well in 6-well cell culture plates, culturing for 24h with the volume of culture medium per well being 1mL, and then starving overnight;
adding HL12-RP1 polypeptide with different concentration gradients (12.5 mu M, 25 mu M and 50 mu M) to culture for 24 hours, 48 hours and 72 hours respectively;
thirdly, collecting the supernatant into a centrifuge tube, and then carefully digesting and collecting the cell culture solution into the centrifuge tube by using pancreatin without EDTA. Centrifuging about 500g for 5 minutes to precipitate cells;
fourthly, washing the cells twice by using precooled PBS, and centrifuging about 500g for 5 minutes to collect the cells;
fifthly, 100 mu L of precooled 1 x annexin V Binding Buffer is added to resuspend the cells;
sixthly, adding 5 mu L annexin V-FITC and 5 mu L PI, mixing evenly, and reacting for 15 minutes in a dark place at room temperature;
seventhly, adding 400 mu L of precooled 1 x annexin V Binding Buffer, gently mixing, placing the sample on ice in a dark place, and detecting by a flow cytometer within 1 hour. And analyzing the detection result.
FIG. 5 shows that HL12-RP1 short peptide can obviously promote the apoptosis of HCT-116 of colorectal cancer cells along with the increase of action concentration through flow cytometry analysis.**P<0.01 compared to the control group. The RHAMM antagonist polypeptide HL12-RP1 promotes the expression of Bax and cleared Caspase-3 protein, inhibits the expression of Bcl-2 protein to activate an apoptosis pathway mediated by Caspase-3 so as to promote the apoptosis of colorectal cancer cells.
Example 4 HL12-RP1 can significantly inhibit the migration of HCT-116 in colorectal cancer cells
(r) Transwell upper ventricular seed 2 x 105Adding HL12-RP1 polypeptide with different concentration gradients (12.5 mu M, 25 mu M and 50 mu M) into each cell, and culturing for 24 hours;
② adding 800ul of culture medium containing 10 percent FBS into the lower chamber;
thirdly, washing the fabric with PBS for 3 times, and fixing the fabric with 4% paraformaldehyde for 30 min;
washing with PBS for 3 times, and dyeing with 0.5% crystal violet for 1 h;
fifthly, carefully scraping the upper layer cells by using a cotton swab, and taking a picture under a microscope
FIG. 6 shows that HL12-RP1 short peptide can obviously inhibit the migration of HCT-116 of colorectal cancer cells along with the increase of action concentration through a cell scratching experiment.**P<0.01 compared to the control group.
Example 5 HL12-RP1 can significantly inhibit the clonogenic capacity of colorectal cancer cell HCT-116 in vitro
Inoculating 1000 colorectal cancer cells HCT-116 into a 6-well cell culture plate, wherein the volume of a culture medium in each well is 1mL, culturing for 24 hours, and then starving overnight;
adding HL12-RP1 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 30 min;
washing with PBS for 3 times, adding 0.5% crystal violet and dyeing for 1 h;
wash with PBS 3 times, take pictures with microscope.
FIG. 7 shows that, through cloning experiments, HL12-RP1 short peptide can obviously inhibit the cloning formation of HCT-116 of colorectal cancer cells along with the increase of action concentration.**P<0.01 compared to the control group.
Example 6 HL12-RP1 can obviously inhibit the angiogenesis capacity of Human Umbilical Vein Endothelial Cells (HUVEC)
Firstly, paving a 24-hole plate by using Matrigel overnight;
② HUVEC cells 5 x 104Inoculating each cell/well into 24-well cell culture plate, adding HL12-RP1 polypeptide with different concentration gradients (25 μ M and 50 μ M) and culturing for 6 h;
wash 3 times with PBS and take pictures with microscope.
FIG. 8 shows the results of HUVEC angiogenesis, and the experimental results show that the RHAMM antagonist polypeptide has the ability to inhibit HUVEC angiogenesis. A: HUVEC angiogenesis test to detect the angiogenesis ability; b: is the average length of blood vessels formed by HUVECs after administration; c, is the ratio of the area of HUVEC vascularized after administration; d, is the branching density of the HUVECs vascularized after administration.
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> RHAMM antagonistic polypeptide, derivative and application thereof
<130> CP121010387
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 12
<212> PRT
<213> Artificial sequence
<400> 1
Leu Ile Leu Arg Cys Arg Arg Ser Phe Ile Gly Tyr
1 5 10
<210> 2
<211> 21
<212> DNA
<213> Artificial sequence
<400> 2
agaaccaact caagcaacag g 21
<210> 3
<211> 23
<212> DNA
<213> Artificial sequence
<400> 3
aggagacgcc acttgttaat ttc 23
Claims (13)
1. A RHAMM antagonist polypeptide comprising the amino acid residue sequence of HL12-RP 1:
Leu-Ile-Leu-Arg-Cys-Arg-Arg-Ser-Phe-Ile-Gly-Tyr SEQ ID NO.1。
2. a derivative of the RHAMM antagonist polypeptide of claim 1, characterized in that:
the derivative of the RHAMM antagonist polypeptide is a product obtained by performing conventional modification on the amino acid side chain group of the RHAMM antagonist polypeptide in claim 1, the amino terminal or the carboxyl terminal of the RHAMM antagonist polypeptide in claim 1, or a product obtained by connecting a tag for polypeptide or protein detection or purification to the RHAMM antagonist polypeptide in claim 1;
the conventional modification is amination, amidation, hydroxylation, carboxylation, carbonylation, alkylation, acetylation, phosphorylation, esterification, glycosylation, cyclization, biotinylation, fluorescent group modification, polyethylene glycol (PEG) modification or immobilization modification;
the label is His6GST, EGFP, MBP, Nus, HA, IgG, FLAG, c-Myc or ProfinityXact.
3. A polynucleotide encoding the RHAMM antagonist polypeptide of claim 1 or the derivative of claim 2.
4. A vector comprising the polynucleotide of claim 3.
5. A host cell transfected with the vector of claim 4.
6. A medicament comprising the RHAMM antagonist polypeptide of claim 1 or the derivative of claim 2.
7. The medicament of claim 6, wherein said medicament comprises one or more pharmaceutically acceptable carriers.
8. The pharmaceutical 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. A test agent comprising the RHAMM antagonist polypeptide of claim 1 or the derivative of claim 2.
10. Use of the RHAMM antagonist polypeptide of claim 1 or the derivative of the RHAMM antagonist polypeptide of claim 2 for the preparation of a medicament for inhibiting proliferation, promoting apoptosis, inhibiting migration or inhibiting angiogenesis of tumor cells highly expressing RHAMM.
11. The use of claim 10, wherein the tumor cells highly expressing RHAMM are selected from the group consisting of rectal cancer cells, colon cancer cells, brain glioma cells, gastric cancer cells, multiple myeloma cells, oral squamous cell carcinoma cells, endometrial tumor cells, and bladder cancer cells.
12. Use of the RHAMM antagonist polypeptide of claim 1 or a derivative of the RHAMM antagonist polypeptide of claim 2 for the preparation of a medicament for the treatment of a tumor disease highly expressing RHAMM.
13. The use of claim 12, wherein the tumor diseases with high expression of RHAMM are selected from rectal cancer, colon cancer, brain glioma, gastric cancer, multiple myeloma, oral squamous cell carcinoma, endometrioma and bladder cancer.
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Non-Patent Citations (2)
| Title |
|---|
| A truncated RHAMM protein for discovering novel therapeutic peptides;Alexandra Hauser-Kawaguchi,et al.;《Bioorg Med Chem》;20181001;第26卷(第18期);第5194-5203页 * |
| 非小细胞肺癌中RHAMM的表达与临床特征及预后的关系;闫明等;《广东医学》;20141130;第35卷(第21期);第3328-3330页 * |
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