CN112920258A - CD44 antagonistic polypeptide and derivative and application thereof - Google Patents
CD44 antagonistic polypeptide and derivative and application thereof Download PDFInfo
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Abstract
The invention relates to a CD44 antagonist polypeptide, a derivative and an application thereof, and particularly discloses a CD44 antagonist polypeptide: the amino acid residue sequence of the CD44 antagonistic polypeptide HL7-ML1 is shown as SEQ ID No:1, SIPDYLFSHTML SEQ ID No: 1. the binding peptide and the derivatives thereof can bind to CD44, block a downstream signal path thereof through the binding with CD44, thereby inhibiting the proliferation of drug-resistant non-small cell cancer cells, promoting the apoptosis of the drug-resistant non-small cell cancer cells, providing effective treatment small molecule drugs for acquired drug-resistant non-small cell cancer patients and the like, and being widely applied in the fields of medicine and biology.
Description
Technical Field
The invention relates to the field of biotechnology and biomedicine, in particular to colorectal cancer target CD44 receptor antagonist polypeptide HL7-ML1 and derivatives and application thereof.
Background
Non small cell carcinoma
According to the investigation of the American cancer society in 2018, lung cancer is the malignant tumor with the highest morbidity (11.6%) and mortality (18.4%) worldwide, and in China, the lung cancer is the first malignant tumor, accounts for 78.7% and is far higher than other cancers.
Non-small cell lung cancer (NSCLC) patients in China account for about 85 percent of all lung cancers. About 75% of patients have been found to be in the middle and advanced stages, with a very low 5-year survival rate. Because the incubation period is long and the mortality rate is high, the non-small cell cancer becomes the cancer which is the most serious harm in China at present, which not only seriously affects the health of people, but also is a deep social problem.
In the face of severe conditions, the research and prevention of NSCLC need to be paid much attention. Early lung cancer treatment is mainly performed by operation, but due to the reasons of unobvious early symptoms and the like, about 80% of patients miss the optimal period of the operation when the patients are diagnosed, and the lung cancer has high recurrence rate and metastasis rate after operation, so that the treatment mainly performed by medicines is long in time and slow in effect, and seriously threatens the life health of the patients.
With the continuous development of NSCLC driver gene mutation research represented by EGFR, molecular targeted drugs, such as epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), have gradually gained medical attention and have been widely applied to clinical treatment of advanced NSCLC. However, acquired resistance developed after a period of treatment with EGFR-TKIs (9-13 months) and was one of the difficulties in treating advanced NSCLC. In recent years, relevant researches show that the enrichment of tumor stem cells and the EMT transformation are important reasons for the NSCLC patients to generate drug resistance to EGFR-TKIs, the mediated malignancy degree of the drug-resistant cells is far higher than that of the drug-resistant cells obtained due to T790M mutation and the like, and an effective targeted treatment method aiming at the tumor stem cells and the EMT is still absent. Therefore, the mechanism of acquired drug resistance of the tumor stem cells and the EMT mediated EGFR-TKIs is clarified, a new target point of reversed drug resistance is found, a targeted treatment strategy aiming at CSCs and EMT is established, the toxic and side effects of traditional treatment are reduced, the curative effect is improved, and the method has important theoretical significance and clinical value.
EMT is one of the main reasons for the acquired drug resistance of NSCLC EGFR-TKIs
EMT is an important biological process for malignant cells of epithelial origin to acquire the ability to migrate and invade. EMT can be excited by TGF-beta, AKT, Notch, NF-kB and other signal channels, and forms a signal network containing a plurality of positive feedback loops together with intracellular transcription factors such as Slug, Snail, Zeb1, Zeb2 and Twist, so as to reduce the expression of epithelial cell markers such as N-Cadherin, E-Cadherin and the like, and up-regulate the expression of mesenchymal markers such as Vimentin, fibronectin and the like, so that the epithelial cells are promoted to be transformed into mesenchymal cells, and the mesenchymal cells are further changed into mesenchymal stem cells.
Studies have demonstrated that EMT transformation is closely associated with acquired resistance to EGFR-TKIs. One recurrent focus puncture sample from erlotinib-acquired resistant patients indicates that acquisition of the interstitial phenotype is the primary cause of resistance; the EMT phenotype is also repeated in EGFR-TKIs drug-resistant NSCLC cell strains HCC827/CLR and HCC4006/ER which are constructed in vitro; in addition, the analysis result of clinical samples of lung cancer patients relapsed after 67 cases of chemotherapy combined with EGFR-TKIs shows that lung cancer tissues insensitive to EGFR-TKIs often highly express mesenchymal markers Vimentin, fibronectin and the like, while patients highly expressing E-cadherin have longer PFS; after EMT-developing lung cancer re-expresses E-cadherin, tumor cells can restore sensitivity to treatment with EGFR-TKIs. Patients positive for EGFR mutations who take oxitinib also face the problem of secondary resistance. An Oscetinic drug-resistant H1975 cell strain (a drug-resistant cell strain containing a T790 mutation) is constructed in vitro, and the EMT transformation is found to be the main reason for generating secondary drug resistance; on the other hand, patients who acquire EGFR-TKI resistance through the EMT pathway progress faster and have worse prognosis than those who are resistant due to the T790M mutation. These results all demonstrate that EMT is the major cause of acquired resistance to EGFR-TKIs in NSCLC patients and is associated with poor prognosis in the patients. However, the mechanism by which EMT mediates resistance to EGFR-TKIs is not clear. The research clarifies the important role of EMT in the process of generating EGFR-TKIs by constructing an EGFR-TKIs drug-resistant NSNLC cell model.
The enrichment of tumor stem cells is the main reason for the drug resistance after EMT transformation
CSCs are a group of malignant tumor cells with characteristics of self-renewal, high tumorigenicity, differentiation potential, high drug resistance, and the like. CSCs have multiple resistance mechanisms, mainly including: 1 high expression of drug efflux caused by the ATP-binding transporters ABCG2 and MDR1, etc.; enhanced DNA damage repair capacity; o 3 high expression of anti-apoptotic genes; o4 is more in the resting stage and insensitive to common chemotherapeutic drugs, which is the root cause that CSCs can escape the existing tumor chemoradiotherapy means, resulting in tumor recurrence and metastasis.
Recent studies have shown that the formation of CSCs is regulated by EMT, suggesting that EMT may be an important factor in the maintenance of the dryness of CSCs. The proportion of tumor stem cells is increased after the immortalized human mammary epithelial cells (HLMEs) are induced to generate EMT; tumor stem cells isolated from HLMEs and human breast cancer cells, both with EMT phenotype [10 ]; the content of CSCs in the tumor cells is increased in the processes of invasion and EMT transformation; TGF-beta 1 induces NSCLC cells which generate EMT and has strong stem cell characteristics. Taken together, the enrichment of CSCs is the underlying cause of EMT-mediated resistance in tumor cells. The targeted elimination of these CSCs and the regulation of the associated links will help to inhibit metastasis of cancer cells. However, due to the lack of effective strategies for targeted killing of CSCs, it is difficult to directly kill CSCs, and the present subject attempts to establish new therapeutic strategies through research on the biological properties of CSCs, and related signal pathways and cell regulation mechanisms, thereby providing new ideas for targeted killing of CSCs.
CD44 plays an important role in the self-renewal of CSCs and the development of multiple drug resistance
CD44 is a transmembrane glycoprotein belonging to the Hyaluronic Acid (HA) receptor among adhesion molecules, widely distributed in the human body, involved in many biological processes, and its high expression is closely related to the poor prognosis of patients. CD44 is mostly in a quiescent state on normal cells, and HAs no binding activity to Hyaluronic Acid (HA), whereas it is in a highly active state on CSCs, and participates in regulating self-renewal, sternness maintenance, EMT transformation, and various drug resistance production processes of CSCs by binding HA. Therefore, in biological and medical research, CD44 is used as a CSCs surface marker for identifying and screening CSCs in malignant tumor tissues such as lung cancer, liver cancer, breast cancer and the like, and the high expression of the CSCs is closely related to the occurrence, development, invasion and metastasis of tumors and is considered as a tumor initiation factor. Research shows that the positive expression rate of CD44 in lung cancer is as high as 85.5%, wherein the positive rates in a lung cancer bone metastasis tissue and an original lung cancer tissue are respectively 92% and 80%, and the difference has statistical significance, which indicates that abnormal high expression of CD44 is related to lung cancer metastasis; the CD44 is remarkably and highly expressed in the residual lung cancer cells after the molecular targeted drug EGFR-TKIs treatment, which indicates that the CD44 positive cells have strong drug resistance; multiple clinical studies have demonstrated that lymph node metastasis is more likely to occur in NSCLC in which high-expression and low-expression of CD44 are expressed, indicating that CD44 positive cells have a strong invasive potential. The research discovers that the CD44 is remarkably and highly expressed in the gefitinib resistant cell strain PC9GR through earlier work; the expression of CD44 in drug-resistant cells is knocked down, so that the expression quantity of CSCs markers such as CD166 and ABCG2 can be reduced, EMT can be reversed, and the sensitivity of the drug-resistant cells to gefitinib can be restored. These results all suggest that CD44 can be a therapeutic target for targeted killing of CSCs, but the current lack of targeted therapeutic strategies for CD44 is a major obstacle to targeted therapy of CD 44. The subject researches the action mechanism of CD44 in the generation and development of tumor, metastasis invasion and drug resistance by using in vivo and in vitro experimental models, confirms the feasibility of the CD44 as a new treatment target, develops a targeted CD44 treatment strategy, and is helpful for inhibiting tumor recurrence and improving curative effect.
Application of phage display technology in research of polypeptide drugs
The polypeptide is a bioactive substance which forms various cell functions in an organism, has the characteristics of small relative molecular mass, high specificity, easy absorption, easy synthesis and modification, capability of improving the immunity of the organism, high safety and the like, and has higher application value in the clinical treatment of tumors. Phage display technology (phase display) uses filamentous phage display proteins and polypeptides to extract polypeptides or proteins of desired properties from a large number of variants. The differential antigen peptides are screened from tumor tissues by a phage display technology, and the polypeptides are often combined with components or surface receptors with high expression of tumor cells, have certain tumor targeting property, and can also be artificially synthesized to obtain high-purity polypeptide monomers. Therefore, like antibodies, polypeptide drugs can also produce ADCC effect and induce apoptosis of tumor cells after binding to tumor antigens. Therefore, the micromolecule polypeptide medicine has excellent clinical application value.
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 CD44 antagonist polypeptide, which has high specific affinity with the receptor CD44, can block the signal pathway of CD44 by binding with CD44, and demonstrates that the polypeptide plays an important role in targeted inhibition of gefitinib-acquired drug-resistant non-small cell cancer cell proliferation, migration, invasion, clone formation, etc., and has great application value in targeted therapy of gefitinib-acquired drug-resistant non-small cell cancer.
Another object of the present invention is to provide derivatives of the above CD44 receptor antagonist polypeptide, which also have high specific affinity for the CD44 receptor and specifically bind to CD 44.
The invention also provides the application of the CD44 antagonist polypeptide and the derivatives thereof.
In order to realize the task, the invention adopts the following technical solution:
one aspect of the present invention provides a CD44 antagonist polypeptide, the amino acid sequence of which is shown in SEQ ID No.1,
SIPDYLFSHTML SEQ ID No：1。
the screening method of the CD44 antagonistic polypeptide utilizes a phage random peptide library, firstly CD44 plasmid is used for transfecting 293T cells to obtain a stable cell line permanently expressing CD44 at a high level, wild type 293T cells are used as control adsorption cells, 5 rounds of whole cell reduction screening are carried out, 50 positive phage amplification is randomly selected, and a clone single-stranded DNA is extracted for sequencing. 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.
In another aspect, the present invention provides a derivative of a CD44 antagonist polypeptide, where the derivative of a CD44 antagonist polypeptide is a product obtained by conventionally modifying an amino acid side chain group of a CD44 antagonist polypeptide, an amino terminus or a carboxyl terminus of a CD44 antagonist polypeptide fragment, or a product obtained by linking a tag for polypeptide or protein detection or purification to a CD44 antagonist polypeptide; the conventional modification is preferably amination, amidation, hydroxylation, carboxylation, carbonylation, alkylation, acetylation, phosphorylation, esterification, glycosylation, cyclization, biotinylation, fluorescent group modification, polyethylene glycol (PEG) modification or immobilization modification and the like; the tag is preferably His6GST, EGFP, MBP, Nus, HA, IgG, FLAG, c-Myc, or ProfinityXact, etc.
The CD44 antagonist polypeptide or derivative thereof can be derived from mammals or birds, such as primates (humans); rodents, including mice, rats, hamsters, rabbits, horses, cattle, canines, cats, and the like.
The CD44 antagonistic polypeptide and the derivative 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 present invention provides a polynucleotide encoding the polypeptide of SEQ ID No. 1.
In a further aspect of the invention, there is provided a vector comprising a nucleotide according to the invention, which can be linked to a promoter sequence by gene technology means.
In a further aspect, the invention provides a host cell transfected with a vector according to the invention.
In a further aspect, the invention provides a medicament comprising a CD44 antagonist polypeptide or a derivative thereof.
In the technical scheme of the invention, the medicine contains one or more pharmaceutically acceptable carriers;
in the technical scheme of the invention, the pharmaceutically acceptable carrier is preferably diluent, excipient, filler, adhesive, wetting agent, disintegrant, absorption enhancer, adsorption carrier, surfactant or lubricant;
in the technical scheme of the invention, the medicine can be further prepared into various forms such as tablets, granules, capsules, oral liquid or injections, and the medicines of various formulations can be prepared according to the conventional method in the pharmaceutical field.
In yet another aspect, the invention provides a test agent comprising a CD44 antagonist polypeptide or a derivative thereof.
In a further aspect, the invention provides antibodies to the aforementioned CD44 antagonist polypeptide or a derivative of the CD44 antagonist polypeptide.
In another aspect, the invention provides the use of the CD44 antagonist polypeptide and the CD44 antagonist polypeptide derivative in the preparation of a medicament for inhibiting the proliferation of tumor cells highly expressing CD 44.
In another aspect, the invention provides the use of the CD44 antagonist polypeptide and the CD44 antagonist polypeptide derivative in the preparation of a medicament for promoting apoptosis of tumor cells highly expressing CD 44.
In the technical scheme of the invention, the tumor cells highly expressing CD44 are selected from gefitinib acquired drug resistant non-small cell cancer cells.
In another aspect, the invention provides the use of the CD44 antagonist polypeptide and the CD44 antagonist polypeptide derivative in the preparation of drugs for treating tumor diseases highly expressing CD 44.
In the technical scheme of the invention, the tumor diseases with high expression of CD44 are selected from non-small cell carcinoma, liver cancer, rectal cancer, colon cancer, bile duct cancer, glioma, endometrial cancer, gastric adenocarcinoma, breast cancer, nasopharyngeal carcinoma and liver cancer.
In the technical scheme of the invention, the tumor disease with high expression of CD44 is selected from drug-resistant tumor diseases, preferably drug-resistant non-small cell carcinoma, drug-resistant liver cancer, drug-resistant rectal cancer, drug-resistant colon cancer, drug-resistant bile duct cancer, drug-resistant glioma, drug-resistant endometrial cancer, drug-resistant gastric adenocarcinoma, drug-resistant breast cancer, drug-resistant nasopharyngeal cancer and drug-resistant liver cancer.
In a specific embodiment of the invention, the tumor disease highly expressing CD44 is selected from gefitinib-resistant non-small cell carcinoma.
In a further aspect, the invention provides the use of the CD44 antagonist polypeptide and the CD44 antagonist polypeptide derivative in the preparation of a medicament for reducing the proliferation, migration, invasion and clonogenic capacity of tumor cells highly expressing CD 44.
In the specific experiment of the invention, the CD44 antagonist polypeptide HL7-ML1 can be specifically combined with the 293T CD44 of the cell with high expression CD44+/+And (4) combining.
In further experiments, the function of the CD44 antagonist polypeptide HL7-ML1 is verified by using gefitinib-acquired drug-resistant non-small cell carcinoma cell PC9GR highly expressing CD44 as a cell model, and HL7-ML1 is found to be capable of obviously inhibiting the migration, invasion and clonogenic capacity of the PC9GR cell compared with a control group.
Advantageous effects
(1) The invention provides a CD44 antagonist polypeptide HL7-ML1 and derivatives thereof, wherein the antagonist polypeptide and the derivatives thereof can be specifically combined with CD44 and specifically combined with CD44 to inhibit a CD44 signal channel.
(2) The invention proves that the high expression of the CD44 is related to the proliferation, infection, migration, invasion and clonogenic capacity of the non-small cell lung cancer for the first time, and proves that the expression of the CD44 is reduced, and the proliferation, infection, migration, invasion and clonogenic capacity of the non-small cell lung cancer can be inhibited.
(3) The CD44 antagonistic polypeptide and the derivative thereof obtained by screening can inhibit migration, invasion and clone forming capability of gefitinib acquired drug-resistant non-small cell cancer cells by blocking a signal channel of CD44, can be used as a biological polypeptide drug of a CD44 binding site, and can be used for preparing a drug for preventing and/or treating non-small cell cancer. Can be widely applied in the medical and biological fields and can generate huge social and economic benefits.
Drawings
FIG. 1: 293T-CD44+/+ cells highly express CD44 protein.
FIG. 2: HPLC detection pattern of HL7-ML1 short peptide.
FIG. 3: example 2 curve of the drug resistance concentration of gefitinib-acquired resistant cells PC9GR cells to doxorubicin;
FIG. 4: example 3 results of CD44 and related drug resistance due to their expression levels in PC9 and PC9GR cells;
FIG. 5: the HL7-ML1 short peptide remarkably inhibits the proliferation of the PC9GR cells. A, protein chip results show that compared with the gefitinib-sensitive strain PC9, the expression level of CD44 in the gefitinib-acquired drug-resistant PC9GR cell strain is higher; b, CCK cell proliferation experiments show that HP7-ML1 short peptides with different concentrations remarkably inhibit the proliferation activity of PC9GR cells.
FIG. 6: with the increase of the acting concentration, the HP7-ML1 short peptide obviously inhibits the clonogenic capacity of PC9GR cells.
FIG. 7: the HP7-ML1 short peptide remarkably inhibits the migration capability of PC9GR cells.
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: panning, amplifying, purifying, sequencing and synthesizing the CD44 antagonistic polypeptide HL7-ML 1.
In the embodiment, positive phage specifically bound with CD44 is obtained by screening, the positive phage is amplified and purified, phage single-stranded DNA (ssDNA) is extracted for sequencing, the obtained sequences are analyzed and compared, and finally, the high-purity antagonist polypeptide HL7-ML1 is synthesized.
The method comprises the following specific steps:
1. establishing a 293T cell line permanently highly expressing CD 44: 293T-CD44+/+
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, collecting slow virus liquid after 48 hours, centrifuging and infecting 273T cells;
seventhly, screening by using a culture medium containing 5 mu g/mL puromycin (puromycin) after 24 hours; obtaining 2 stably expressing CD44 after the cell is not dead any more93T cell line: 293T-CD44+/+。
Extracting total protein, detecting with protein chip, comparing with cell infected with blank carrier, 293T-CD44+/+The cells significantly highly expressed CD44 protein, and the results are shown in fig. 1, i.e. can be used for positive phage selection.
2. Performing panning, amplification, purification, sequencing and synthesis of the CD44 antagonist 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)。
Panning of CD44 antagonist peptides: high expression CD44 cell is expressed as 105The culture dish is inoculated on 60X 15mm which is coated with polylysine in advance2In a culture dish, when the cells are cultured to 80-90% of the cell density, 1 mu L of eluent is firstly taken for each round of elutriation (meanwhile, a cell line which does not express CD44 is used as a blank control), the rest eluent is added into 20mL of LB culture solution for amplification, then purification and the titer after amplification are detected again, the amplified product is stored for a short time at 4 ℃, the same number of orders are taken for the next round of elutriation, and the rest amplified product is stored at-20 ℃ by using 50% of glycerol.
Measuring the titer of the phage by taking 4 sterilized 10mL centrifuge tubes, preparing 1 sterilized centrifuge tube for each phage dilution, and melting Top agar (agar Top) in a microwave oven) 3mL of 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 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 for 10min at 4 ℃. Transferring the supernatant into another clean centrifugal tube, and centrifuging again at 10,000rpm for 10min at 4 ℃; 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 12000rpm 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, centrifuging at 4 ℃ and 10000rpm 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 ℃ and 12000rpm for 20min, and removing the 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. Amplification of the monoclonal phage, which comprises adding the overnight cultured ER2738 host bacterial liquid into 2mL LB liquid culture medium according to the proportion of 1:100, culturing at 37 ℃ and 250rpm for 2h with vigorous shaking; selecting a plate with less than 100 plaques from the fourth round of titer plates by using a sterilization 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 SIPDYLFSHTML, which was expressed as HL7-ML1, and the final short peptide was obtained from Hefei peptide Biotech Co. The synthesized short peptide is detected by HPLC, and the detection map is shown in figure 2.
Example 2 gefitinib-acquired resistant cell PC9GR cell has a significant increase in doxorubicin-resistant concentration
Firstly, PC9 and gefitinib resistant cell PC9GR are mixed in a ratio of 5 multiplied by 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;
② adding gefitinib (PC9:50,25,12.5,6.25,3.125,1.5625,0nM) with different concentration gradients (PC9GR:50, 25,12.5,6.25,3.125,1.5625,0uM) and adriamycin (2500,1250,625,312.5,156.25, 0nM) with different concentrations for culturing for 72 hours respectively;
thirdly, abandoning the supernatant in the culture plate, adding 100 mu L of CCK8 working solution into each hole, and continuously putting the cells into a carbon dioxide incubator for culture for 4 hours;
selecting 450nm wavelength to test on the enzyme labeling instrument, and drawing the cell growth curve. The results are shown in FIGS. 3A and B, and show that: IC50 of PC9 for gefitinib was 6.798nM, and IC50 of PC9GR cells for gefitinib was 11.79 uM; the results are shown in fig. 3C, which shows: the drug-resistant concentration of PC9GR cells to doxorubicin was also significantly increased compared to PC9 cells.
Example 3 high expression of CD44 and drug resistance related proteins by PC9GR cells
Experiments were conducted on the drug-resistant non-small cell carcinoma cell PC9GR and the non-drug-resistant non-small cell carcinoma cell PC9, respectively, and the expression levels of CD44 and the related drug-resistant agents in PC9 and PC9GR cells were compared by using a protein chip. The results are shown in FIG. 4, which shows: the expression of CD44 is shown in both cell strains, and compared with the Gefitinib-sensitive strain PC9, the expression level of CD44 in the Gefitinib-acquired drug-resistant PC9GR cell strain is higher; CD166, ALDH1A1 and ABCG2 tumor stem cell markers and a mesenchymal cell marker Vimentin are remarkably and highly expressed in PC9GR cells, and the results prove that the PC9GR cells generate EMT and obtain the characteristics of the tumor stem cells.
Example 4 HL7-ML1 significantly inhibited the proliferation of the gefitinib-acquired drug-resistant non-small cell carcinoma cell PC9GR
Firstly, gefitinib resistant cell PC9GR and adriamycin resistant colorectal cancer cellHCT116-ADR and Adriamycin resistant hepatoma cell HepG2-ADR at 5X 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;
② adding HL7-ML1 polypeptide with different concentration gradients (10mM,7.5mM,5mM,2.5mM,1.25mM,0.625mM,0.3125mM) to culture for 40 hours respectively;
thirdly, abandoning the supernatant in the culture plate, adding 100 mu L of CCK8 working solution into each hole, and continuously putting the cells into a carbon dioxide incubator for culture for 4 hours;
selecting 450nm wavelength to test on the enzyme labeling instrument, and drawing the cell growth curve. The results are shown in FIG. 5, which shows: HP7-ML1 short peptide with different concentrations obviously inhibits the proliferation activity of cells PC9GR, HCT116-ADR and HepG 2-ADR; and showed dose-dependent, with increasing HP7-ML1, the inhibitory activity was also significantly increased. Combining the results of examples 2 and 3, it is known that HP7-ML1 short peptide can inhibit various drug-resistant cells by inhibiting EMT produced by PC9GR cells.
Example 5 HL7-ML1 inhibits clonogenic potential of PC9GR cells and promotes apoptosis
Inoculating gefitinib-acquired drug-resistant non-small cell cancer cell PC9GR into a 6-well cell culture plate at 2000 cells/well, wherein the volume of a culture medium in each well is 1mL, culturing for 24h, and starving overnight;
adding HL7-ML1 polypeptide with different concentration gradients (5mM,2.5mM and 0mM) for respective culture for 12 days;
thirdly, cell supernatant is discarded, the cells are washed twice by PBS, and 4 percent PFA is fixed for 10 minutes;
and fourthly, adding 0.5 percent crystal violet working solution to dye for 30 minutes, photographing and analyzing the result.
The results are shown in FIG. 6, which shows: the HP7-ML1 short peptide has the capability of inhibiting the clonogenic capacity of PC9GR cells, and the inhibition effect is very obvious along with the increase of the action concentration.
Example 6 HL7-ML1 inhibits the migratory capacity of PC9GR cells
secondly, after culturing for 48 hours, drawing a transverse line at the bottom of the culture hole by using a gun head;
③ sucking the cell supernatant, washing the cell supernatant for 2 to 3 times by PBS, and removing the scratched cells;
adding HL7-ML1 polypeptide (serum-free culture medium diluted polypeptide) with different concentration gradients (5mM,2.5mM,1.25mM,0.625mM,0mM) into the culture box for culture;
sampling and photographing after 24 hours and 48 hours respectively, and observing the change of the cell migration.
The results are shown in FIG. 7, which shows: the HP7-ML1 short peptide remarkably inhibits the migration capability of PC9GR cells.
SEQUENCE LISTING
<110> Shenzhen International institute for graduate of Qinghua university
<120> CD44 antagonistic polypeptide, derivative and application thereof
<130> CP121010181C
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 12
<212> PRT
<213> Artificial sequence
<400> 1
Ser Ile Pro Asp Tyr Leu Phe Ser His Thr Met Leu
1 5 10
Claims (10)
1. A CD44 antagonist polypeptide, wherein: the amino acid residue sequence of the CD44 antagonist polypeptide is shown as SEQ ID No.1,
SIPDYLFSHTML SEQ ID No：1。
2. a derivative of the CD44 antagonist polypeptide of claim 1, wherein:
the derivative of the CD44 antagonist polypeptide is a product obtained by performing conventional modification on an amino acid side chain group of the CD44 antagonist polypeptide of claim 1, an amino terminal or a carboxyl terminal of the CD44 antagonist polypeptide fragment of claim 1, or a product obtained by connecting a tag for polypeptide or protein detection or purification to the CD44 antagonist polypeptide of 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 CD44 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. An agent against a high expression CD44 tumor disease, said agent comprising a combination of one or more of the CD44 antagonist polypeptide of claim 1, the derivative of claim 2, the polynucleotide of claim 3, the vector of claim 4, and the host cell of claim 5;
preferably, the medicament contains one or more pharmaceutically acceptable carriers;
more preferably, the pharmaceutically acceptable carrier is a diluent, excipient, filler, binder, wetting agent, disintegrant, absorption enhancer, adsorption carrier, surfactant or lubricant.
7. A test agent comprising the CD44 antagonist polypeptide of claim 1 or the derivative of claim 2.
8. An antibody to a CD44 antagonist polypeptide or a derivative of a CD44 antagonist polypeptide, wherein the CD44 antagonist polypeptide is the CD44 antagonist polypeptide of claim 1, and wherein the CD44 antagonist polypeptide derivative is the derivative of claim 2.
9. The use of the CD44 antagonist polypeptide of claim 1 or a derivative of the CD44 antagonist polypeptide of claim 2 for the preparation of a medicament for inhibiting proliferation of tumor cells with high expression of CD44, promoting apoptosis of tumor cells with high expression of CD44, and reducing migration, invasion and clonogenic capacity of tumor cells with high expression of CD 44;
preferably, the tumor cells highly expressing CD44 are selected from non-small cell carcinoma, liver cancer, rectal cancer, colon cancer, cholangiocarcinoma, glioma, endometrial cancer, gastric adenocarcinoma;
preferably, the tumor disease with high expression of CD44 is selected from drug-resistant tumor diseases, more preferably drug-resistant non-small cell carcinoma, drug-resistant liver cancer, drug-resistant rectal cancer, drug-resistant colon cancer, drug-resistant bile duct cancer, drug-resistant glioma, drug-resistant endometrial cancer, drug-resistant gastric adenocarcinoma, drug-resistant breast cancer, drug-resistant nasopharyngeal carcinoma and drug-resistant liver cancer.
10. Use of the CD44 antagonist polypeptide of claim 1, or a derivative of the CD44 antagonist polypeptide of claim 2, for the preparation of a medicament for the treatment of a tumor disease that is highly expressing CD 44;
preferably, the tumor diseases with high expression of CD44 are selected from non-small cell carcinoma, liver cancer, rectal cancer, colon cancer, bile duct cancer, glioma, endometrial cancer and gastric adenocarcinoma;
preferably, the tumor disease with high expression of CD44 is selected from drug-resistant tumor diseases, more preferably drug-resistant non-small cell carcinoma, drug-resistant liver cancer, drug-resistant rectal cancer, drug-resistant colon cancer, drug-resistant bile duct cancer, drug-resistant glioma, drug-resistant endometrial cancer, drug-resistant gastric adenocarcinoma, drug-resistant breast cancer, drug-resistant nasopharyngeal carcinoma and drug-resistant liver cancer.
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