CN112920258B - CD44 antagonistic polypeptide, derivative and application thereof - Google Patents
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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 antagonist polypeptide HL7-ML1 is shown as SEQ ID No.1, the amino acid residue sequence of SIPDYLFSHTML is shown as 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 the CD44, inhibit the proliferation of drug-resistant non-small cell cancer cells, promote the apoptosis of the drug-resistant non-small cell cancer cells, provide effective treatment small molecule drugs for acquired drug-resistant non-small cell cancer patients and the like, and can be 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 a 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 from T790M mutation and other reasons, and an effective targeted treatment method aiming at the tumor stem cells and the EMT is still absent at present. 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 acquired drug resistance of EGFR-TKIs in NSCLC
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 paths, and forms a signal network containing a plurality of positive feedback loops together with intracellular transcription factors such as Slug, snail, zeb1, zeb2, twist and the like, 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, promote the transformation of epithelial cells to mesenchymal cells, and further change the mesenchymal cells into mesenchymal stem cells.
The study 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 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 the sensitivity to EGFR-TKIs treatment. Patients positive for EGFR mutations who take oxicetinib also face the problem of secondary resistance. An Osteinib drug-resistant H1975 cell line (a drug-resistant cell line 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 of 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 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 inducing the immortalized human mammary epithelial cells (HLMEs) to generate EMT; isolating tumor stem cells 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 segment will help to suppress 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 a 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 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 is involved 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 the abnormal high expression of CD44 is related to lung cancer metastasis; the CD44 is obviously 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-expressing CD44 is expressed than in those with low expression, indicating that CD 44-positive cells have a strong invasive potential. The research discovers that the CD44 is obviously 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 is 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 for CD 44. The subject researches the action mechanism of CD44 in the occurrence and development, metastasis invasion and drug resistance generation 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 beneficial to 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 weight, 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 in 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 and can block the signal pathway of CD44 by binding with CD44, thus proving that the polypeptide plays an important role in targeted inhibition of gefitinib-acquired drug-resistant non-small cell carcinoma cell proliferation, migration, invasion, clone formation, etc., and has great application value in targeted therapy of gefitinib-acquired drug-resistant non-small cell carcinoma.
It is another object of the present invention to provide derivatives of the aforementioned antagonist polypeptides for CD44 receptor, which derivatives also have specific high affinity for CD44 receptor and specifically bind to CD 44.
It is still another object of the present invention to provide the use of the above-mentioned CD44 antagonist polypeptide and its derivatives.
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 method for screening the CD44 antagonistic polypeptide utilizes a phage random peptide library, firstly CD44 plasmids are used for transfecting 293T cells to obtain a stable cell line with permanent high expression CD44, wild type 293T cells are used as control adsorption cells, 5 rounds of whole cell subtraction screening are carried out, 50 positive phages are randomly selected for amplification, 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 invention provides a derivative of a CD44 antagonist polypeptide,the CD44 anti-polypeptide derivative is a product obtained by performing conventional modification on an amino acid side chain group of the CD44 antagonist polypeptide and an amino terminal or a carboxyl terminal of a CD44 antagonist polypeptide segment, or is a product obtained by connecting a label for polypeptide or protein detection or purification on the 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 His 6 GST, EGFP, MBP, nus, HA, igG, FLAG, c-Myc, or ProfinityXact, etc.
The CD44 antagonist polypeptide and derivatives 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 adopting a known method in the prior art, and can be chemically synthesized by using 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 encoding a polypeptide as set forth in 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 injection, and the medicines of various formulations can be prepared according to a 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 derivatives of the CD44 antagonist polypeptide.
In still another aspect, the present invention provides the use of the CD44 antagonist polypeptide, or a derivative of the CD44 antagonist polypeptide, in the preparation of a medicament for inhibiting the proliferation of tumor cells that highly express CD 44.
In another aspect, the invention provides the use of the CD44 antagonist polypeptide and the derivative of the CD44 antagonist polypeptide in the preparation of a medicament for promoting apoptosis of tumor cells with high expression of 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 a medicament for treating tumor diseases with high CD44 expression.
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 diseases with high expression of CD44 are 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 carcinoma and drug-resistant liver cancer.
In a specific embodiment of the present 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 a CD44 antagonist polypeptide, or a derivative of a CD44 antagonist polypeptide, as described herein, in the preparation of a medicament for reducing the proliferation, migration, invasion and clonogenic capacity of a tumor cell that expresses CD 44.
In the specific experiment of the invention, CD44 antagonist polypeptide HL7-ML1 can be used for specificity and high expression of CD44 cells 293T 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 can be 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 antagonist polypeptide and the derivative thereof obtained by screening can inhibit migration, invasion and clone forming capability of gefitinib acquired drug-resistant non-small cell carcinoma cells by blocking a signal path 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 carcinoma. Can be widely applied in the medical and biological fields and 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 resistance concentration of gefitinib-acquired resistant cells PC9GR cells to doxorubicin;
FIG. 4: example 3 results of CD44 and related drug resistance agents due to their expression levels in PC9 and PC9GR cells;
FIG. 5: HL7-ML1 short peptide obviously inhibits the proliferation of 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 obviously inhibit the proliferation activity of PC9GR cells.
6, figure 6: the HP7-ML1 short peptide obviously inhibits the clonogenic capacity of PC9GR cells along with the increase of the acting concentration.
FIG. 7: the HP7-ML1 short peptide obviously 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 without specifying the specific conditions is a conventional method and a conventional condition well known in the art, or a condition suggested by an instrument manufacturer.
Example 1: panning, amplifying, purifying, sequencing and synthesizing the CD44 antagonist polypeptide HL7-ML1.
In the embodiment, a 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 expressing CD 44: 293T-CD44 +/+
(1) Vigorous growing human 293T cells were selected at 5X 10 days before transfection 5 One cell/well, inoculated in 6-well plate, cultured until the next day, the cell fusion degree is 60％；
(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 for 20 minutes at room temperature, and gently adding 600. Mu.L of opti-MEM medium to the mixed dilution;
(4) gently rinsing the cells to be transfected once by using 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) collecting slow virus liquid after 48 hours, centrifuging and infecting 273T cells;
(7) after 24 hours, selection was carried out using a medium containing 5. Mu.g/mL puromycin (puromycin); obtaining the 293T cell line which stably expresses CD44 after the cell is not dead any more: 293T-CD44 +/+ 。
(8) Extracting total protein, performing protein chip detection, comparing with cell infected with blank vector, 293T-CD44 +/+ The cells significantly highly expressed the CD44 protein, and the results are shown in fig. 1, i.e. can be used for positive phage screening.
2. Performing panning, amplification, purification, sequencing and synthesis of the CD44 antagonist polypeptide
(1) Preparation of ER2738 host bacterial liquid: performing aseptic technique operation, namely taking 200 mu L of LB-Tet liquid culture medium in a 1.5mL sterile centrifuge tube, taking 0.2 mu L of bacterial liquid from the glycerol frozen product of E.coli ER2738, fully and uniformly mixing the bacterial liquid with the glycerol frozen product, completely absorbing and coating the bacterial liquid on an LB-Tet plate, marking the plate, standing the plate at room temperature for 3min, and then placing the plate in a 37 ℃ constant temperature incubator for inversion overnight culture. Observing the next day, sealing with sealing film after the clone grows out, and storing at 4 deg.C in dark for use. Single colonies were picked aseptically with a sterile pipette tip and placed into 10mL sterile centrifuge tubes pre-filled with 3mL LB-Tet broth, labeled and shake-cultured overnight on a constant temperature shaker at 37 ℃ and 300 rpm/min. The next day, bacterial amplificationThe solution was stored at 4 ℃ until use. Taking 10mL of a sterilized centrifuge tube, adding 3mL of LB-Tet liquid culture medium in an aseptic operation, inoculating 30 mu L of overnight-cultured bacteria, carrying out shake culture at constant temperature of 37 ℃ and 300rpm/min for 2-3 h, wherein the bacteria are in an exponential growth phase and are in a fog shape (OD) through visual observation 600 ～0.5)。
(2) Panning of CD44 antagonist peptides: high expression CD44 cells are expressed as 10 5 The culture dish is inoculated on 60X 15mm which is coated with polylysine in advance 2 In a culture dish, when the cells are cultured to 80-90% in a conventional way, 1 mu L of eluent is firstly taken for 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 measured, an amplified product is stored at 4 ℃ for a short time, the same magnitude is taken for the 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 the Top agar (agar Top) in a microwave oven, adding 3mL Top agar per tube, and using a water bath at 45 ℃. For each dilution of phage, 1 LB/IPTG/Xgal plate was prepared and pre-warmed in a 37 ℃ incubator for use. Will OD 600 E.coli ER2738 E.coli of 0.5 was aliquoted at phage dilution 200. Mu.L/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 detected 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 And 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 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: adding the overnight-cultured ER2738 host bacterium liquid into 20mL of LB liquid culture medium in a 250mL conical flask according to a ratio 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 at 4 ℃ for 10min. Transferring the supernatant into another clean centrifugal tube, and centrifuging again at 10,000rpm at 4 deg.C for 10min; transferring 80% of the supernatant into another clean centrifugal tube, adding 1/4 volume of PEG/NaCl, reversing, mixing uniformly, and precipitating at 4 ℃ overnight; the next day, the pellet was centrifuged at 12000rpm for 20min at 4 ℃. Carefully sucking the supernatant with a clean gun head, centrifuging at 12,000rpm for 1min at 4 ℃, 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 into the supernatant, and incubating on ice for 60min for reprecipitation; taking out the centrifuge tube, centrifuging at 12000rpm at 4 deg.C for 20min, and removing supernatant; the pellet was resuspended in 200. Mu.L TBS and centrifuged at 10,000rpm for 1min at 4 ℃. The supernatant was transferred to another centrifuge tube. Short-term storage at 4 deg.C, or long-term storage at-20 deg.C with 50% glycerol. 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; 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 4.5h; 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 a fresh centrifuge tube and stored at 4 ℃ or stored for a long period at-20 ℃ with 50% glycerol.
(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 Erlenmeyer flask, adding 100mL 1 xTAE buffer solution, uniformly mixing, putting the flask into a microwave oven, heating and boiling until the agarose is completely dissolved; and (3) closing the induction cooker, taking out the triangular flask, cooling the triangular flask to room temperature (the triangular flask 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 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 sample sequence was SIPDYLFSHTML expressed as HL7-ML1, and the short peptide was finally obtained from Hefei peptide Biotech Ltd. The synthesized short peptide is detected by HPLC, and the detection map is shown in figure 2.
Example 2 Gefitinib-acquired resistant cell PC9GR cells also show a significant increase in doxorubicin-resistant concentrations
(1) PC9 and gefitinib resistant cell PC9GR at 5X 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) gefitinib (PC 9:50,25,12.5,6.25,3.125,1.5625,0 nM) and adriamycin (2500, 1250,625,312.5,156.25,0 nM) were added at different concentration gradients for 72 hours;
(3) discarding the supernatant in the culture plate, adding 100 mu L of CCK8 working solution into each hole, and continuously putting the culture plate into a carbon dioxide incubator for culturing for 4 hours;
(4) and (4) selecting a wavelength of 450nm on a microplate reader for detection, and drawing a growth curve of the cells. The results are shown in FIGS. 3A and B, and show that: PC9 has an IC50 for gefitinib of 6.798nM, and PC9GR cells have an IC50 for gefitinib of 11.79uM; the results are shown in fig. 3C, which shows: compared with PC9 cells, the drug resistance concentration of PC9GR cells to adriamycin is also obviously increased.
Example 3 high expression of CD44 and drug resistance-related proteins in PC9GR cells
Experiments were performed 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 agent 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; the CD166, ALDH1A1 and ABCG2 tumor stem cell markers and the 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 inhibits the proliferation of gefitinib-acquired drug-resistant non-small cell carcinoma cell PC9GR
(1) The gefitinib resistant cell PC9GR, the adriamycin resistant colorectal cancer cell HCT116-ADR and the adriamycin resistant liver cancer cell HepG2-ADR are mixed by 5 multiplied by 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 HL7-ML1 polypeptides with different concentration gradients (10mM, 7.5mM,5mM,2.5mM,1.25mM,0.625mM, 0.3125mM) for 40 hours respectively;
(3) discarding the supernatant in the culture plate, adding 100 mu L of CCK8 working solution into each hole, and continuously putting the culture plate into a carbon dioxide incubator for culturing for 4 hours;
(4) and (4) detecting on a microplate reader by selecting the wavelength of 450nm, and drawing a growth curve of the cells. The results are shown in FIG. 5, which shows: HP7-ML1 short peptide with different concentrations obviously inhibits the proliferation activity of cell PC9GR, HCT116-ADR and HepG2-ADR cells; and showed dose-dependent, with increasing HP7-ML1, the inhibitory activity was also significantly increased. The results of examples 2 and 3 show that HP7-ML1 short peptide can inhibit various drug-resistant cells by inhibiting EMT produced by PC9GR cells.
Example 5 HL7-ML1 inhibits clonogenic potency of PC9GR cells promoting their apoptosis
(1) 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 then starving overnight;
(2) adding HL7-ML1 polypeptide with different concentration gradients (5 mM,2.5mM,0 mM) for respective culture for 12 days;
(3) discard cell supernatant, wash cells twice with PBS, 4% pfa fixation for 10min;
(4) adding 0.5% crystal violet working solution for dyeing 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
(1) 3X 10 inoculations per well in six-well plates 5 Each well of the cell culture medium has a volume of 2mL;
(2) after culturing for 48 hours, drawing a transverse line at the bottom of the culture hole by using a gun head;
(3) sucking cell supernatant, washing with PBS for 2-3 times, and removing scratched cells;
(4) adding HL7-ML1 polypeptide (serum-free medium diluted polypeptide) with different concentration gradients (5 mM,2.5mM,1.25mM,0.625mM,0 mM) and putting the polypeptide into an incubator for culture;
(5) samples were taken after 24 hours and 48 hours, respectively, and photographed to observe changes in cell mobility.
The results are shown in FIG. 7, which shows: the HP7-ML1 short peptide obviously inhibits the migration capability of PC9GR cells.
SEQUENCE LISTING
<110> Shenzhen International institute for graduate of Qinghua university
<120> CD44 antagonist polypeptide, derivatives and applications 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 (13)
1. A CD44 antagonist polypeptide, which polypeptide comprises: 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 connecting a label for polypeptide or protein detection or purification to the CD44 antagonist polypeptide of claim 1;
the label is His 6 GST, EGFP, MBP, nus, HA, 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, 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.
7. The drug against highly expressed CD44 tumor disease of claim 6, which comprises one or more pharmaceutically acceptable carriers.
8. The medicament of claim 7 for treating tumor diseases with high expression of CD44, wherein the pharmaceutically acceptable carrier is diluent, binder, wetting agent, disintegrant or lubricant.
9. The drug against highly expressed CD44 tumor disease as claimed in claim 7, wherein the pharmaceutically acceptable carrier is excipient.
10. The drug for treating tumor diseases with high expression of CD44 as claimed in claim 7, wherein the pharmaceutically acceptable carrier is absorption enhancer, adsorption carrier, surfactant.
11. A detection reagent comprising the CD44 antagonist polypeptide of claim 1 or the derivative of claim 2.
12. The use of the CD44 antagonist polypeptide of claim 1 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;
the tumor cells with high expression of CD44 are selected from non-small cell cancer, liver cancer and rectal cancer.
13. Use of a CD44 antagonist polypeptide of claim 1 in the preparation of a medicament for the treatment of a neoplastic disease that is highly expressing CD 44;
the tumor diseases with high CD44 expression are selected from non-small cell cancer, liver cancer and rectal cancer;
the tumor diseases with high expression of CD44 are selected from drug-resistant non-small cell cancer, drug-resistant liver cancer and drug-resistant rectal cancer.
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