阅读说明：本技术 一种靶向识别膜联蛋白a2的亲和短肽及其制备方法与用途 (Affinity short peptide for targeted recognition of annexin A2 and preparation method and application thereof ) 是由 魏敏杰 于 2019-08-20 设计创作，主要内容包括：本发明涉及一种靶向识别膜联蛋白A2的亲和短肽及其制备方法与用途,其可以特异性靶定膜联蛋白A2(Annexin A2),尤其是基于Annexin A2在大多数肿瘤中高表达的特点,进而为高效,靶向识别肿瘤组织,预测和提高肿瘤靶向治疗提供新的可能性；本发明还涉及了该多肽作为多肽分子探针或肿瘤导向多肽能与抗肿瘤的药物偶联,作为靶头增加药物或载有药物的载体如纳米材料、脂质体等在过表达Annexin A2的细胞中的含量,再添加药学上可接受的辅料或佐剂制成新型的更有效的靶向抗癌药物。本发明的多肽还可制成显像剂,用于多种高表达Annexin A2肿瘤的靶向治疗和成像,另外,还可制成多肽抑制剂,阻断Annexin A2与相关的蛋白相互作用。本发明所述多肽能对膜联蛋白A2具有特异性靶向作用,选择性强,且本发明涉及的肽可以采用化学合成的方法制备得到,其纯度高、分子量小、特异性强、无免疫原性及安全可靠。(The invention relates to an affinity short peptide for targeted identification of Annexin A2, a preparation method and application thereof, which can specifically target Annexin A2(Annexin A2), and particularly provides new possibility for efficient targeted identification of tumor tissues and prediction and improvement of tumor targeted therapy based on the characteristic that Annexin A2 is highly expressed in most tumors; the invention also relates to the polypeptide which can be coupled with anti-tumor drugs as a polypeptide molecular probe or a tumor-oriented polypeptide, and can be used as a target to increase the content of drugs or drug-loaded carriers such as nano materials, liposomes and the like in cells over-expressing Annexin A2, and then pharmaceutically acceptable auxiliary materials or adjuvants are added to prepare the novel more effective targeted anti-cancer drugs. The polypeptide can also be prepared into an imaging agent for targeted therapy and imaging of various tumors with high expression of Annexin A2, and can also be prepared into a polypeptide inhibitor for blocking the interaction between Annexin A2 and related proteins. The polypeptide can have a specific targeting effect on annexin A2, is high in selectivity, can be prepared by a chemical synthesis method, and is high in purity, small in molecular weight, high in specificity, free of immunogenicity, safe and reliable.)

1. An affinity short peptide for targeted recognition of annexin A2, a preparation method and application thereof are characterized in that the amino acid sequence is as follows: YWRGYN.

2. The Annexin A2 specific binding peptide of claim 1, wherein the amino acid sequence of the binding peptide is shown in a sequence table.

3. A polypeptide molecular probe comprises the polypeptide provided by the invention, and the encoded nucleotide sequence is shown in a sequence table so as to accurately screen tumors which over-express Annexin A2.

4. A tumor-targeting polypeptide is characterized in that an amino acid sequence of the binding peptide is shown in a sequence table, the binding peptide is coupled with an anti-tumor drug, the binding peptide is used as a target to increase the content of the drug or a drug-loaded carrier such as a nano material, a liposome and the like in cells over expressing Annexin A2, and pharmaceutically acceptable auxiliary materials or adjuvants are added to prepare a novel more effective targeted anti-cancer drug.

5. The imaging agent is characterized in that the amino acid sequence of the binding peptide is shown in a sequence table, and the imaging agent is used for targeted therapy and imaging of various tumors over-expressing Annexin A2.

6. A polypeptide inhibitor is characterized in that the amino acid sequence of the binding peptide is shown in a sequence table, and the binding peptide can block the interaction between Annexin A2 and related proteins.

Technical Field

The invention relates to the field of biotechnology, in particular to application of a polypeptide with high affinity with annexin A2 and good binding specificity and sensitivity.

Background

Cancer is the second leading cause of death worldwide, second only to cardiovascular disease. At present, the clinical treatment of malignant tumor mainly comprises surgery, chemotherapy and radiotherapy, and the satisfactory curative effect is difficult to achieve, wherein the chemotherapy is the fastest method developed in tumor treatment in recent years. However, the chemotherapy drugs kill the tumor cells and the normal cells of the human body at the same time, and have large toxic and side effects. Drug therapy is one of the important methods for systemic treatment of malignant tumors. The traditional anti-tumor chemotherapy drugs have poor targeting property and cannot distinguish normal tissues from tumor tissues. The medicine is eliminated from blood quickly, and only a small amount of medicine is distributed to the target position, so that the accumulating capacity in the tumor is poor, and the medicine has great toxicity and adverse reaction. These drugs have low antitumor therapeutic effects and are unsatisfactory in therapeutic effects. With further research, the traditional cytotoxic chemotherapy is shifted to molecular targeted drugs and immunotherapy drugs, wherein the molecular targeted therapy has stronger specificity, pertinence and effectiveness, better tolerance of patients, lower toxic and side effects compared with cytotoxic drugs, and remarkable advantages compared with the traditional chemotherapeutic drugs.

Targeted therapy has seen significant efficacy in treating certain types of cancer starting late 90 s, as effective as chemotherapy, but with much less side effects than chemotherapy. Targeted therapy is also a very active area of research at present. The targeted therapy is to design a corresponding therapeutic drug aiming at a well-defined carcinogenic site (the site can be a protein molecule inside a tumor cell or a gene fragment), and the drug enters into the body and specifically selects the carcinogenic site to combine to take effect, so that the tumor cell is specifically killed without affecting normal tissue cells around the tumor, and the molecular targeted therapy is also called as 'biological missile'. The principle of this treatment is that non-small cell lung cancer, which has sensitive mutations to EGFR (epidermal growth factor receptor), is treated with significant efficacy using small molecules, e.g., tyrosine phosphatase inhibitors, that specifically target abnormal or deregulated proteins of cancer cells, but the emergence of drug resistance genes is currently a major obstacle that prevents further improvement in efficacy. Therefore, the research of targeting antitumor drugs and molecular probes is imperative. The premise for successful cancer targeted therapy is to find the target of the therapy.

Annexin A2(Annexin A2) is a protein with calcium ion-mediated phospholipid binding property, belongs to an Annexin family member, is widely distributed in various eukaryotic cell membranes, cytoplasm and extracellular fluid, and is mainly expressed in cells such as vascular endothelial cells, monocytes, macrophages, dendritic cells, trophoblast cells, epithelial cells, tumor cells and the like. Its function is mainly involved in membrane transport and a series of activities on membrane surface depending on calmodulin, including membrane fusion in exocytosis, vesicle trafficking, cell adhesion, cell proliferation, apoptosis, replication, signaling and ion channel formation, and many diseases and in vivo regulation are related to annexin A2. Annexin A2 is also called Annexin II, Calpacin I heavy chain, Lipocortin 1I, P36. It was first found in Rous sarcoma virus transformed chicken embryo fibroblasts. They are involved in a range of cellular activities such as exocytosis, endocytosis, cell proliferation, differentiation and apoptosis. Annexin a2 was first found to be highly expressed in acute promyelocytic leukemia, while Annexin v, another member of its family, was associated with antiphospholipid syndrome, thus suggesting the concept of annexnopathiy. The research finds that Annexin A2 has the characteristics of RNA combination, is related to DNA synthesis and replication and plays a role in the occurrence, infiltration and metastasis of tumors, unlike other Annexins. It plays a positive regulatory role in various types of tumors and also plays multiple roles in regulating cell function, including angiogenesis, proliferation, apoptosis, cell migration, invasion, and adhesion. In recent years, Annexin a2 has been found to be abnormally expressed in tumors such as gastric cancer, colon cancer, prostatic adenocarcinoma, hepatocellular carcinoma, and pancreatic ductal adenocarcinoma, and to be involved in tumor infiltration, metastasis, or prognosis. Annexin a2 has become an important target for the treatment of cancer.

The polypeptide is a compound formed by connecting a plurality of amino acids through peptide bonds, and generally consists of 10-100 amino acid molecules, wherein the connection mode is the same as that of protein, and the relative molecular mass is less than 10000. Polypeptides are ubiquitous in organisms, and several tens of thousands of polypeptides have been found in organisms so far, and they are widely involved in and regulate functional activities of various systems, organs, tissues and cells in the organism, and play an important role in life activities. The polypeptide as an important bioactive substance has the characteristics of high activity, low immunogenicity, low toxicity, easy loading and the like, and is widely applied to cancer treatment. The targeted drug delivery system can selectively concentrate and position drugs in target organs, target tissues and target cells, and modify small molecular polypeptides on the surface of the targeted drug delivery system, so that the toxic and side effects of traditional chemotherapeutic drugs can be reduced, and the therapeutic index can be improved. Tumor targeting peptides refers to a class of peptides that are capable of targeting a tumor or the tumor microenvironment. Due to advances in phage display technology, a large number of peptides have been discovered that have strong affinity for specific receptors/markers present on tumors and tumor vessels. Tumor targeting peptides have a strong affinity for specific receptors or markers present on tumors or tumor vessels and are therefore capable of targeting tumors or tumor microenvironments, which are also commonly referred to as tumor homing peptides. In recent years, polypeptide drugs synthesized by modern biotechnology have become one of hot spots in drug research and development, have wide adaptation diseases, high safety and obvious curative effect, are widely applied to prevention, diagnosis and treatment of diseases such as tumors, hepatitis, diabetes, AIDS and the like at present, and have wide development prospects.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The invention aims to provide an affinity short peptide for targeted recognition of annexin A2, a preparation method and application thereof, wherein the amino acid sequence of the affinity short peptide is as follows: YWRGYN, in particular to the application of the peptide in preparing anticancer drugs.

The invention provides a preparation method of an affinity short peptide for targeted recognition of annexin A2, which comprises the following steps:

(1) preparation of polypeptide YW7

The Fmoc solid phase synthesis polypeptide strategy is adopted, and Fmoc-Asn (pbf) -OH is used as a raw material to be bonded with Wang resin. Then after removing the Fmoc group, carrying out condensation reaction with Fmoc-typ (tBu) -OH in the presence of a condensing agent DIC and HOBt to complete the connection of a second amino acid; then removing Fmoc, and carrying out condensation reaction with a third amino acid Fmoc-Val (Boc) -OH; synthesizing from C end to N end (the used amino acids comprise Fmoc-Gly-OH, Fmoc-Arg-OH and Fmoc-Trp-OH) in sequence according to the program until the synthesis is finished to obtain the resin with the side chain protection of the Fmoc group removed;

adding a cutting reagent into the obtained peptide resin, reacting for 2 hours at 20 ℃ in a dark place, and filtering; collecting the filtrate, concentrating by a rotary evaporator, adding precooled anhydrous ether with the volume about 10 times that of the filtrate, precipitating for 3 hours at the temperature of minus 20 ℃, separating out white powder, collecting the precipitate, washing the precipitate by the anhydrous ether, and drying in vacuum to obtain polypeptide, wherein a cutting reagent is prepared by mixing TFA, water and TIS (triisopropylchlorosilane) according to the mass ratio of 96:1.5: 2.5;

purifying by using a C18 reversed-phase preparation column, collecting a main peak, and freeze-drying to obtain a white solid, namely a product;

the product is identified by mass spectrum, and the molecular weight shown in the mass spectrum is basically consistent with the theoretical molecular weight.

(2) Preparation of FITC-Positive polypeptide fragment FITC-YW7

Adopting Fmoc solid phase synthesis polypeptide strategy, taking Fmoc-YW7-Wang resin as a raw material, removing Fmoc group, and then carrying out condensation reaction with Fmoc-Acp-OH in the presence of a condensing agent DIC and HOBt; then removing Fmoc, and carrying out condensation reaction with FITC-Cl;

adding a cutting reagent into the obtained peptide resin, reacting for 2 hours at 20 ℃ in a dark place, and filtering; collecting the filtrate, concentrating by a rotary evaporator, adding precooled anhydrous ether with the volume about 10 times that of the filtrate, precipitating for 3 hours at the temperature of minus 20 ℃, separating out white powder, collecting the precipitate, washing the precipitate by the anhydrous ether, and drying in vacuum to obtain polypeptide, wherein a cutting reagent is prepared by mixing TFA, water and TIS (triisopropylchlorosilane) according to the mass ratio of 96:1.5: 2.5;

purifying by using a C18 reversed-phase preparation column, collecting a main peak, and freeze-drying to obtain a white solid, namely a product;

the product is identified by mass spectrum, and the molecular weight shown in the mass spectrum is basically consistent with the theoretical molecular weight.

The invention provides a polypeptide molecular probe, which comprises the polypeptide provided by the invention, and the amino acid sequence of the polypeptide is YWRGYN.

Preferably, the polypeptide probe has the advantages of strong selectivity, high purity, small molecular weight, strong penetrability, no immunogenicity, safety and reliability.

More preferably, the probe can become a molecular probe with high sensitivity and specificity, which is introduced into the body and then is specifically combined with a specific target protein Annexin A2 in the cell to generate a certain signal, thereby realizing highly specific diagnosis or targeted therapy.

The invention provides a tumor-targeted polypeptide, which comprises an amino acid sequence of a binding peptide shown in a sequence table, is coupled with an anti-tumor drug, is used as a target to increase the content of the drug or a drug-loaded carrier such as a nano material, a liposome and the like in cells over-expressing Annexin A2, and is added with pharmaceutically acceptable auxiliary materials or adjuvants to prepare a novel more effective targeted anti-cancer drug.

The invention provides an imaging agent, which comprises a binding peptide with an amino acid sequence shown in a sequence table and is used for targeted therapy and imaging of various tumors over-expressing Annexin A2.

The invention provides a polypeptide inhibitor, which comprises the amino acid sequence of the binding peptide shown in a sequence table and can block the interaction between Annexin A2 and related proteins.

The invention has the beneficial effects that: (1) the polypeptide provided by the invention has the specific targeting property of Annexin A2, so that the polypeptide can be widely applied to tumors over-expressing Annexin A2 in practical application. Therefore, the tumor targeting polypeptide and the derivative thereof have important application values in diagnosis, screening and targeted therapy of over-expressed Annexin A2 tumor molecules.

(2) The polypeptide has specific binding effect on over-expressed Annexin A2 tumor cells, and brings more hopes for the fields of further searching for a new ligand of Annexin A2, researching binding sites of interaction between macromolecules, searching for ligand molecules with high affinity and biological activity, screening medicines, developing vaccines and novel diagnostic reagents and the like.

(3) Compared with the traditional medicine, the short peptide has the advantages of smaller relative molecular mass, weak immunogenicity, high activity and the like, the short peptide medicine is easier to produce simply and conveniently in large scale, and the selected polypeptide is expected to become a new-age medicine with good effect and high benefit.

Preferably, the cancer of the present invention is a cancer in which Annexin a2 is overexpressed, and preferably pancreatic cancer.

Drawings

FIG. 1 is a diagram showing the results of the recombination and purification of Annexin A2; wherein, FIG. 1 (A) is the whole map of the constructed Annexin A2 expression plasmid, and FIG. 1 (B) is the result map of the purification of Annexin A2 protein.

FIG. 2 shows the results of a screening for the specific binding of Annexin A2 to a positive polypeptide; wherein, FIG. 2 (A) is a three-round target screening titer test result chart of the Annexin A2 specific binding positive polypeptide by using the phage display technology, and A, B, C is a first, a second and a third round screening titer test blue-white spot flat chart respectively. FIG. 2 (B) is a diagram showing the result of DNA sequencing of the phage having the highest repetition rate.

FIG. 3 is a mass spectrometric identification chart of polypeptide YW7 and FITC-positive polypeptide fragment FITC-YW7 synthesized in solid phase; wherein, FIG. 3 (A) is the mass spectrum of polypeptide YW7 synthesized by solid phase, and FIG. 3 (B) is the mass spectrum of FITC-positive polypeptide fragment FITC-YW7 synthesized by solid phase.

FIG. 4 is the result of the verification of the affinity of the positive polypeptide YW7 with Annexin A2, wherein FIG. 4 (A) is the result of the identification of the affinity of the MOE docking polypeptide YW7 with Annexin A2. FIG. 4 (B) is a diagram showing the result of MST experiment to identify the affinity of polypeptide YW7 with Annexin A2.

FIG. 5 is a graph showing the in vitro identification of the ability of the polypeptide FITC-YW7 to specifically target binding to pancreatic cancer cells overexpressing Annexin A2; wherein FIG. 5 (A) is a fluorescence result graph and a bar graph of the immunofluorescence validation polypeptide FITC-YW7 for the ability to specifically target and bind to pancreatic cancer Panc-1 cells. FIG. 5 (B) is a graph and a bar graph of the experimental results of flow cytometry to identify the ability of the polypeptide FITC-YW7 to specifically target-bind pancreatic cancer Panc-1 cells. FIG. 5 (C) is a graph and bar graph showing the fluorescence results of the co-localization of FITC-YW7 and Annexin A2 on the cell surface of pancreatic cancer Panc-1.

Detailed Description

The technical solution of the present invention is further illustrated by the following specific examples.

EXAMPLE 1 recombination and purification of Annexin A2

1.1 transformation of the constructed Annexin A2 expression plasmid

Competent cells were thawed on ice. And adding plasmids into the competent cells, gently mixing uniformly, and standing on ice for 5 min. The 42 degree heat shock was applied for 90 seconds. Quickly transferred to ice and cooled for 3 min. Non-resistant LB medium was added at 37 degrees. The mixture was dipped with a glass rod and spread evenly onto AMP plate medium. The plates were placed in a 37 ℃ incubator overnight. The next day, the colony growth was checked and stored in a 4 degree refrigerator.

1.2 Mass amplification of Annexin A2

The overnight bacteria Annexin A2 were shaken and AMP and LB (1: 1000) were added to the Erlenmeyer flask. Shake bacteria at 37 degrees, 200rpm overnight. The next day, overnight bacteria Annexin A2 were stored and frozen at-20 ℃. LB, inoculum and AMP were added to the Erlenmeyer flask and shaken at 37 ℃ and 180rpm for 4 hours. IPTG was added to the bacterial solution for induction, and the bacteria were shaken at 22 ℃ and 180rpm for 5 hours. And (4) taking a sterile centrifuge tube, and subpackaging the bacteria liquid. Centrifuging at 12000g at 4 ℃ for 10min, discarding the supernatant, and freezing and storing at-20 ℃.

1.3 purification of Annexin A2

4ml/g of lysis buffer was added to the bacterial pellet and resuspended. Adding lysozyme to the mixture to a final concentration of 1mg/ml, and freezing for 30 min. The ultrasound was performed 6 times at 200w for 10 seconds each, with 10 seconds intervals. Centrifugation is carried out at 4 ℃ for 10000g for 20min, and the supernatant is kept on ice, and 100ul of each sample is kept. 0.3ml of a Beyogold ™ GST-tag Purification Resin was centrifuged at 4 ℃ and 1000g for 10 seconds, the supernatant was discarded, 0.5ml of lysis buffer was added, and the equilibration was repeated 3 times and centrifugation at 4 ℃ and 1000g for 10 seconds. Beyogold GST-tag Purification Resin was added to the supernatant, and bound at 4 ℃ for 1 hour. The mixture was applied to an affinity column and 100ul of the effluent was retained. The column was washed 4 times with 0.2ml of lysis buffer, leaving 100ul of sample for the 4 th time. Eluting with 0.5ml of eluent respectively, reserving 100ul of sample for each tube, and freezing and storing the residual-80.

The results are shown in FIG. 1, FIG. 1 is a diagram of the recombination and purification results of Annexin A2, wherein FIG. 1 (A) is a full map of the constructed Annexin A2 expression plasmid. FIG. 1 (B) is a graph showing the results of the purification of Annexin A2 protein, which shows that there is a significant purification of Annexin A2 protein at 62 kd.

Example 2 Targeted screening of Annexin A2 protein for specific binding to Positive Polypeptides

2.1 recovery and culture of host bacterium E.coli ER2738

Preparing an escherichia coli plate, taking an LB-TET culture plate, preheating the LB-TET culture plate in a 37-degree incubator for 1 hour, dipping a small amount of bacterial liquid by using an inoculating loop after the E.coli ER2738 bacterial liquid is melted, uniformly paving the bacterial liquid on the culture plate, and then inversely placing the bacterial liquid in a constant-temperature incubator at 37 ℃ for overnight culture. Preparing host bacterial liquid, picking single colony from a culture plate with good growth, placing the colony in LB bacterial culture solution containing tetracycline, and oscillating at 37 ℃ and 180rpm overnight for culturing to enable the bacteria to be in logarithmic phase. The prepared LB-Tet plate containing the escherichia coli is placed in a refrigerator at 4 ℃ for storage and standby, and the host bacterium liquid is placed in a refrigerator at-80 ℃ for storage and standby.

2.2 phage display heptapeptide library targeting screening

Annexin A2 is used as a target protein, a phage display technology targeted screening method is utilized, the operational instructions of a phage display heptapeptide library kit are referred, three rounds of targeted screening are performed, the input total amount of each round of phage is ensured to be the same as much as possible, and the screening pressure is increased progressively for each round, so that the highly enriched phage is finally obtained. The specific screening steps are as follows:

2.2.1 preparation of Annexin A2

Preparing Annexin A2 with a final concentration of 5ug/ml, dissolving in 0.1M NAHCO₃(pH 8.6), spread on 96-well plates, shake for 30min, 4 ℃ overnight.

2.2.2 preparation of bacterial liquid

On the day of screening, overnight cultured host cells were added to LB medium. Shaking at 180rpm for about 3 hours at 37 ℃. The above is the bacterial solution used for the titer determination and amplification.

2.2.3 protein immobilization

The liquid in the 96-well plate was pipetted clean and patted clean on sterile filter paper. Blocking with 0.5% BSA and standing at 37 ℃ for 1 hour.

2.2.4 peptide library binding

The blocking solution in the 96-well plate was discarded. A100-fold dilution of the Ph.D.TM-7 primary phage-displayed heptapeptide library with TBST was added and shaken microscopically at 120rpm for 1 hour at room temperature.

2.2.5 washing

The first round of unbound phage fluid was aspirated, and the 96-well plate was placed on sterile filter paper and patted dry vigorously. The 96-well plate was washed 10 times with 0.1% TBST for about 30 seconds each time, and the bottom and edges of the 96-well plate were washed, the wash was decanted, and blotted dry on sterile filter paper.

2.2.6 elution

The eluent (0.2M Glycine-HCl, pH 2.2) was added to a 96-well plate and shaken gently at 80rpm for 15min at room temperature. After the elution is finished, the eluent is gently blown and sucked out, added into an EP tube containing a neutralization buffer solution and mixed evenly.

2.3 determination of phage titer

The strain is shaken in advance, TET is added into LB, the temperature is 37 ℃ and 180rpm, and after 3 hours, the logarithmic growth phase is reached (the OD value is about 0.6 is better). Before the titer is determined, the LB/IPTG/Xgal plate is preheated for more than 1 hour in an incubator at 37 ℃. Agarose gel is prepared and heated by microwave to melt. Taking the eluate after neutralization after adsorption (or the supernatant of the amplified phage), and diluting the phage by the multiple of LB culture medium. The dilution range is as follows: neutralization of the eluate after adsorption 10²-10⁵(ii) a Phage supernatant dilution after amplification 10⁹-10¹². Prepared bacterial solution was added to each EP tube, and 10. mu.l of phage solution at different dilution was added to each tube. Shaking on shaking instrument for 5min and mixing. The infected phage solution was added to room temperature thawed agar and the suspension was immediately added to pre-warmed LB/IPTG/Xgal plates and spread evenly with a cooled coated glass rod (one plate per dilution and marked). The coated plates were placed upside down in an incubator at 37 ℃ for overnight culture. The next day the plates were examined for phage blue spot growth and counted (i.e., number of phage spots per plate).

2.4 phage amplification purification

And amplifying and purifying the rest eluent after adsorption for subsequent screening. And (3) taking a sterile centrifuge tube, and inoculating the prepared overnight host bacteria into an LB culture medium to form pre-logarithmic host bacteria liquid. All the post-adsorption eluents were added to the pre-logarithmic host bacterial solution and amplified by rapid oscillation at 37 ℃ and 200rpm for 5 hours. The amplified phage solution was centrifuged at 12000rpm for 10min at 4 ℃ and the supernatant was transferred to a new centrifuge tube and centrifuged again under the same conditions. Carefully taking the supernatant of 80% of the upper part of the centrifuge tube into a new centrifuge tube, adding one sixth volume of PEG/NaCl into the centrifuge tube, and standing in a refrigerator at 4 ℃ for overnight precipitation. The next day, the 50ml tube from the previous day was centrifuged at 12000rpm at 4 ℃ for 15min, the supernatant was discarded, and the same conditions were repeated for 2min, and the remaining supernatant was discarded. The pellet was resuspended in 1ml TBS and transferred to a sterile EP tube and centrifuged at 14000rpm for 5min at 4 ℃. The supernatant was transferred to a new EP tube, added again one sixth volume of PEG/NaCl and allowed to settle on ice for 1 hour. Centrifuging at 14000rpm for 10min at 4 deg.C, removing supernatant, and retaining precipitate. The pellet was resuspended in 200ul TBS, centrifuged at 1000rpm for 1min at 4 ℃ and the supernatant was kept in fresh EP tubes (this is the post-amplification phage solution) and stored at-20 ℃ in a freezer.

2.5 second to third rounds of screening

The basic steps of the screening are the same as the first round. Phage liquid amplified in the previous round is selected as a secondary peptide library in each next round of screening, the phage input amount basically consistent with that in the first round is kept as much as possible in each round, and three rounds of targeted screening are carried out totally. The phage titer was determined for each round of screened eluents and phage recovery was calculated.

2.6 Positive monoclonal phage selection and Single-stranded DNA extraction and sequencing

2.6.1 Positive monoclonal phage selection

The phage liquid obtained after the third round of screening is subjected to titer measurement, LB plates are paved, and 40 blue spots which grow well at intervals of 5mm are randomly picked on the plates with the number of growing spots less than 100. 40 randomly picked blue spots were added to 1ml of the logarithmic precursor host bacterial solution (amplified with phage) and amplified at 37 ℃ for 4.5 hours with rapid oscillation at 200 rpm.

2.6.2 extraction of Positive monoclonal phage Single-stranded DNA

And (3) centrifuging the amplified monoclonal phage liquid at 4 ℃ and 14000rpm for 30 seconds respectively, taking the supernatant to transfer to a new tube, centrifuging at 4 ℃ and 1000rpm for 30 seconds, taking 80 percent of the supernatant to transfer to a new nuclease-free centrifuge tube, adding 300ul of glycerol into 300ul of bacterial liquid according to the proportion of 1:1, and freezing and storing in a refrigerator at-20 ℃, thus obtaining the amplified monoclonal phage liquid. Adding 200ul PEG into the remaining 500ul of bacterial liquid, uniformly mixing, standing at room temperature for 20min, centrifuging at 4 degrees for 14000rpm, 10min, discarding the supernatant, centrifuging at 4 degrees for 14000rpm for 3min, discarding the supernatant, adding 100ul NaI, uniformly mixing, adding 250ul absolute ethyl alcohol, standing for 10min, centrifuging at 4 degrees for 10000rpm for 10min, discarding the supernatant, slightly washing with precooled 70% ethyl alcohol for 3 times, air drying for 30min, centrifuging at 10000rpm for 5min, discarding the supernatant, and adding 60ul TE.

2.6.3DNA purification

Taking the last step of 60ul of TE tube, adding 40ul of TE to make up to 100 ul. 500ul of Buffer B3 was added to the tube and mixed well. The mixture was transferred to an adsorption column at room temperature at 8000g, centrifuged for 30 seconds, and the filtrate was added to the adsorption column again and passed through the column again. The liquid in the collecting tube is poured off, and the adsorption column is put back into the collecting tube. Mu.l of Wash Solution, 9000g, was added to the column and centrifuged for 30 seconds. The liquid in the collecting pipe is poured out, and the adsorption column is put into the collecting pipe again. Repeating the above steps, placing the empty adsorption column and collection tube into a centrifuge, and centrifuging for 1min at 9000 g. Add 40. mu.l of Elution Buffer to the center of the adsorption membrane and let stand at room temperature for 2 min. The DNA solution obtained was subjected to centrifugation at 9000g for 1min for sequencing.

The results are shown in fig. 2, fig. 2 is the targeted screening result of Annexin a2 specific binding positive polypeptide, wherein fig. 2 (a) is a diagram of three rounds of targeted screening titer experiments on Annexin a2 specific binding positive polypeptide by using phage display technology, and A, B, C is a first, second and third rounds of screening titer experiments blue-white spot plate respectively. Table 1 shows the phage input amount, the recovered phage titer and the phage recovery rate of three rounds of targeted screening, and the results show that the positive monoclonal phage is significantly enriched in the third round. FIG. 2B is a diagram showing the result of DNA sequencing of the phage having the highest repetition rate positive, and the amino acid sequence thereof is YWRGYN (YW 7). The sequence is compared with the known protein polypeptide sequence to analyze the homology and the similarity, and the result shows that the sequence has no homology with the known protein polypeptide sequence and no similarity with the nucleotide sequence.

TABLE 1 phage input, recovered phage titer and recovery for three rounds of subtractive selection

Example 3 solid phase Synthesis and identification of Positive Polypeptides YW7 and FITC-YW7

According to the determined amino acid sequence, carrying out homology comparison analysis on the amino acid sequence and bioinformatics analysis on the nucleic acid sequence. Polypeptide YW7 (with the sequence of YWRGYN) and FITC-positive polypeptide fragment FITC-YW7 (with the sequence of YWRGYN) were synthesized by solid phase synthesis, and the synthesized polypeptides were identified. Meanwhile, the polypeptide YW7 and the FITC-positive polypeptide fragment FITC-YW7 are synthesized by the company of Biotechnology engineering (Shanghai) GmbH.

3.1 preparation of polypeptide YW7

3.1.1 adopting Fmoc solid phase synthesis polypeptide strategy, and bonding Fmoc-Asn (pbf) -OH as raw material with Wang resin. Then after removing Fmoc group, carrying out condensation reaction with Fmoc-Tyr (tBu) -OH in the presence of a condensing agent DIC and HOBt to complete the connection of a second amino acid; then removing Fmoc, and carrying out condensation reaction with a third amino acid Fmoc-Val (Boc) -OH; according to the procedure, the side chain-protected Fmoc group-free resin was obtained by sequential synthesis from the C-terminus to the N-terminus, (amino acids used include Fmoc-Gly-OH, Fmoc-Arg-OH and Fmoc-Trp-OH) until the synthesis was completed.

3.1.2 adding a cutting reagent into the obtained peptide resin, reacting for 2 hours at 20 ℃ in the dark, and filtering; collecting the filtrate, concentrating by a rotary evaporator, adding precooled anhydrous ether with the volume about 10 times of that of the filtrate, precipitating for 3 hours at the temperature of minus 20 ℃, separating out white powder, collecting the precipitate, washing the precipitate by the anhydrous ether, and drying in vacuum to obtain the polypeptide, wherein the cutting reagent is prepared by mixing TFA, water and TIS (triisopropylchlorosilane) according to the mass ratio of 96:1.5: 2.5.

3.1.3 purifying by using a C18 reversed-phase preparation column, collecting a main peak, and freeze-drying to obtain a white solid, namely a product; the product is identified by mass spectrum, and the molecular weight shown in the mass spectrum is basically consistent with the theoretical molecular weight.

3.2 preparation of FITC-Positive polypeptide fragment FITC-YW7

3.2.1 adopting Fmoc solid phase synthesis polypeptide strategy, taking Fmoc-YW7-Wang resin as raw material, removing Fmoc group, and then carrying out condensation reaction with Fmoc-Acp-OH in the presence of a condensing agent DIC and HOBt; then removing Fmoc, and carrying out condensation reaction with FITC-Cl.

3.2.2 adding a cutting reagent into the obtained peptide resin, reacting for 2 hours at 20 ℃ in the dark, and filtering; collecting the filtrate, concentrating by a rotary evaporator, adding precooled anhydrous ether with the volume about 10 times of that of the filtrate, precipitating for 3 hours at the temperature of minus 20 ℃, separating out white powder, collecting the precipitate, washing the precipitate by the anhydrous ether, and drying in vacuum to obtain the polypeptide, wherein the cutting reagent is prepared by mixing TFA, water and TIS (triisopropylchlorosilane) according to the mass ratio of 96:1.5: 2.5.

3.2.3 purifying by using a C18 reversed-phase preparation column, collecting a main peak, and freeze-drying to obtain a white solid, namely a product; the product is identified by mass spectrum, and the molecular weight shown in the mass spectrum is basically consistent with the theoretical molecular weight.

The results are shown in FIG. 3, FIG. 3 is a mass spectrometric identification chart of polypeptide YW7 synthesized in solid phase and FITC-positive polypeptide fragment FITC-YW 7; wherein, FIG. 3 (A) is the mass spectrum of polypeptide YW7 synthesized by solid phase, and FIG. 3 (B) is the mass spectrum of FITC-positive polypeptide fragment FITC-YW7 synthesized by solid phase. The mass spectrum result shows that the polypeptide YW7 synthesized by the solid phase and the FITC-positive polypeptide fragment FITC-YW7 are respectively consistent with the theoretical values thereof, and are consistent with the molecular weights and other results of the polypeptide synthesized by the biological engineering (Shanghai) corporation and the FITC-positive polypeptide fragment. The polypeptide synthesized by solid phase synthesis meets the quality requirement and can be used for subsequent effect evaluation.

Example 4 verification of the affinity of the Positive polypeptide YW7 to Annexin A2 protein

4.1 MOE docking polypeptide YW7 with Annexin A2 protein identification affinity

Based on the induced fit effect existing in the process of protein-ligand interaction, the Annexin A2 in the PDB database is placed at the active site of a target molecule one by one, the optimal conformation of the action of the Annexin A2 and the polypeptide YW7 is searched by continuously optimizing the position and conformation of the polypeptide YW7, the binding mode and the affinity of the ligand are predicted, and the ligand which is close to the natural conformation and has the optimal affinity with a receptor is selected through a scoring function.

4.2 MST (microcalorimetric surge apparatus) identification of the affinity of the polypeptide YW7 to Annexin A2

In the MST detection, the concentration of the polypeptide YW7 is kept unchanged, meanwhile, the unlabeled Annexin A2 protein is diluted in a gradient way, and the reaction solution is MST Buffer (contain 0.05% Tween-20). After a short period of binding reaction, the sample was loaded into a nt.115 standard capillary and measured by Monolith nt.115.

The results are shown in fig. 4, fig. 4 is the verification result of the affinity of the positive polypeptide YW7 and Annexin A2, wherein fig. 4 (A) is a graph of the identification result of the affinity of the MOE docking polypeptide YW7 and Annexin A2, the MOE molecular docking result shows that the polypeptide YW7 can be well combined with the protein Annexin A2, and the S score is-66.078. The Tyr1-Trp2-Arg3 of the polypeptide YW7 forms a conformation similar to that of cyclic peptide and is positioned at the inner side of an active pocket, and the benzene ring of the Tyr1 forms hydrophobic interaction with Leu 300; gly4-Val5-Tyr6 then assumes a stretched linear conformation, lying outside the active pocket, in which the amide NH of Val5 forms a hydrogen bond with the terminal carbonyl of Asn 61. FIG. 4 (B) is a MST chart showing the affinity of polypeptide YW7 with Annexin A2, and shows that polypeptide YW7 has affinity with Annexin A2, and Kd value is 12 μ M.

Example 5 in vitro identification of the Capacity of the polypeptide FITC-YW7 to specifically target binding to pancreatic cancer cells overexpressing Annexin A2

5.1 immunofluorescence identification of binding Capacity of polypeptide FITC-YW7 to pancreatic cancer Panc-1 cells

HPDE6-C7 and PANC-1 cells were plated in 24-well plates with slides, plated in cell incubators for cell attachment and plating. After 24 hours, the medium was discarded and washed 3 times with PBS, with gentle shaking for 5min each time. Fixing with 4% paraformaldehyde for 10 min. PBS was washed 3 times, and the mixture was shaken slightly for 5min each time. Permeabilize with PBS containing 0.5% TritonX-100 for 10 min. PBS was washed 3 times, and the mixture was shaken slightly for 5min each time. Blocking with 3% BSA, and standing at room temperature for 15 min. Adding 0 μ M, 0.75 μ M, 3 μ M polypeptide FITC-YW7, standing at 37 deg.C for 5 min. PBS was washed 3 times, and the mixture was shaken slightly for 5min each time. Adding DAPI dye solution, and standing at 37 ℃ for 15 min. The slide glass in the 24-well plate is taken out, inverted and covered on the slide glass with the anti-fluorescence quenching sealing tablet, and stored in a dark 4-DEG wet box. Confocal microscopy locates FITC-YW7 in the cell location and identifies the specific targeting of the polypeptide FITC-YW7 to pancreatic cancer cells overexpressing Annexin A2.

5.2 flow cytometry to identify the binding Capacity of the polypeptide FITC-YW7 to pancreatic cancer Panc-1 cells

HPDE6-C7 and PANC-1 cells were plated in 6-well plates with slides, plated in cell incubators for cell attachment and monolayer plating. After 24 hours, the medium was discarded, trypsinized for 1min, centrifuged at 1000rpm for 5min, the digestion was stopped with 10% serum, the supernatant was discarded, washed 1 time with PBS, centrifuged at 1000rpm for 5min, and the supernatant was discarded. Adding 0.75 μ M polypeptide FITC-YW7, mixing well, and incubating at 37 deg.C for 5 min. Centrifuge at 1000rpm for 5min and discard the supernatant. PBS was added for washing, and the mixture was centrifuged at 1000rpm for 5min at room temperature, and the supernatant was discarded. 300ul PBS was added and mixed well. And (4) detecting by using a flow cytometer.

5.3 immunofluorescence identification of Co-localization of polypeptide FITC-YW7 and Annexin A2 on pancreatic cancer Panc-1 cell surface

And (3) paving the PANC-1 cells in a 24-well plate with a glass slide, putting the cells in a cell incubator to culture the cells to adhere to the wall and paving a monolayer. After 24 hours, the medium was discarded and washed 3 times with PBS, with gentle shaking for 5min each time. Fixing with 4% paraformaldehyde for 10 min. PBS was washed 3 times, and the mixture was shaken slightly for 5min each time. Permeabilize with PBS containing 0.5% TritonX-100 for 10 min. PBS was washed 3 times, and the mixture was shaken slightly for 5min each time. Blocking with 3% BSA, and standing at room temperature for 15 min. The Annexin A2 antibody is added in a proportional dilution, and the mixture is incubated at 4 ℃ and standing overnight. PBS was washed 3 times, and the mixture was shaken slightly for 5min each time. Goat anti-rabbit IgG fluorescent secondary antibody (TRITC) is added in proportion, and the mixture is incubated for 1.5 hours at a standing temperature of 37 ℃. 0.75 μ M of the polypeptide FITC-YW7 was added, and the mixture was incubated at 37 ℃ for 5 min. PBS was washed 3 times, and the mixture was shaken slightly for 5min each time. Adding DAPI dye solution, and standing at 37 ℃ for 15 min. The slide glass in the 24-well plate is taken out, inverted and covered on the slide glass with the anti-fluorescence quenching sealing tablet, and stored in a dark 4-DEG wet box. Confocal microscopy detects the positions of green fluorescence and red fluorescence on cells, and identifies whether polypeptides FITC-YW7 and Annexin A2 are co-localized on the cell surface.

The results are shown in FIG. 5, and FIG. 5 is a graph showing the in vitro identification result of the specific targeting binding capacity of the polypeptide FITC-YW7 to pancreatic cancer Panc-1 cells. Wherein, FIG. 5 (A) is a fluorescence result chart and a bar chart for verifying the specific targeting binding capacity of the polypeptide FITC-YW7 and pancreatic cancer Panc-1 cells by immunofluorescence, the fluorescence result chart and the bar chart show that when pancreatic cancer cells PANC-1 and HPDE6-C7 are respectively incubated with 0 mu M, 0.75 mu M and 3 mu M FITC-YW7 for 5min, the fluorescence intensity of the polypeptide FITC-YW7 bound with pancreatic cancer cells over-expressing Annexin A2 is increased along with the increase of the concentration of FITC-YW7, and higher fluorescence intensity and difference (P) can be shown at 0.75 mu M (P is P.<0.05）。*P<0.05，**P<0.01，***P<0.001. FIG. 5 (B) is an experimental result chart and a bar chart for identifying the specific targeting binding capacity of the polypeptide FITC-YW7 to pancreatic cancer Panc-1 cells by flow cytometry, and the experimental result chart and the bar chart show that the polypeptide FITC-YW7 has the specific targeting binding capacity to pancreatic cancer Panc-1 cells. PANC-1 and HPDE6-C7 were incubated with 0.75. mu.M FITC-YW7, respectively, for 5min, and strong fluorescence signals were observed on PANC-1 cells, while weaker fluorescence signals were detected on HPDE6-C7 cells. *P <0.05，**P<0.01，***P<0.001. FIG. 5 (C) is a fluorescence result graph and a bar graph for immunofluorescence to identify that polypeptide FITC-YW7 and Annexin A2 are co-localized on the surface of pancreatic cancer Panc-1 cells, wherein the fluorescence result graph and the bar graph show that polypeptide FITC-YW7 and Annexin A2 are co-localized on the surface of cells and the cross coverage rate is more than 60% when the Annexin A2 antibody and polypeptide FITC-YW7 are used for co-incubation with pancreatic cancer cells. P<0.05，**P<0.01，***P<0.001。

It can be concluded from the experimental examples that the polypeptide of the invention has the property of targeting annexin a 2. Can be used as a polypeptide molecular probe to accurately screen tumors over-expressing Annexin A2; can also be used as tumor-oriented polypeptide, coupled with anti-tumor drugs, used as a target to increase the content of drugs or drug-loaded carriers such as nano materials, liposomes and the like in cells over-expressing Annexin A2, and then added with pharmaceutically acceptable auxiliary materials or adjuvants to prepare novel more effective targeted anti-cancer drugs. For targeted therapy and imaging of a variety of Annexin a2 overexpressing tumors; the polypeptide can also be optimized to be used as a polypeptide inhibitor to block the interaction of Annexin A2 and related proteins.

<110> Liaoning science and technology research and development center Limited

<120> affinity short peptide for targeted recognition of annexin A2, and preparation method and application thereof

<140> 2019107668539

<141> 2019－08－20

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 7

<212> PRT

<213> Artificial sequence

<400> 1

Tyr Trp Arg Gly Val Tyr Asn

1 5