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Affinity short peptide for targeted recognition of Annexin A2 and preparation method and use of affinity short peptide

An annexin and target recognition technology, which is applied in the preparation methods of peptides, chemical instruments and methods, peptide/protein components, etc., can solve problems such as hindering the improvement of curative effect, and achieve simple and large-scale production, good effect and high benefit. Effect

Active Publication Date: 2021-02-26
辽宁医学诊疗科技研发中心有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This treatment is based on the use of small molecules with specificity against abnormal or dysregulated proteins in cancer cells, for example, tyrosine phosphatase inhibitors, in the treatment of non-small cell lung cancer with sensitive mutations in EGFR (Epidermal Growth Factor Receptor) , the curative effect is remarkable, but the emergence of drug resistance genes is currently the main obstacle to further improve the curative effect

Method used

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  • Affinity short peptide for targeted recognition of Annexin A2 and preparation method and use of affinity short peptide
  • Affinity short peptide for targeted recognition of Annexin A2 and preparation method and use of affinity short peptide
  • Affinity short peptide for targeted recognition of Annexin A2 and preparation method and use of affinity short peptide

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Experimental program
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Effect test

Embodiment 1

[0035] Example 1 Recombination and purification of Annexin A2

[0036] 1.1 Transformation of the constructed Annexin A2 expression plasmid

[0037] Thaw the competent cells on ice. Add plasmid to competent cells, mix gently, and let stand on ice for 5 minutes. Heat shock at 42 degrees for 90 seconds. Quickly transfer to ice and cool for 3 min. Add non-resistant LB medium, 37 degrees. Dip the mixture with a glass rod and spread it evenly on the AMP plate medium. Place the plate in a 37 degree incubator overnight. The next day, check the growth of the colonies and store them in a 4-degree refrigerator.

[0038] 1.2 Massive amplification of Annexin A2

[0039] Shake the bacteria Annexin A2 overnight, add AMP and LB (1:1000) in the Erlenmeyer flask. 37 degrees, 200rpm shake overnight. The next day, store Annexin A2 overnight and freeze at -20°C. Add LB, bacterial solution and AMP into the Erlenmeyer flask, and shake the bacteria at 37 degrees and 180 rpm for 4 hours. Ad...

Embodiment 2

[0043] Example 2 Targeted screening of Annexin A2 protein specific binding positive polypeptide

[0044] 2.1 Recovery and cultivation of host bacteria E.coli ER2738

[0045] To prepare an E. coli plate, take the LB-TET culture plate and preheat it in a 37-degree incubator for 1 hour. After the E.coli ER2738 bacterial liquid melts, use an inoculation loop to dip a small amount of bacterial liquid evenly on the culture plate, and then place it upside down at 37°C overnight in a constant temperature incubator. Prepare the host bacterial solution, pick a single colony from a well-grown culture plate, place it in LB bacterial culture solution containing tetracycline, and cultivate overnight at 37°C with shaking at 180rpm, so that the bacteria are in the logarithmic growth phase. The prepared LB-Tet plate containing Escherichia coli was stored in a 4°C refrigerator for later use, and the host bacterial solution was stored in a -80°C refrigerator for later use.

[0046] 2.2 Targete...

Embodiment 3

[0076] Example 3 Solid phase synthesis and identification of positive polypeptide YW7 and FITC-YW7

[0077] According to the measured amino acid sequence, the homology comparison analysis of its amino acid sequence and the bioinformatics analysis of its nucleic acid sequence are carried out. Polypeptide YW7 (sequence YWRGVYN) and FITC-positive polypeptide fragment FITC-YW7 (sequence YWRGVYN) were synthesized by solid-phase synthesis, and the synthesized peptides were identified. At the same time, the above polypeptide YW7 and FITC-positive polypeptide fragment FITC-YW7 were synthesized at Sangon Bioengineering (Shanghai) Co., Ltd.

[0078] 3.1 Preparation of polypeptide YW7

[0079] 3.1.1 The strategy of Fmoc solid-phase synthesis of peptides was adopted, and Fmoc-Asn(pbf)-OH was used as raw material to bond with Wang resin. Then take off the Fmoc group, then carry out condensation reaction with Fmoc-Tyr(tBu)-OH in the presence of condensing agents DIC and HOBt, and complete...

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Abstract

The invention relates to affinity short peptide for the targeted recognition of Annexin A2 and a preparation method and use of the affinity short peptide. The affinity short peptide can specifically target Annexin A2, and especially the new possibility is provided for the efficient and targeted recognition of tumor tissues and the prediction and improvement of tumor targeted therapy based on the characteristic of high expression of Annexin A2 in most tumors. The polypeptide can be used as a polypeptide molecular probe, or a tumor-directing polypeptide which can be coupled with an anti-tumor drug, is used as a target head to increase the content of the drug or drug-loaded carriers such as a nano-material, liposome and the like in cells over-expressing Annexin A2, and is added with a pharmaceutically acceptable auxiliary material or adjuvant to prepare a novel and more effective targeted anti-cancer drug. The polypeptide provided by the invention 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 has the specific targeting effect on Annexin A2 and is high in selectivity. The peptide involved in the invention can be prepared through a chemical synthesis method, andis high in purity, small in molecular weight, high in specificity, free of immunogenicity, and safe and reliable.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application of a polypeptide with high affinity to annexin A2 and good binding specificity and sensitivity. Background technique [0002] Cancer is the second leading cause of death worldwide, after cardiovascular disease. At present, the clinical treatment of malignant tumors is mainly based on surgery, chemotherapy and radiotherapy, and it is difficult to achieve satisfactory curative effect. Among them, chemotherapy is the fastest-growing method in tumor treatment in recent years. However, while chemotherapy drugs kill tumor cells, they also kill normal cells in the human body, which has severe side effects. Drug therapy is one of the important methods for the systemic treatment of malignant tumors. Traditional anti-tumor chemotherapeutic drugs have poor targeting and cannot distinguish between normal tissue and tumor tissue. The drug is quickly eliminated from the blood, a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/06C07K1/20C07K1/06C07K1/04A61K47/62A61K38/08A61P35/00
CPCC07K7/06A61K47/62A61P35/00A61K38/00
Inventor 魏敏杰
Owner 辽宁医学诊疗科技研发中心有限公司
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