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EDB-FN protein targeting peptide with high affinity and application thereof

An EDB-FN, targeting peptide technology, applied in the fields of molecular biology and immunology, can solve the problems affecting the application of short peptides and low specificity, and achieve the effect of overcoming delivery difficulties, high specificity, and easy delivery and distribution.

Active Publication Date: 2018-04-13
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the molecular structure of short peptides is relatively simple, and the specificity is usually not high when used as targeting markers. The lack of specificity also affects the application of short peptides as targeting molecules.

Method used

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  • EDB-FN protein targeting peptide with high affinity and application thereof
  • EDB-FN protein targeting peptide with high affinity and application thereof
  • EDB-FN protein targeting peptide with high affinity and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1. Screening of EDB-FN Protein Targeting Peptides

[0049] This method utilizes the immobilized recombinant EDB-FN protein and the dodecapeptide pool (NEB company) to mix and screen, and the steps are as follows:

[0050] 1. Prokaryotic expression and purification of EDB-FN protein

[0051] The constructed pGEX-4T-3-EDB-FN plasmid was transferred into the expression strain of BL21, the protein was induced to express by IPTG at 16°C, the protein was purified by GST column, and the recombinant EDB-FN protein with a purity greater than 90% was obtained.

[0052] 2. The screening of the phage dodecapeptide of EDB protein, the steps are as follows:

[0053] (1) Using the solid-phase screening method, dilute the EDB protein to 30-100ug / uL with 1xPBS, coat it on the ELISA strip, add 100uL to each well, and coat overnight at 4°C;

[0054] (2) Wash three times with PBS, add 300uL 4% skimmed milk to each well, and block at 37°C for 2 hours;

[0055] (3) After washing t...

Embodiment 2

[0067] Example 2. Determination of the Binding Ability of Dodecapeptide A16 and EDB-FN Protein

[0068] The nucleic acid sequence of dodecapeptide A16 was inserted into the PET28a plasmid, fused with the EGFP coding sequence for expression, transferred to BL21 for recombinant expression, and purified to obtain the fusion protein of dodecapeptide A16 and EGFP.

[0069] The EDB-FN protein recombinantly expressed in Example 1 was biotinylated with Thermo Scientific's EZ-link Sulfo-NHS-LC-Biotin and BiothnylationKits biotinylation kit, so that each EDB-FN protein was labeled with 2 biological white.

[0070] The instrument used: Octet RED / QK was used to determine the binding force of biotinylated EDB-FN protein to recombinant A16-EGFP.

[0071] Dilute the biotinylated EDB protein to 10ug / ml (200 μL per well), and dilute dodecapeptide A16 to 80 μg / mL, 40 μg / mL and 20 μg / mL (200 μL per well). The dilution solution is PBS+0.1% Tween -20+0.1% BSA, add sample according to Table 2:

...

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Abstract

The invention relates to a fibronectin EDB subtype specific targeting short peptide A16 with an amino acid sequence of ATYRLFQGVEVV. The targeting short peptide A16 can specific recognize EDB-FN protein expressed by tumor cells, has high affinity to EDB-FN and is capable of carrying functional molecules with diagnosis or therapeutic activity to tumor cells to perform diagnosis or treatment effect.

Description

technical field [0001] The invention belongs to the technical field of molecular biology and immunology, and specifically relates to phage display peptide library technology and recombinant expression technology, in particular to the screening, identification, preparation and application of a high-affinity EDB-FN protein targeting peptide. Background technique [0002] As we all know, malignant tumors are in a unique microenvironment, which promotes the growth, divergence and metastasis of tumor cells. Tumor-related proteins are usually abnormally expressed in the extracellular matrix of tumors, among which fibronectin is one of the most important proteins in the extracellular matrix of tumors. play a key role. In addition, long-term scientific research has found that an important subtype of fibronectin, extradomain-B fibronectin (EDB-FN), is highly related to the growth process of tumors in patients, and has become a recognized tumorigenesis in the medical field. Importan...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C12N15/11G01N33/574A61K47/64A61P35/00
CPCC07K7/08G01N33/57484G01N2333/78
Inventor 林坚陈航宇李承鹏周斌陈鹏
Owner PEKING UNIV
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