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Damaged DNA-nanometer gold compound and its preparation method and use

A nano-gold and complex technology, which is applied in the fields of protein enrichment, separation and detection, can solve a large number of specificity problems, and achieve the effect of simplified operation and high protein capture specificity

Inactive Publication Date: 2015-08-26
INST OF CHEM CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The purpose of the present invention is to overcome the defects of the prior art that a large amount of cell lysate is required and the specificity is low, and to provide a damaged DNA-gold nanocomposite with high specificity that can be captured using a small amount of cell lysate and its preparation method , the method for capturing drug-damaged DNA-responsive proteins by the damaged DNA-nano-gold complex and the application of the damaged DNA-nano-gold complex in the capture and / or identification of drug-damaged DNA-responsive proteins

Method used

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  • Damaged DNA-nanometer gold compound and its preparation method and use
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  • Damaged DNA-nanometer gold compound and its preparation method and use

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preparation example Construction

[0034]The method for preparing the damaged DNA-nano gold complex provided by the present invention comprises contacting the drug-damaged double-strand DNA with the nano-gold, so that the drug-damaged double-strand DNA is connected with the nano-gold.

[0035] According to the present invention, in the contact system, the molar ratio of drug-damaged double-stranded DNA to nano-gold is preferably 250-500:1.

[0036] According to the present invention, the contacting conditions can be conventional conditions for linking DNA to gold nanoparticles. Preferably, the contacting method is: incubate at 25-38° C. for 1-2.5 h, add sodium chloride and Make the final concentration of sodium chloride in the contact system 0.05-0.15M, and then stand at 3-5°C for 10-15h. In order to better suppress the non-specific adsorption of the exposed gold nanometer surface to the protein, the method of the present invention also includes adding thiol-modified polyethylene glycol (commercially available)...

Embodiment 1

[0049] This example is used to illustrate the preparation and characterization of the cisplatin-damaged DNA-nano-gold complex of the present invention.

[0050] The sequence of the double-stranded DNA is: 5'-CCTCTCTGGACCTTCC-3' (DNA1, SEQ ID NO:1), HS-5'-GGAAGGTCCAGAGAGG-3' (DNA2, SEQ ID NO:2).

[0051] Cisplatin and single-stranded DNA1 were incubated at 37°C for 2 days at a molar ratio of 0.8:1. The reaction mixture was separated by HPLC to obtain pure cisplatin-crosslinked DNA1. The chromatographic column used was C 8 Column (4.6mm×250mm, 5μm, Agilent), the mobile phase is water and acetonitrile both containing 20mM TEAA. Purified cisplatin-crosslinked DNA1 was dialyzed in aqueous solution (dialysis bag molecular weight cut-off 1kD) for 5 hours to remove acetonitrile and TEAA, and the amount of cisplatin-crosslinked DNA1 was determined by ultraviolet light. 7.5 anneal in a buffer containing 10mM Tris, 50mM NaCl, and 1mM EDTA to form double-stranded DNA damaged by cisplatin...

Embodiment 2

[0055] This example is used to illustrate the method of the present invention for capturing cisplatin-damaged DNA response proteins.

[0056] (1) Tumor cell culture and whole protein extraction

[0057] MCF-7 was cultured, cultured in a 100mm petri dish until the dish was covered, the medium was removed, and the cells were collected with trypsin for cell lysis and protein extraction. After the cells were washed with PBS buffer, 200 μL of RIPA lysate containing newly added PMSF was added, lysed at 4°C for 0.5h, and then centrifuged at 13000g for 4min at 4°C, the supernatant was the cell lysate, and the protein content was determined by BCA kit , the specific operation can also refer to the literature "Akins, R.E.; Tuan, R.S. BioTechniques.1992, 12, 469-499".

[0058] (2) Capture cisplatin-damaged DNA response proteins

[0059] Add 0.2mL of the prepared positive probe (the cisplatin-damaged DNA-nanogold complex obtained in Example 1) and the control probe into PBS buffer and d...

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Abstract

The invention discloses a damaged DNA-nanometer gold compound and its preparation method and use in drug-damaged DNA response protein capture and / or identification, and a method for capturing a drug-damaged DNA response protein by the damaged DNA-nanometer gold compound. In the damaged DNA-nanometer gold compound, a mole of nanometer gold and 7-12 moles of the drug-damaged double-chain DNA are bonded. The damaged DNA-nanometer gold compound can realize high-specificity capture of the drug-damaged DNA response protein only by a less amount of a cell lysate. In capture of the drug-damaged DNA response protein, through the damaged DNA-nanometer gold compound, the captured drug-damaged DNA response protein can be separated from other proteins by a simple centrifugation process so that processes are simplified.

Description

technical field [0001] The invention belongs to the field of protein enrichment, separation and detection, and in particular relates to a damaged DNA-nano-gold complex and a preparation method thereof, a method for the damaged DNA-nano-gold complex to capture drug-damaged DNA responsive proteins, and the damaged Application of DNA-nanogold complexes in the capture and / or identification of drug-damaged DNA-responsive proteins. Background technique [0002] Platinum antineoplastic drugs represented by cisplatin are typical representatives of cytotoxic antineoplastic drugs. Since Rosenberg discovered the biological activity of cisplatin (Cisplatin) to inhibit cell growth in 1969, cisplatin has become the most widely used anti-tumor drug in clinical practice, and the second-generation platinum drugs carboplatin and oxaliplatin have also entered clinical use. After entering the human body, cisplatin can diffuse through the charged cell membrane, enter the cytoplasm, be transpor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/14G01N33/68
Inventor 汪福意杜支凤罗群郭伟
Owner INST OF CHEM CHINESE ACAD OF SCI
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