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Polypeptide for enrichment, separation and detection of circulating tumor cells and application thereof

A tumor cell and polypeptide solution technology, which is used in the enrichment, separation and detection of circulating tumor cell polypeptides and its application field, can solve the problem of high cost of detecting CTCs

Active Publication Date: 2014-07-16
北京中科纳泰科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to provide a new idea and new method for enriching, separating and detecting CTCs in view of the current high cost of detecting CTCs

Method used

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  • Polypeptide for enrichment, separation and detection of circulating tumor cells and application thereof
  • Polypeptide for enrichment, separation and detection of circulating tumor cells and application thereof
  • Polypeptide for enrichment, separation and detection of circulating tumor cells and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0108] Example 1. Verification of polypeptide-specific recognition of EpCAM

[0109] 1. The sequence of the peptide used, reagents and verification cells

[0110] Peptide fragments (synthesized by Shanghai Keyept Biotechnology Co., Ltd., with a purity of 98 mass%) are shown in Table 1 below. Magnetic nanoparticles: streptavidin-magnetic nanoparticles (solulink, ST022912, USA).

[0111] Table 1

[0112]

[0113] MCF-7 cells, which are known to be consistent with reports, were regarded as EpCAM-positive cells, and HL-60 cells were regarded as EpCAM-negative cells to verify the specific recognition of the polypeptide fragment of EpCAM.

[0114] 2. Specific method

[0115] 1) Verification of EpCAM-positive specific peptides.

[0116] Firstly, a series of FITC-peptides designed and synthesized were made into 1 mg / ml mother solution with PBS with pH=7.2; a certain amount (≥10 6 1) MCF-7 and HL-60 cells in a 1.5 μl centrifuge tube, add 7 μl of each polypeptide mother soluti...

Embodiment 2

[0127] Example 2. Specific recognition of polypeptides Pep7-2 and Pep7-3 by biotin-streptavidin Interaction and magnetic nanoparticle coupling

[0128] 1. Take 250 μl of 5 mg / ml magnetic nanoparticles, mix them on a vortex mixer (WH-866, Taicang Hualida Experimental Equipment Co., Ltd.) for 3~5 minutes, collect them with a strong magnetic magnet, and remove the solvent. Then take 500 μl of 1 mg / ml FITC-Pep7-2-biotin (Biotin) and 1 mg / ml FITC-Pep7-3-biotin (Biotin) in PBS and mix them with magnetic nanoparticles (polypeptides and magnetic nanoparticles The mass ratio of the particles is 2:5), and then placed on a shaker (THZ-D, Taizhou Wanda Gas Spring Co., Ltd.) and mixed for 40min at 25°C and 150rpm. Then use a strong magnetic magnet to enrich the magnetic nanoparticles on one side of the centrifuge tube, absorb the liquid from the other side of the centrifuge tube after about 10 minutes, then add a certain amount of PBS buffer, resuspend the magnetic nanoparticles, enric...

Embodiment 3

[0130] Example 3. Characterization of polypeptide-magnetic nanoparticles

[0131] Take the FITC-Pep7-2-biotin (Biotin)-magnetic nanoparticle and FITC-Pep7-3-biotin (Biotin)-magnetic nanoparticle solution prepared in 1 in Example 2 (the mass of the polypeptide and the magnetic nanoparticle The ratio is 2:5) a little, take pictures under the microscope, the result is as follows figure 2 As shown, A and B are photos of Pep7-2-magnetic nanoparticles under bright field and blue light excitation respectively, and the magnetic nanoparticles modified with Pep7-2 emit green light under blue light (the absorption and emission wavelengths of FITC are 495nm and 535nm); C and D are the photos of Pep7-3-magnetic nanoparticles excited by bright field and blue light, respectively, and the magnetic nanoparticles modified with pep7-3 emit green light under blue light; indicating that the peptide and magnetic nanoparticles are successfully coupled .

[0132] Use FITC-Pep7-2-biotin (Biotin) ...

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Abstract

The invention provides a polypeptide for enrichment, separation and detection of circulating tumor cells and application thereof. The amino acid sequence of the polypeptide is shown as SEQ ID NO:1. The invention also includes a polypeptide-magnetic nanoparticle, which contains the magnetic nanoparticle and the polypeptide that is coupled to the magnetic nanoparticle and used for specific recognition of tumor cells. In addition, the invention discloses application of the polypeptide and the polypeptide-magnetic nanoparticle in preparation of products for enriching, separating or detecting circulating tumor cells or drugs for treating tumor metastasis related diseases. The invention provides a new method for utilizing a polypeptide cell surface antigen to conduct specific recognition, puts forward the new polypeptide-magnetic nanoparticle substance for enrichment, separation and detection of CTC, provides a fast, accurate and low-cost detection technology for enrichment, separation and detection of CTC, and provides an effective method for diagnosis, treatment and prognosis of clinical tumor metastasis patients.

Description

technical field [0001] The invention belongs to the technical field of biomedicine and relates to a polypeptide, in particular to a polypeptide-magnetic nanoparticle that can be used for enrichment, separation and detection of circulating tumor cells (Circulating Tumor Cell, CTC) and its preparation method and application. The method is accurate and fast, and greatly reduces the cost of enrichment, separation and detection of CTC cells with high expression of Epithelial Cell Adhesion Molecule (EpCAM). Background technique [0002] Cancer is one of the major diseases that threaten human beings. Invasion and metastasis are one of the most prominent features of malignant tumors. Tumor cells detach from the primary tumor spontaneously or due to diagnostic and therapeutic operations, and undergo Epithelial-Mesenchymal Transition (EMT) , so that it has flow characteristics and enters the peripheral blood to form circulating tumor cells (CTC). Therefore, the detection of CTCs in p...

Claims

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Application Information

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IPC IPC(8): C07K14/00C12N5/09G01N33/68G01N21/64A61K38/16A61P35/04
CPCA61K38/00C07K14/00C07K17/14C12N5/0693
Inventor 王琛白林灵杜一萌杨延莲
Owner 北京中科纳泰科技有限公司
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