Nucleic acid aptamer sequence for recognizing and being combined with human transferrin receptor and application of nucleic acid aptamer sequence

A nucleic acid aptamer and transferrin technology, applied in the field of biochemical analysis, can solve the problems of large batch differences in antibody activity, prone to interactive reactions, and high price, achieving the effects of no immunogenicity, short cycle, and easy storage.

Active Publication Date: 2016-12-07
INST OF CHEM CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the main detection method of transferrin receptor is enzyme-linked immunoassay (ELISA), but its steps are cumbersome, time-consuming, expensive, and the antibody activity varies widely among batches, and it is prone to interactive reactions. Intuitive and specific detection of human transferrin receptor has promising applications in clinical diagnosis and treatment

Method used

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  • Nucleic acid aptamer sequence for recognizing and being combined with human transferrin receptor and application of nucleic acid aptamer sequence
  • Nucleic acid aptamer sequence for recognizing and being combined with human transferrin receptor and application of nucleic acid aptamer sequence
  • Nucleic acid aptamer sequence for recognizing and being combined with human transferrin receptor and application of nucleic acid aptamer sequence

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Example 1, Screening and Preparation of Nucleic Aptamers

[0079] 1. Culture of paclitaxel-resistant colon cancer cells and sensitive colon cancer cells

[0080] 1. Culture of paclitaxel-resistant colon cancer cells

[0081] Paclitaxel-resistant colon cancer cells (HCT-8T) (purchased from Jiangsu KGI Biotechnology Co., Ltd. (Nanjing)) were treated with RPMI 1640 (containing 10% fetal bovine serum, 1% penicillin / streptomycin) supplemented with 0.1 μg / mL paclitaxel Cultured, inoculated into RPMI 1640 (containing 10% fetal bovine serum, 1% penicillin / streptomycin) without adding paclitaxel and cultured for one generation before use.

[0082] 2. Culture of sensitive colon cancer cells

[0083] Sensitive colon cancer cells (HCT-8) (purchased from Jiangsu KGI Biotechnology Co., Ltd. (Nanjing)) were cultured with RPMI1640 (containing 10% fetal bovine serum, 1% penicillin / streptomycin).

[0084] All the above cells were routinely cultured in an incubator (37°C, 5% CO 2 ), p...

Embodiment 2

[0116] Example 2. Binding Ability of Nucleic Aptamer to Paclitaxel-resistant Colon Cancer Cell HCT-8T

[0117] The nucleic acid aptamer 2, nucleic acid aptamer 3, nucleic acid aptamer 4, nucleic acid aptamer 5 and nucleic acid aptamer 6 obtained in Example 1 were respectively labeled with fluorescein (FAM) molecules at the 5' end to obtain FAM labeled nucleic acid aptamers 2. FAM-labeled aptamer 3, FAM-labeled aptamer 4, FAM-labeled aptamer 5 and FAM-labeled aptamer 6 (synthesized by Shanghai Sangong Co., Ltd.). Each single-stranded DNA (FAM-labeled DNA sequence) was dissolved in binding buffer, and the concentration was calibrated according to ultraviolet absorption, heated at 95°C for 5 minutes, placed on ice for 10 minutes, and left at room temperature for 30 minutes. The denatured-refolded DNA is diluted with binding buffer to a DNA solution with a concentration gradient of 1nmol / L, 2nmol / L, 5nmol / L, 10nmol / L, 20nmol / L, 50nmol / L, 100nmol / L and 200nmol / L (Molecular Probe S...

Embodiment 3

[0121] Example 3. Mass Spectrometry Identification of Aptamer 2 Specific Recognition and Binding to Transferrin Receptor

[0122] 1. Preparation of isotope-labeled Jurkat E6-1 cells and nucleic acid aptamer 2 derivatives

[0123] 1. Preparation of isotope-labeled Jurkat E6-1 cells

[0124] Heavy isotope-labeled Jurkat E6-1 cells (Institute of Basic Medical Sciences, Academy of Medical Sciences (Beijing)) using heavy isotope-labeled lysine ([ 13 C 6 , 15 N 2 ]-L-lysine) and heavy isotope-labeled arginine ([ 13 C 6 ]-L-arginine) culture medium of RPMI 1640; light isotope-labeled Jurkat E6-1 cells use light-isotope-labeled lysine ([ 12 C 6 , 14 N 2 ]-L-lysine) and light isotope-labeled arginine ([ 12 C 6 ]-L-arginine) PMI1640 medium (Thermo company, culture medium product number: 89982). Cells were cultured for 6-7 passages before use.

[0125] 2. Preparation of aptamer 2 derivatives

[0126] (1) Biotin-labeled aptamer 2

[0127] The biotin-labeled nucleic acid apt...

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Abstract

The invention discloses a nucleic acid aptamer sequence for recognizing and being combined with a human transferrin receptor and application of the nucleic acid aptamer sequence. A nucleic acid aptamer and derivatives thereof can be applied in kits, molecular probes and targeted media for recognizing or preparing and detecting transferrin receptor high-expression tumors and applied in nucleic acid probes for designing, preparing and detecting transferrin receptors. The nucleic acid aptamer has the advantages that the affinity and specificity are higher than those of the prior art, the aptamer is free of immunogenicity, chemical synthesis can be achieved, and the nucleic acid aptamer is small in molecular weight, stable, easy to store and mark and the like.

Description

technical field [0001] The invention belongs to the technical field of biochemical analysis, and specifically relates to a nucleic acid aptamer sequence that recognizes and binds to human transferrin receptor and its application. Background technique [0002] Transferrin receptor (TfR), also known as CD71, is a transmembrane glycoprotein widely present in animal cells. It is a type II homodimeric glycoprotein (180kDa), which is Its main function is to mediate the transmembrane transport and endocytic absorption of iron by interacting with transferrin. Iron is involved in multiple physiological processes such as DNA synthesis in cells, cell proliferation, immune regulation, and electron transfer in the intracellular respiratory chain. The level of expression of transferrin receptor is closely related to the demand of cells for iron. The expression of transferrin receptor is very low on normal cells, and the cells and tissues with vigorous metabolism and need a large amount ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115G01N33/68
CPCC12N15/115C12N2310/16G01N33/68G01N2333/70582
Inventor 上官棣华张楠邴涛刘祥军沈璐瑶
Owner INST OF CHEM CHINESE ACAD OF SCI
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