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Aptamer for osteosarcoma detection and kit for osteosarcoma detection

A kit, a technology for bone cancer, applied in the directions of measuring devices, biochemical equipment and methods, instruments, etc., can solve the problems of limited detection concentration and improved accuracy, and achieve the effects of simple method, time saving, and rapid separation effect

Inactive Publication Date: 2016-07-13
曹帅
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the detection of bone cancer is generally carried out by blood drawing using colloidal gold technology, such as the test paper detection disclosed in CN103267856B, but this detection method has certain limitations, the detection concentration is limited, and the accuracy needs to be improved

Method used

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  • Aptamer for osteosarcoma detection and kit for osteosarcoma detection
  • Aptamer for osteosarcoma detection and kit for osteosarcoma detection
  • Aptamer for osteosarcoma detection and kit for osteosarcoma detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Embodiment 1: nucleic acid aptamer screening

[0073] From a random oligo DNA library synthesized in vitro, 5'-ATTCGACATGCCTGGACATA(N36)CATGCCACATGACCAAGGCA-3'.

[0074] Primers used for enrichment:

[0075] F: 5, -FAM-ATTCGACATGCCTGGACATA-3

[0076] R: 5, -biotin-TGCCTTGGTCATGTGGCATG-3

[0077] After measuring the OD260 of the oligomeric DNA library, centrifuge and dry; dissolve the library with 300ul binding buffer (containing 4.5g / L glucose in PBS, 5mMMgCl2, 2mg / mLBSA, 0.2mg / mL yeast tRNA), and take 250pmol (the first round of 10nmol ), 95°C for 5min, put it on ice quickly, and centrifuge quickly later.

[0078] Reverse screening: 250 pmol of the library was sucked into a culture dish with a diameter of 6 cm of human bone marrow mesenchymal stem cells with a growth coverage of 85%, supplemented to 1 mL with binding buffer; shaken at 4°C for 1.2 h; the supernatant was taken.

[0079] Positive sieve: Add the reverse sieve supernatant to a 6 cm culture dish with a g...

Embodiment 2

[0083] Example 2 Affinity Performance Verification

[0084] Take the target cell myeloma cell line XG7 and incubate with the aptamer GSL1-25 at different concentrations of 0, 25nM, 50nM, 100nM, 150nM, 200nM, 250nM, 300nM, 500nM, 800nM, 1000nM for 20min, wash and perform flow cytometry Experiments showed that as the concentration of the aptamer increased, its binding force to the target cells became stronger, and eventually the binding tended to be saturated. When the binding rate of the aptamer to the cell reaches 50%, the concentration of the aptamer required at this time is the equilibrium dissociation constant Kd, from which we can obtain the binding force constants of the aptamers to the cells as follows:

[0085]

[0086]

Embodiment 3

[0087] Adapter specificity analysis described in embodiment 3

[0088] 50nM FITC-labeled aptamer GSL1-25 was combined with target cells myeloma cell line XG7 or counter-screened bone marrow mesenchymal stem cells and oral epithelial cells for 20min, washed, fixed with 4% paraformaldehyde and confocal Microscopic experiments showed that for the myeloma cell line XG7, the aptamer GSL1-25 could well bind to it, and obvious fluorescence could be seen on the cell membrane, while for bone marrow mesenchymal stem cells and oral cavity For epithelial cells, none of the aptamers GSL1-GSL25 were obviously bound to it, so we could hardly see the fluorescence in the fluorescent field.

[0089] The following conclusions can be drawn through the above experiments: the aptamer sequences of the present invention can well bind to target cells, and the targeting is also very good.

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Abstract

The invention relates to an aptamer capable of binding osteosarcoma specifically.The aptamer is high in binding property and stability and is screened out from a random library by targeting the osteocarcinoma through application of a novel combinatorial chemistry technology SELEX (systematic evolution of ligands by exponential enrichment).The invention further relates to a kit for osteocarcinoma cell separation.The kit is capable of identifying osteocarcinoma cells quickly and is extremely high in application value.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an aptamer for bone cancer detection and a kit thereof. Background technique [0002] Bone cancer (Osteosarcoma), which is a malignant tumor that grows from the skeletal system, is the most common type of bone tumor. It will invade adjacent tissues and organs, and may also spread to other distant organs. Occurs in children and adolescents between the ages of ten and twenty, and the incidence rate is slightly higher in males than in females. The cause of bone cancer is unknown so far. Its primary site and its occurrence at a fast-growing age suggest that it may be related to the increase in the activity of osteoblasts. Genetic factors also seem to play a role in bone cancer. May be associated with chronic inflammation, radiation or viral infection. [0003] At present, the detection of bone cancer is generally performed by blood drawing using colloidal gold technology, such as the t...

Claims

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Application Information

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IPC IPC(8): C12N15/115G01N33/574G01N33/569
CPCC12N15/115C12N2310/16C12N2320/13G01N33/56966G01N33/57407
Inventor 曹帅
Owner 曹帅
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