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Sequence and application of a nucleic acid aptamer cga02 that specifically recognizes chromogranin a

A nucleic acid aptamer, chromogranin technology, applied in DNA/RNA fragments, recombinant DNA technology, biochemical equipment and methods, etc., to achieve the effect of good reproducibility, simple operation and short cycle

Active Publication Date: 2022-08-05
丽水君弘生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

CGA02 can specifically recognize chromogranin A, but not bind to other proteins. There are no related research reports on nucleic acid aptamers that can specifically bind chromogranin A.

Method used

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  • Sequence and application of a nucleic acid aptamer cga02 that specifically recognizes chromogranin a
  • Sequence and application of a nucleic acid aptamer cga02 that specifically recognizes chromogranin a
  • Sequence and application of a nucleic acid aptamer cga02 that specifically recognizes chromogranin a

Examples

Experimental program
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Effect test

example 1

[0026] Example 1: Gel migration retardation assay to identify the specific recognition of chromogranin A by fluorescently labeled CGA02

[0027] (1) Synthesize the sequence of CGA02, label the fluorophore FITC at its 5' end (synthesized by Shanghai Shenggong Company), and the sequence is: 5'-TGCAGGGTGGAGACACTTCCCGCTTGGCTGACCGGG-3'.

[0028] (2) Dissolve a certain concentration of FITC-labeled nucleic acid aptamer CGA02 in an appropriate volume of buffer (50 mM Tris-HCl, 100 mM NaCl, 1 mM MgCl 2 , 5 mM KCl, pH 7.4), followed by thermal activation. The method of heat activation treatment is as follows: after denaturation at 95°C for 5 min, immediately placed in an ice-water bath for 10 min, and then placed at room temperature for 10 min.

[0029] (3) The heat-activated FITC-labeled CGA02 was incubated with chromogranin A, albumin, carcinoembryonic antigen (CEA), and prostate specific antigen (PSA) for 1 h in a dark box at room temperature.

[0030] (4) The co-incubation system...

Embodiment 2

[0033] Example 2: Identification of Biotin-labeled CGA02 Specific Recognition of Chromogranin A by ELISA

[0034] (1) Synthesize the CGA02 sequence, label it with biotin at its 5' end (synthesized by Shanghai Sangong Company), and the sequence is: 5'-TGCAGGGTGGAGACACTTCCCGCTTGGCTGACCGGG-3'.

[0035] (2) Chromogranin A, albumin, carcinoembryonic antigen (CEA), and prostate specific antigen (PSA) were dissolved in carbonate buffer at pH 9.7, and added to the enzyme-linked strip at 100 μl / well at 4°C Coat protein overnight.

[0036] (3) Discard the coating solution, add 100 μl of maleic acid blocking solution containing 1% casein to each well, and block at room temperature for 1 hour.

[0037] (4) Dissolve different concentrations of biotin-labeled CGA02 in appropriate volumes of buffer (50 mM Tris-HCl, 100 mM NaCl, 1 mM MgCl) 2 , 5 mM KCl, pH 7.4), followed by thermal activation. The method of heat activation treatment is as follows: after denaturation at 95°C for 5 min, imme...

example 3

[0043] Example 3: Determination of the dissociation constant (KD value) of the binding of nucleic acid aptamer CGA02 to chromogranin A

[0044] (1) Coupling of chromogranin A with carboxyl magnetic beads: the purity of the chromogranin A was >98%, and it was purchased from ProSpec Company, and the carboxyl magnetic beads and their coupling reagents were purchased from Bangs Laboratories Company in the United States. The coupling procedure of chromogranin A to magnetic beads was carried out according to the manufacturer's instructions. The protein concentration in the chromogranin A solution before and after coupling was measured by the BCA method, and the coupling efficiency of the magnetic beads was calculated to be 72%; the chromogranin A magnetic beads were dispersed in PBS buffer at 4°C save.

[0045] (2) Mix solutions of FITC-labeled nucleic acid aptamer CGA02 with different concentrations with chromogranin A magnetic beads respectively, and incubate for 1 h at room temp...

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Abstract

The invention relates to a sequence of a nucleic acid aptamer CGA02 that specifically recognizes chromogranin A and its application. The sequence of the nucleic acid aptamer CGA02 is: 5'-TGCAGGGTGGAGACACTTCCCGCTTGGCTGACCGGG-3'; the nucleic acid aptamer CGA02 of the present invention It can recognize chromogranin A with high affinity and high specificity; as part of the kit, the nucleic acid aptamer CGA02 is suitable for the auxiliary diagnosis of neuroendocrine tumors and other cancers or for the clinical and basic research of tumor occurrence, development and other related mechanisms research, etc. The method of the invention is simple, rapid, and has good characteristics, is suitable for auxiliary diagnosis of clinical malignant tumor specimens, and has wide clinical application and basic application prospects.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to the sequence and modification of a nucleic acid aptamer CGA02 that specifically recognizes chromogranin A, and the application of the modified CGA02 in recognizing chromogranin A. The present invention relates to the application of nucleic acid aptamer CGA02 in biomedicine. The nucleic acid aptamer CGA02 and the modified CGA02 are used as components of the kit for auxiliary diagnosis of malignant tumors or basic research related to the occurrence, development and process of malignant tumors. Background technique [0002] Aptamers are also known as "synthetic antibodies" and "chemical antibodies", and their chemical essence is that a single-stranded oligonucleotide molecule (ssDNA or RNA) folds into a specific three-dimensional structure and binds to the target substance with high affinity and high specificity. Nucleic acid aptamers have the characteristics of high affinity, high spe...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/115G01N33/68G01N33/574
CPCC12N15/115G01N33/6893G01N33/57488C12N2310/16C12N2310/531
Inventor 吴冬王开宇
Owner 丽水君弘生物科技有限公司
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