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Nucleic acid aptamer of affinity viral hepatitis C core protein and application of nucleic acid aptamer

A nucleic acid aptamer and core protein technology, applied in the direction of DNA/RNA fragments, measuring devices, instruments, etc., can solve the problems of high cost of HCV virus, harsh storage conditions, and high price, and achieve high application value, easy preparation, and low cost low effect

Active Publication Date: 2014-08-27
INST OF CHEM CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these kits can detect HCV antigens, because these kits are detected by double-antibody sandwich enzyme-linked assay (ELISA), a large amount of monoclonal antibody preparation is required
However, the preparation process of monoclonal antibodies is complicated, expensive, and the storage conditions are harsh.
This makes the cost of detecting HCV virus higher, and the detection sensitivity is also seriously affected

Method used

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  • Nucleic acid aptamer of affinity viral hepatitis C core protein and application of nucleic acid aptamer
  • Nucleic acid aptamer of affinity viral hepatitis C core protein and application of nucleic acid aptamer
  • Nucleic acid aptamer of affinity viral hepatitis C core protein and application of nucleic acid aptamer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1, preparation of related proteins and related solutions

[0039] 1. Preparation of HCV core protein (target protein) with histidine tag

[0040] 1. Amplification of the coding gene of HCV core protein

[0041] Prepare the DNA shown in the sequence 4 of the sequence listing (the coding gene of the hepatitis C virus core protein, GENBANK ACCESSION NO.HM566118.1, the hepatitis C virus core protein shown in the sequence 3 of the coding sequence listing), as PCR amplification The template is used for PCR amplification with a primer pair consisting of primer 1 and primer 2 to obtain a PCR amplification product.

[0042] Primer 1 (upstream primer): 5'-CGCGC GAATTC ATGAGCACGAATCCT-3';

[0043] Primer 2 (downstream primer): 5'-CTGCAG GGATCC AGAGGCCGGGACGGTCA-3';

[0044] EcoRI restriction site and BamHI restriction site were introduced into the 5' ends of the upstream and downstream primers respectively.

[0045] PCR amplification conditions: 95°C for 2min; 3...

Embodiment 2

[0074] Example 2, Screening and Preparation of Nucleic Aptamers

[0075] 1. Protein immobilization

[0076] 1. Take Ni-NTA agarose microbeads and place them in a 5ml centrifuge tube, remove the supernatant, and wash with PBS buffer three times;

[0077] 2. Disperse the microbeads in step 1 in the target protein (or control protein), incubate at room temperature for 1 hour, and centrifuge and wash with PBS buffer three times;

[0078] 3. Redisperse the microbeads from step 2 in 1ml of PBS buffer and store at 4°C for later use.

[0079] 2. Design of Random Nucleic Acid Library

[0080] A random nucleic acid library comprising 20 nucleotides at both ends and 40 nucleotides in the middle is designed as follows: 5'-ACGCTCGGATGCCACTACAG(N 40 ) CTCATGGACGTGCTGGTGAC-3'; N 40 Represents 40 random nucleotides.

[0081] 3. Screening of nucleic acid aptamers

[0082] 1. DNA library pretreatment

[0083] The random nucleic acid library is dissolved in binding buffer.

[0084] 2. An...

Embodiment 3

[0092] Example 3, Binding Characterization of Nucleic Aptamer and Hepatitis C Virus Core Protein

[0093] 1. FITC labeling of nucleic acid aptamers and random nucleic acid libraries

[0094] The nucleic acid aptamer HCV-1 prepared in Example 2 was labeled with fluorescein isothiocyanate (FITC).

[0095] The random nucleic acid library prepared in Example 2 was labeled with fluorescein isothiocyanate (FITC).

[0096] 2. Binding characterization of nucleic acid aptamer and hepatitis C virus core protein

[0097] 1. Immobilization of target protein

[0098] (1) Take 200 μl Ni-NTA agarose beads and place them in a 5ml centrifuge tube, remove the supernatant, and wash with PBS buffer three times;

[0099] (2) Disperse the microbeads in step (1) in 1 mL of the target protein, incubate at room temperature for 1 h, and centrifuge and wash with PBS buffer three times;

[0100] (3) The microbeads in step (2) were redispersed in 1 ml of PBS buffer, and kept at 4°C for later use.

[...

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Abstract

The invention discloses a nucleic acid aptamer of an affinity viral hepatitis C core protein and an application of the nucleic acid aptamer. The nucleic acid aptamer is the following (a) or (b): (a) single-stranded DNA represented by sequence 2 in a sequence table; (b) single-stranded DNA containing the nucleic acid aptamer described in (a). Due to the adoption of the nucleic acid aptamer disclosed by the invention, the viral hepatitis C core protein in a solution can be captured and the viral hepatitis C core protein in a solution can also be detected, which are conductive to hepatitis C serology diagnosis and blood screening. By virtue of the nucleic acid aptamer disclosed by the invention, the monoclonal antibody can be partially replaced to capture core protein for detecting hepatitis C and the nucleic acid aptamer has the advantages of high sensitivity, low cost, and convenience in production and preservation. The nucleic acid aptamer has a relatively high application value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a nucleic acid aptamer affinity to the core protein of hepatitis C virus and its application, in particular to a nucleic acid aptamer affinity to the core protein of hepatitis C virus and its role in assisting the identification of hepatitis C application in patients. Background technique [0002] Viral hepatitis C is an infectious disease caused by the hepatitis C virus (HCV), which can be transmitted through blood or blood products. At present, there are nearly 200 million people infected with HCV in the world, and about 40 million to 50 million people are infected with HCV in my country, second only to the number of hepatitis B carriers. Since hepatitis C is caused by an RNA virus, no effective HCV vaccine has been developed worldwide. Only early detection and early treatment of hepatitis C can reduce the damage of the virus to liver cells. Therefore, it is of great significance ...

Claims

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Application Information

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IPC IPC(8): C12N15/115G01N33/577G01N33/576G01N33/569G01N33/543
Inventor 方晓红张振赵子龙徐丽董再再赵立波
Owner INST OF CHEM CHINESE ACAD OF SCI
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