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Aptamer with affinity to viral hepatitis C core antigen and application thereof

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

Active Publication Date: 2016-04-13
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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  • Aptamer with affinity to viral hepatitis C core antigen and application thereof
  • Aptamer with affinity to viral hepatitis C core antigen and application thereof
  • Aptamer with affinity to viral hepatitis C core antigen and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1, preparation of related proteins and related solutions

[0049] 1. Preparation of HCV core antigen (target protein) with histidine tag

[0050] 1. Amplification of the gene encoding HCV core antigen

[0051] Prepare the DNA shown in sequence 4 of the sequence listing (the coding gene of the hepatitis C virus core antigen, GENBANKACCESSIONNO.HM566118.1, the hepatitis C virus core antigen shown in the sequence 3 of the coding sequence listing), as a template for PCR amplification , using a primer pair consisting of primer 1 and primer 2 to perform PCR amplification to obtain a PCR amplification product.

[0052] Primer 1 (upstream primer): 5'-CGCGCGAATTCATGAGCACGAATCCT-3',

[0053] Primer 2 (downstream primer): 5'-CTGCAGGGATCCAGAGGCCGGGACGGTCA-3',

[0054] PCR amplification conditions: 95°C for 2min; 35 cycles of 95°C for 30s, 66°C for 30s, 72°C for 1min; 72°C for 7min.

[0055] 2. Construction of prokaryotic expression vector

[0056] ①Use restriction e...

Embodiment 2

[0085] Example 2, Screening and Preparation of Nucleic Aptamers

[0086] 1. Protein immobilization

[0087] 1. Take Ni-NTA agarose microbeads and place them in a 5ml centrifuge tube, remove the supernatant, and wash with PBS buffer three times;

[0088] 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;

[0089] 3. Redisperse the microbeads from step 2 in 1ml of PBS buffer and store at 4°C for later use.

[0090] 2. Design of Random Nucleic Acid Library

[0091] 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, ie N is A, T, C or G.

[0092] 3. Screening of nucleic acid aptamers

[0093] 1. DNA library pretreatment

[0094] The random nucleic acid library is dissolved in binding buf...

Embodiment 3

[0104] Example 3. Binding Characterization of Nucleic Aptamer and Hepatitis C Virus Core Antigen

[0105] 1. FITC labeling of nucleic acid aptamers and random nucleic acid libraries

[0106] The nucleic acid aptamer HCV103 prepared in Example 2 was labeled with fluorescein isothiocyanate (FITC).

[0107] The random nucleic acid library prepared in Example 2 was labeled with fluorescein isothiocyanate (FITC).

[0108] 2. Binding characterization of nucleic acid aptamer to HCV core antigen

[0109] 1. Immobilization of target protein

[0110] (1) Take 200 μl of Ni-NTA agarose beads and place them in a 5ml centrifuge tube, remove the supernatant, and wash with PBS buffer three times;

[0111] (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;

[0112] (3) The microbeads in step (2) were redispersed in 1 ml of PBS buffer and kept at 4°C for use.

[0113] 2. Binding c...

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Abstract

The invention discloses an aptamer with affinity to viral hepatitis C core antigen and application thereof. The aptamer provided by the invention is following (a) or (b): single-stranded DNA as shown in sequence 2 of following sequence table (a); single-stranded DNA containing the aptamer described in (a). The aptamer can be used to capture the viral hepatitis C core antigen in a solution and to detect the viral hepatitis C core antigen in the solution, facilitating serological diagnosis and blood screening of viral hepatitis. The aptamer can be used to partially replace a monoclonal antibody, capturing a core antigen to detect viral hepatitis C, and the aptamer has the advantages of high sensitivity, low cost, ease of preparation and ease of storage. The aptamer has high applicable value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a nucleic acid aptamer affinity to the core antigen of hepatitis C virus and its application, in particular to a nucleic acid aptamer affinity to the core antigen 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): C12N15/115G01N33/577G01N33/576G01N33/569
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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