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Nucleic acid aptamer specifically combined with hepatitis C virus core protein and application thereof

A technology of hepatitis C virus and nucleic acid aptamer, which is applied in the determination/testing of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of harsh storage conditions, high cost, and high price, and achieve high application value , low cost, easy to store

Active Publication Date: 2011-07-13
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 immunosorbent 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, which makes the cost of detecting HCV virus high and the detection sensitivity is also seriously affected.

Method used

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  • Nucleic acid aptamer specifically combined with hepatitis C virus core protein and application thereof
  • Nucleic acid aptamer specifically combined with hepatitis C virus core protein and application thereof
  • Nucleic acid aptamer specifically combined with hepatitis C virus core protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1, preparation of related proteins and related solutions

[0036] 1. Preparation of HCV core protein (target protein) with histidine tag

[0037] 1. Amplification of the coding gene of HCV core protein

[0038] Prepare the DNA shown in the sequence 4 of the sequence listing (the coding gene of the hepatitis C virus core protein, GENBANKACCESSION NO.HM566118.1, the hepatitis C virus core protein shown in the sequence 3 of the coding sequence listing), as PCR amplified As a template, a primer pair composed of primer 1 and primer 2 is used for PCR amplification to obtain a PCR amplification product.

[0039] Primer 1 (upstream primer): 5'-CGCGC GAATTC ATGAGCACGAATCCT-3';

[0040] Primer 2 (downstream primer): 5'-CTGCAG GGATCC AGAGGCCGGGACGGTCA-3';

[0041] EcoR1 restriction site and BamH1 restriction site were introduced into the 5' ends of the upstream and downstream primers respectively.

[0042] PCR amplification conditions: 95°C for 2min; 35 cycles of ...

Embodiment 2

[0071] Example 2, Screening and Preparation of Nucleic Aptamers

[0072] 1. Protein immobilization

[0073] 1. Take Ni-NTA agarose microbeads and place them in a 5ml centrifuge tube, remove the supernatant, and wash with PBS buffer three times;

[0074] 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;

[0075] 3. Redisperse the microbeads from step 2 in 1ml of PBS buffer and store at 4°C for later use.

[0076] 2. Design of Random Nucleic Acid Library

[0077] 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.

[0078] 3. Screening of nucleic acid aptamers

[0079] 1. DNA library pretreatment

[0080] The random nucleic acid library is dissolved in binding buffer.

[0081] 2. An...

Embodiment 3

[0102] Embodiment 3, the specificity of nucleic acid aptamer (detected by fluorescein)

[0103] 1. FITC labeling of nucleic acid aptamers and random nucleic acid libraries

[0104] The aptamer HCVs prepared in Example 2 were labeled with fluorescein isothiocyanate (FITC).

[0105] The nucleic acid aptamer HCVs1 prepared in Example 2 was labeled with fluorescein isothiocyanate (FITC).

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

[0107] 2. The specificity of nucleic acid aptamers

[0108] 1. Protein immobilization

[0109] (1) Immobilization of target protein

[0110] ①Put 200ul Ni-NTA agarose microbeads 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 Re-disperse the microbeads from step ...

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Abstract

The invention discloses a nucleic acid aptamer specifically combined with a hepatitis C virus core protein and application thereof. The nucleic acid aptamer is a single stranded DNA shown in a sequence 2 of a sequence table or single stranded DNA containing the nucleic acid aptamer. The nucleic acid aptamer has better affinity with a hepatitis C virus core protein. By using the nucleic acid aptamer disclosed by the invention, the hepatitis C virus core protein in a solution can be captured and can be also detected, and hepatitis C serodiagnosis and blood screening can be realized. Part of thenucleic acid aptamer disclosed by the invention can be used to replace a monoclonal antibody to capture the core protein for hepatitis C detection. The invention has the advantages of high sensitivity, low cost, easiness of preparation and storage, and high application value.

Description

technical field [0001] The invention relates to a nucleic acid aptamer specifically binding to the core protein of hepatitis C virus and its application. Background technique [0002] Hepatitis C virus is an infectious disease caused by hepatitis C virus (Hepatitis C virus, HCV), which can be infected 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 to explore and develop new methods for early detection of HCV. [0003] The only way to determine whether you are infected with hepatitis C is to do a hepatitis C serum test. Current serologica...

Claims

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Application Information

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IPC IPC(8): C12N15/115C12Q1/70
Inventor 方晓红张振赵子龙徐丽朱海珍
Owner INST OF CHEM CHINESE ACAD OF SCI
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