Aptamer capable of identifying hepatitis C virus (HCV) nonstructural 5A (NS5A) protein, derivatives thereof and screening method and use thereof

A nucleic acid aptamer and screening method technology, applied in the field of genetic engineering, can solve the problems of patient interruption of treatment, expensive treatment, and serious side effects of combination therapy, and achieve the effects of inhibiting HCV replication, specifically inhibiting HCV infection, and being easy to preserve.

Inactive Publication Date: 2011-11-02
HUNAN UNIV
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Problems solved by technology

Although the addition of ribavirin can enhance the level of sustained antiviral response, it is also associated with serious side effects (such as hemolytic anemia), which often lead to discontinuation of treatment in 20% of patients
Due to th...

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  • Aptamer capable of identifying hepatitis C virus (HCV) nonstructural 5A (NS5A) protein, derivatives thereof and screening method and use thereof
  • Aptamer capable of identifying hepatitis C virus (HCV) nonstructural 5A (NS5A) protein, derivatives thereof and screening method and use thereof
  • Aptamer capable of identifying hepatitis C virus (HCV) nonstructural 5A (NS5A) protein, derivatives thereof and screening method and use thereof

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Embodiment

[0053] Example: nucleic acid aptamers capable of recognizing HCV NS5A protein, derivatives of nucleic acid aptamers, screening methods and applications thereof.

[0054] 1. Preparation of related proteins.

[0055] 1.1 Preparation of HCV NS5A protein (ie target protein) with histidine tag.

[0056] 1.1.1 Amplification of the gene encoding HCV NS5A protein

[0057] Using the pJFH1 plasmid as a template for PCR amplification, the NS5A gene was amplified by PCR with the first primers consisting of the following primers 1 and 2 (PCR reagents were purchased from Roche, and 100 microliters of PCR tubes were purchased from Eppendorf):

[0058] Primer 1 (upstream primer):

[0059] 5'-CGCGCCATATGTCCGGATCCTGGCTCCGCGAC-3';

[0060] Primer 2 (downstream primer):

[0061] 5'-CTGCAGAAGCTTTCAGCAGCACACGGTGGTATCG-3';

[0062] The 5' ends of the upstream and downstream primers were respectively introduced with Nde I restriction site and HindIII restriction site;

[0063] The PCR amplifica...

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Abstract

The invention discloses an aptamer capable of identifying a hepatitis C virus (HCV) nonstructural 5A (NS5A) protein, which is a DNA fragment represented by any of sequences from SEQ ID No.1 to SEQ ID No.5. The derivatives of the aptamer comprise nucleotide substituted RNA aptamers, skeleton-restructured derivatives, restructured peptide nucleic acid derivatives and radioactive molecule connected derivatives. The screening method of the aptamer comprises: preparing HCV NS5A protein with histidine marker, and preparing protein pET200/D/LacZ; immobilizing the proteins, and designing a random nucleic acid library; and finally, screening the aptamer by pretreating the DNA library, performing reverse screening, screening and repeated screening and other steps, wherein the obtained aptamer has high competition power and optimized sequence length. The aptamer and the derivatives thereof can be used in the preparation of detection kit or diagnosis reagent for the HCV NS5A protein and can also be used in preparation of reagents for inhibiting replication of hepatitis C virus in liver cells.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a nucleic acid aptamer, a derivative of the nucleic acid aptamer and a screening method and application thereof. Background technique [0002] Hepatitis C virus (HCV) infection is a serious global health problem, with 200 million infected people worldwide. It is estimated that there are 3-4 million new infections each year. About 40 million to 50 million people in my country are infected with HCV, second only to the number of hepatitis B carriers. [0003] Currently, the standard treatment for hepatitis C virus is interferon or interferon combined with ribavirin. Treatment of patients infected with genotypes 2 and 3 has an HCV eradication rate of 75% to 90%, however, about 50% of patients infected with HCV types 1 and 4 remain insensitive. Although the addition of ribavirin can enhance the level of sustained antiviral response, it can also bring about s...

Claims

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Application Information

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IPC IPC(8): C12N15/115C12N15/10C12Q1/70C12Q1/68G01N33/68A61K48/00A61P31/14C12R1/93
Inventor 朱海珍方晓红杨大荣徐丽刘斌雷少华刘念礼
Owner HUNAN UNIV
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