Method for efficiently and quickly screening HIV P24 antigen nucleic acid aptamer

A nucleic acid aptamer and antigen technology, applied in the field of biotechnology detection, can solve the problem of not screening out target aptamers, and achieve the effects of shortening the screening cycle, reducing screening costs, and high sensitivity

Inactive Publication Date: 2017-02-22
YANSHAN UNIV
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

Existing literature shows that there have been studies on the screening of HIV P24 antigen

Method used

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  • Method for efficiently and quickly screening HIV P24 antigen nucleic acid aptamer
  • Method for efficiently and quickly screening HIV P24 antigen nucleic acid aptamer
  • Method for efficiently and quickly screening HIV P24 antigen nucleic acid aptamer

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Embodiment

[0032] A method for efficiently and rapidly screening HIV P24 antigen nucleic acid aptamers, using carboxylated agar magnetic beads as a medium, carrying out 6 rounds of screening to select highly specific nucleic acid aptamers for HIV P24 antigens, the 6 rounds of screening are Obtain the highly specific nucleic acid aptamer of HIV P24 antigen with two rounds of positive screening and four rounds of reverse screening, specifically including the following steps:

[0033] (1) Processing of ssDNA library;

[0034] (2) Put 0.3ml magnetic beads in a 1.5mLEP tube, add 300ul HIV P24 antigen, incubate at 37°C for 2h, discard the antigen in the EP tube, add 500uL blocking solution, 37°C, 1h, bind the primary ssDNA library, 37 ℃, incubate for 1h;

[0035] (3) Discard the unbound ssDNA library, wash with screening buffer 3 times, 1 min each time, add 200ul deionized water, 95°C, 10 min, collect the supernatant, and use it as a secondary library for the next round of screening;

[0036...

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Abstract

The invention discloses a method for efficiently and quickly screening HIV P24 antigen nucleic acid aptamer. The method includes that subduction index enriched ligand system evolution (SELEX) technology and qPCR technology are utilized, streptavidin is adopted to prepare single-stranded DNA, TPBS is used to wash away nonspecific single-stranded DNA, comerical carboxylated agar magnetic beads are used as a separation medium for selex screening, and the HIV P24 antigen nucleic acid aptamer is obtained by screening from a random oligonucleotide library, so that screening efficiency is improved. Therefore, the method can serve as early detection technology, a method which is high in sensitivity, low in cost and simple and convenient to operate is provided for clinical detection, a new specific carrier is provided for tumor treatment, and a foundation is laid for screening other single target protein and tumor serum marker nucleic acid aptamer. The method is high in screening efficiency, short in period and low in cost.

Description

technical field [0001] The invention belongs to the field of biotechnology detection, and relates to a novel nucleic acid aptamer screening method, in particular to a method for efficiently and rapidly screening HIV P24 antigen nucleic acid aptamers. Background technique [0002] In 2004, an estimated 35.9 to 44.3 million people worldwide were living with HIV, of which 4.3 to 6.4 million were new infections, and 2.8 to 3.5 million died of AIDS. These figures are constantly increasing, among which East Asia, Eastern Europe, Central Asia and other regions have the fastest growth rate. HIV infection can stimulate the body to produce envelope protein (Gp120, Gp41) antibodies and core protein (P24) antibodies. Compared with antibody / antigen detection, DNA aptamers have the following advantages: (1) DNA aptamers are more stable, not easy to degrade, and can be stored for a long time; (2) The binding force to target molecules is stronger, even stronger than natural ligands; (3) T...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12N15/115
CPCC12N15/1048C12N15/115C12N2310/16C12N2320/13C12Q2531/113C12Q2541/101C12Q2563/143C12Q2563/149
Inventor 栗坤修尘林
Owner YANSHAN UNIV
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