Hepatoma cell-specific aptamer and preparation method thereof

A liver cancer cell and aptamer technology, applied in the field of genetic engineering, can solve the problems of low binding force or specificity of liver cancer cells, achieve sensitive early diagnosis methods, and improve the effect of diagnosis and treatment

Active Publication Date: 2015-08-26
JIANGHAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] In order to solve the problem of low binding force or specificity between aptamers and liver cancer cells in the prior art, an embodiment of the present invention provides a liver cancer cell-specific aptamer and a preparation method thereof

Method used

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  • Hepatoma cell-specific aptamer and preparation method thereof
  • Hepatoma cell-specific aptamer and preparation method thereof
  • Hepatoma cell-specific aptamer and preparation method thereof

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Embodiment 1

[0050] The embodiment of the present invention provides a liver cancer cell-specific aptamer, which may include: the H2c aptamer shown in the sequence listing SEQ ID NO: 1, the H2c aptamer shown in the sequence listing SEQ ID NO: 2 At least one of the H4c adapter and the H5b adapter shown in the sequence table SEQ ID NO: 3, wherein the structures of the three adapters are as follows Figure 1-3 shown.

[0051] Specifically, the 5' end of the aptamer can be connected with the fluorescent group FAM, Cy5 or Cy3, and the 3' end of the aptamer can be connected with the quenching group BHQ1 or BHQ2. Among them, when the above three aptamers are used for fluorescence experiments, the 5' end can be connected to the fluorescent group Cy5, and when the above-mentioned aptamers are used as fluorescent probes, the 5' end can be connected to the fluorescent group Cy5 or Cy3 .

[0052] The aptamer provided in the embodiment of the present invention can recognize liver cancer cells with hi...

Embodiment 2

[0054]The embodiment of the present invention provides a method for preparing the aptamer provided by the first embodiment of the present invention, such as Figure 21 As shown, the method includes:

[0055] Step 1: Synthesizing the first library of random single-stranded oligonucleotides and primers, wherein the first library of single-stranded oligonucleotides is 5′-ACCGACCGTGCTGGACTCT-N 40 - AGTATGAGCGAGCGTTGCG-3', the primers include a forward primer and a reverse primer, the forward primer is shown in SEQ ID NO: 4 in the sequence listing, and the reverse primer is shown in SEQ ID NO: 5 in the sequence listing.

[0056] Step 2: Carry out forward screening, which includes: co-incubating the first library with target cells, wherein the incubation temperature is 37°C, and the target cells can be at least one of Huh7.5.1 liver cancer cells and HepG2 liver cancer cells , the target cells selected in this implementation are Huh7.5.1 liver cancer cells.

[0057] Step 3: Separat...

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Abstract

The invention discloses a hepatoma cell-specific aptamer and a preparation method thereof. The hepatoma cell-specific aptamer comprises at least one of an H2c aptamer, an H4c aptamer and an H5b aptamer. The method comprises the following steps: a first library and a primer are synthesized; the first library and a target cell are incubated; nucleic acid that is not combined with the target cell is separated and purified, a first template is obtained, and asymmetric amplification is performed on the first template to obtain a second library; the second library and an inverse screening cell are incubated; nucleic acid combined with the inverse screening cell is separated and purified, a second template is obtained, and asymmetric amplification is performed on the second template to obtain a third library; operation is repeated until a twelfth template is obtained, symmetric amplification is performed on the twelfth template, an amplification product is obtained, cloned and sequenced, and single aptamers are obtained through separation; secondary structures of the single aptamers are analyzed through simulation, the binding rate is determined, and the aptamer is picked out. The aptamer prepared with the method can recognize hepatoma cells and hepatoma tissue in a high-specificity manner.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a liver cancer cell-specific aptamer and a preparation method thereof. Background technique [0002] Systematic Evolution of Ligands by Exponential Enrichment (SELEX) is a combined technology. Cell-SELEX technology is a new technology developed in recent years. It uses the whole cell as the target for screening. , The aptamers screened by this cell-SELEX technology have a stronger resolution ability and can distinguish small differences between cells. [0003] Liver cancer is a very common malignant disease in the digestive system, and its morbidity and mortality are very high. It is the number one "killer" that threatens human health. Although the current treatment methods for liver cancer have been greatly improved, the survival rate of patients is still not ideal. Therefore, early detection and early treatment are the most effective ways to improve the survival r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115C12N15/10
Inventor 丁菲
Owner JIANGHAN UNIVERSITY
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