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Hepatoma cell aptamer sequence and application thereof

A nucleic acid aptamer and liver cancer cell technology, applied in the field of cell biology, can solve the problem of lack of effective therapeutic drugs for chemotherapy of liver cancer

Active Publication Date: 2013-10-30
AFFILIATED HUSN HOSPITAL OF FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the clinical treatment plan, although surgery can cure liver cancer, it is limited to a small number of patients. For most patients, either because of large lesions or due to factors such as postoperative recurrence and metastasis, chemotherapy is still the main treatment method. There is still a lack of effective therapeutic drugs. In order to improve the chemotherapy effect of liver cancer, researchers in this field are committed to finding effective drug targets against liver cancer.

Method used

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  • Hepatoma cell aptamer sequence and application thereof
  • Hepatoma cell aptamer sequence and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1 Nucleic acid aptamer screening (one round)

[0018] First round oligo DNA library sequence: 5'-acgctcggatgccactacagNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNctcatggacgtgctggtgac-3'

[0019] Primers used for enrichment:

[0020] Aptamer_L: 5'-FAM-acgctcggatgccactacag-3'

[0021] Aptamer_R: 5'-biotin-gtcaccagcacgtccatgag-3'

[0022] Determination of oligomeric DNA library O D260 Afterwards, it was centrifuged and dried. Dissolve the library with 300ul binding buffer (4.5g / L glucose, 5mMMgCl2, 2mg / mLBSA, 0.2mg / mL yeast tRNA in PBS), take 250pmol (10nmol in the first round), put it on ice at 95°C for 5min, and put it on ice for a while. Then centrifuge quickly.

[0023] Reverse screening: 250pmol of the library was drawn into a 6cm-diameter petri dish with a growth coverage of HL-7702 reaching 70-80%, and made up to 1mL with binding buffer. Shake at 4°C for 1 h. Take the supernatant.

[0024] Positive sieve: Add the supernatant of the reverse sieve into a 6c...

Embodiment 2

[0028] Example 2 Nucleic Aptamer TA Cloning

[0029] Prepare the following solutions in a microcentrifuge tube, the total amount is 5 ul: pMD19-TVector 1 ul, aptamer PCR product 1 ul, dH2O 3 ul. Add 5ul of SolutionI. React at 16°C for 30 minutes. The whole amount (10ul) was added to 100ul DH5a competent cells, and placed in ice for 30 minutes. After heating at 42°C for 45 seconds, place in ice for 1 minute. Add 890ul LB medium, shake and culture at 37°C for 60 minutes. Cultivate on LB agar plate medium containing X-Gal, IPTG, and Amp to form a single colony.

Embodiment 3

[0030] Colony PCR and sequencing of embodiment 3 nucleic acid aptamer clone

[0031] pMD19-T colony PCR and sequencing primers:

[0032] RV-M: 5′-GAGCGGATAACAATTTCACACAGG-3′

[0033]M13(-47): 5'-CGCCAGGGTTTTTCCCAGTCACGAC-3'

[0034] Use a sterilized toothpick to pick white monoclonal colonies and add them to 4ml LB (Amp50ng / L) liquid medium, shake the bacteria overnight at 180rpm at 37°C ( The bacterial liquid was used as a PCR template for PCR amplification. Water was used as a negative control.

[0035] Configure the PCR system: bacteria solution Primer RV-M (10uM) 0.5ul, primer M13(-47) 0.5ul, 2×TaqMix 7.5ul, water 5.5ul (total system 15ul). Amplify on a PCR machine under the following conditions: pre-denaturation at 95°C for 5 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 45 s, a total of 33 cycles; total extension at 72°C for 10 min.

[0036] After the reaction, take 3-6ul bacterial liquid PCR product and carry out agarose gel...

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Abstract

The invention belongs to the technical field of cell biology and in particular relates to a hepatoma cell aptamer sequence and an application thereof. A characteristic sequence TTTTTT specifically bound with HepG2 is prepared by screening an aptamer specifically bound with a human hepatoma cell line HepG2. The characteristic sequence is the basis of specific binding of the aptamer and the human hepatoma cell line HepG2, can be used for design and preparation of targeted drugs, preparations or other products for resisting hepatoma, and can serve as a molecular probe or target for resisting hepatoma. The characteristic sequence provides a powerful support for molecular diagnosis and targeted therapy of hepatoma and has important clinical values.

Description

technical field [0001] The invention belongs to the technical field of cell biology. The invention relates to a nucleic acid aptamer sequence of liver cancer cells, in particular to the sequence and application of a nucleic acid aptamer of liver cancer cells. Background technique [0002] Statistics show that my country is the largest country in the occurrence of liver cancer, its incidence rate accounts for 55% of the world, and its mortality rate accounts for 45% of the world, and it is increasing at a rate of more than 300,000 cases per year. According to reports, liver cancer is the sixth most common cancer in the world and the third leading cause of cancer-related deaths in the world, seriously threatening human health. In the clinical treatment plan, although surgery can cure liver cancer, it is limited to a small number of patients. For most patients, chemotherapy is still the main treatment method due to the large lesions or consideration of postoperative recurrence...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115C12Q1/68A61K48/00A61P35/00
Inventor 刘杰张骏
Owner AFFILIATED HUSN HOSPITAL OF FUDAN UNIV
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