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Nucleic acid aptamer specifically bound with human epidermal growth factor receptor III-type mutant and application of nucleic acid aptamer

A technology of epidermal growth factor and nucleic acid aptamer, which is applied in the field of biomedicine, can solve the problem of no observed bone marrow suppression, and achieve the effect of broad application prospects

Active Publication Date: 2013-09-11
SOUTHERN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although 188 Re and 90 Y has a similar beta energy, but no observations like 90 myelosuppression ［48］

Method used

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  • Nucleic acid aptamer specifically bound with human epidermal growth factor receptor III-type mutant and application of nucleic acid aptamer
  • Nucleic acid aptamer specifically bound with human epidermal growth factor receptor III-type mutant and application of nucleic acid aptamer
  • Nucleic acid aptamer specifically bound with human epidermal growth factor receptor III-type mutant and application of nucleic acid aptamer

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Experimental program
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Embodiment 1

[0055] Experiment 1. Preparation of nucleic acid aptamers that specifically bind to a glioma cell line (U87Δ) overexpressing human epidermal growth factor receptor type III mutant (EGFRvIII). The screening process is as follows: figure 1 As shown, the results of 11 rounds of sequencing are as follows image 3 shown.

[0056] 1. Design and synthesize initial ssDNA library and primers for PCR amplification

[0057] Design and synthesize the ssDNA initial GN library with a length of 76 bases: 5'-ATCCAGAGTGACGCAGCA (N40) TGGACACGGTGGCTTAGT-3', the two ends of the initial ssDNA library are fixed sequences, and the middle is 40 random sequences, N stands for A, T, C , G four random bases.

[0058] Design and synthesize primers for amplifying double-stranded dsDNA:

[0059] 5'-FITC-Pn1:5'-ATCCAGAGTGACGCAGCA-3';

[0060] 5'-Biotin-Pn2: 5'-ACTAAGCCACCGTGTCCA-3'

[0061] The above-mentioned initial ssDNA library and primers were synthesized by biological companies.

[0062] Using th...

Embodiment 2

[0098] Experiment 1. The role of aptamers in inhibiting tumor growth in nude mouse tumor-bearing animal models

[0099] Specific method: U87Δ cells were treated with DMEM containing 10% FBS in 5% CO 2 , Cultured at 37°C, and nude mice were raised in the SPF experimental barrier environment for 4 to 6 weeks. A total of 28 nude mice were divided into blank group and in vivo jet-PEI TM Transfection reagent group, original library GN group and aptamer U2 treatment group, 4 groups, 7 animals in each group; U87Δ cells were resuspended in modified RPMI-1640 culture medium, and 1.5×10 6 ~2.0×10 6 Use a 1ml syringe to subcutaneously inject 100 μl into the right buttocks of male nude mice (BALB / c) at 4 to 6 weeks old; observe the tumor formation after 2 weeks, and measure the tumor volume with a vernier caliper; use in vivo jet-PEI TM The reagents were transfected with the aptamer U2 and the original library GN (initial ssDNA library) for intratumoral injection (1 injection every ot...

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Abstract

The invention discloses a nucleic acid aptamer specifically bound with a human epidermal growth factor receptor III-type mutant and an application of the nucleic acid aptamer. The nucleotide sequence of the nucleic acid aptamer is any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4. The nucleic acid aptamer specifically bound with the human epidermal growth factor receptor (EGFRv) III-type mutant is only specifically bound with U87-delta cell lines rather than U87 cell lines, so that the nucleic acid aptamer can be effectively applied to preparing a sensor or a kit for diagnosing tumor cells for expressing EGFRv III. Furthermore, the nucleic acid aptamer can be adopted as a medicine carrying tumor cells resistant to express EGFRv III or a carrier of siRNA (Small Interfering RNA) for entering tumor cells or tissues capable of expressing EGFRv III so as to play a role of a targeted matter, thereby having broad application prospects.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a nucleic acid aptamer specifically combined with a human epidermal growth factor receptor type III mutant and an application thereof. Background technique [0002] The systematic evolution of ligands by exponential enrichment (SELEX) technology is a new combinatorial chemistry technology developed in the early 1990s. It was inspired by combinatorial chemistry by Gold and Ellington in the United States. A novel in vitro screening technique constructed [1-3] , The oligonucleotides screened by this technique are called aptamers. It synthesizes a large-capacity random oligonucleotide library in vitro, combines in vitro PCR amplification technology, and undergoes multiple rounds of screening with target molecules to obtain high-affinity and specific oligonucleotide aptamers (aptamers). Target molecules include metal ions, drugs, amino acids, base analogs, nucleotides and poly...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115C12Q1/68A61K47/26A61K48/00A61P35/00
Inventor 张兴梅伍锡栋谭燕梁惠玉高天明
Owner SOUTHERN MEDICAL UNIVERSITY
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