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Method for establishing finite cell strain for screening read-though promoting drug

A cell line, limited technology, applied in the field of bioengineered animal cell lines

Inactive Publication Date: 2013-05-01
SHANXI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is a lack of high-throughput cell-level drug screening system at home and abroad, so there is an urgent need to establish corresponding cell lines for in vitro screening of potential drugs for diseases caused by nonsense mutations, and it can also be used for clinical screening of genetic diseases caused by nonsense mutations. medicine

Method used

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  • Method for establishing finite cell strain for screening read-though promoting drug
  • Method for establishing finite cell strain for screening read-though promoting drug
  • Method for establishing finite cell strain for screening read-though promoting drug

Examples

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Embodiment Construction

[0027] 1) Construction of recombinant dual fluorescent mammalian expression vector

[0028] The DsRed fluorescent protein gene was double digested with Xho I and EcoR I to obtain a 0.8kb gene fragment, which was recovered by electrophoresis. The mammalian high-efficiency expression vector pEGFP-N1 was also cut into a linear vector with Xho I and EcoR I double enzymes, and recovered by electrophoresis. Ligate it with the recovered DsRed fluorescent protein gene fragment using T4 DNA ligase to obtain the recombinant plasmid pDsRed-EGFP, which was identified by enzyme digestion. (See figure 2 )

[0029] Design a pair of completely complementary mutation primers, use high-fidelity enzymes to carry out PCR amplification with the recombinant plasmid pDsRed-EGFP as a template, digest the template with Dpn I, take 10 μL of the product to transform bacteria, pick clones to extract plasmids, and sequence them for identification (see image 3 ), to obtain the recombinant dual fluores...

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Abstract

The invention discloses a eukaryote expression vector for expressing a green fluorescent protein (EGFP) and a red fluorescent protein (DsRed); a mutant contains a premature termination condon in a 445 amino acid position of the green fluorescent protein EGFP; and the expression of the green fluorescent protein EGFP can be effectively stopped by a mutational site. A constructed double fluorescent expression vector and a mutant thereof are introduced into an African green monkey kidney cell Cos-7 and treated by a read-though promoting drug PTC124; expression intensities of the green fluorescent protein and the red fluorescent protein are detected by using a flow cytometry; and the read-though promoting efficiency of the read-though promoting drug to the vector can be efficiently detected. In addition, different nonsense mutations and upstream and downstream correlation sequences can be introduced into a multiple clone site of the expression vector, so that targeted drugs for diseases initiated by the different nonsense mutations can be specifically screened.

Description

Technical field: [0001] The present invention relates to the field of bioengineering animal cell lines, in particular to a method for establishing a limited cell line for promoting read-through drug screening, and more specifically to a dual fluorescent mammalian expression vector and its PTC mutant limited cell line The establishment method and the dual fluorescent mammalian expression vector and its PTC mutant limited cell line established according to the method. Background technique: [0002] According to the authoritative statistics of the Human Genome Mutation Database (Human Genome Mutation Database, HGMD, http: / / www.hgmd.org) in 2012, due to frameshift mutations, alternative splicing or direct nonsense mutations, the human genome has formed Premature termination codons (PTC), the existence of PTC causes the body to produce truncated proteins or proteins with negative dominant effects, one-third of human genetic diseases are caused by this; 11.2% of human Genetic dis...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/85C12N5/10C12R1/91
Inventor 申泉李毳柴宝峰王刚
Owner SHANXI UNIV
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