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Plasmid for gene therapy of cardiac muscle

A gene therapy and plasmid technology, applied in gene therapy, genetic material components, cardiovascular system diseases, etc., can solve the problems of uncontrollable specific expression of target genes, reducing the specificity and effectiveness of gene therapy

Inactive Publication Date: 2005-12-28
GUANGDONG GENERAL HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the general eukaryotic expression plasmid has no cardiomyocyte-specific promoter, it cannot control the specific expression of the target gene in cardiomyocytes, thus greatly reducing the specificity and effectiveness of gene therapy

Method used

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  • Plasmid for gene therapy of cardiac muscle
  • Plasmid for gene therapy of cardiac muscle

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Construction of pEGFP-MLC-VP22 plasmid

[0029] The CMV enhancer-MLC-2V junction fragment was prepared by PCR amplification ligation method, and the original pEGFP-N fragment was replaced by directional cloning method 3 The CMV adder and promoter of the vector were made into pEGFP-MLC vector; the VP22 gene with Bgl II and Hind III recognition sequences were introduced into the 5' and 3' ends respectively, and inserted into the pEGFP-MLC vector downstream of the MLC-2V promoter by directional cloning Between the single Bgl II and Hind III sites, the pEGFP-MLC-VP22 vector was constructed.

[0030] The specific method is as follows:

[0031] According to the known human MLC-2V promoter sequence, design synthetic PCR primers: upstream primer, 5'-AATGGGAGTTTGTTTTGGACCCAGAGCACAGAG-3', downstream primer, 5'-TAATA GCTAGC GGCCGGCCCCTGCTGT-3' (Nhe I), using human genomic DNA as a template, was amplified to obtain the MLC-2V fragment (250bp). The sequence SEQ ID NO: ...

Embodiment 2

[0068] Example 2 Plasmid amplification, extraction and purification

[0069] Using protozoa Escherichia coli as the host cell, pEGFP-MLC-VP22 was amplified, extracted and purified by conventional molecular biology methods. For specific operations, see the literature "Molecular Cloning Experiment Guide" ([US] J. Sambrook, E.F. Fritsch, T. Maniartis, second edition).

Embodiment 3

[0070] Example 3 Cytology experiment

[0071] The same amount of pEGFP-N was transferred by liposome method 3 , pEGFP-MLC and pEGFP-MLC-VP22 were respectively transfected into cultured human Hela (human uterine cancer cell line), Ea.hy926 (human embryonic umbilical vein endothelial cells), IMA (human inferior mesenteric artery medial smooth muscle cell line) and Hep2 (human liver cancer cell line), after 24 hours of transfection, observe the expression of GFP in the cells of each group. It was found that: pEGFP-N 3 GFP can be expressed in the above cells, but neither pEGFP-MLC nor pEGFP-MLC-VP22 expresses GFP.

[0072] The same amount of pEGFP-N was transferred by liposome method 3 , pEGFP-MLC and pEGFP-MLC-VP22 were transfected into isolated and cultured neonatal mouse cardiomyocytes, and after 24 hours of transfection, the expression of GFP in the cells of each group was observed. It was found that: pEGFP-N 3 GFP was not expressed, but both pEGFP-MLC and pEGFP-MLC-VP22 ...

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Abstract

A myocardial gene therapy plasmid is characterized in that the eukaryotic expression carrier has a cardiomyocyte-specific promoter (CMV-MLC-2V) and a herpes simplex virus structural protein VP22 gene with cell shuttling function. The plasmid of the invention can be specifically expressed in cardiomyocytes, can be fused and expressed with the target gene, and can conveniently detect the transfection efficiency and intracellular location of the target gene.

Description

technical field [0001] The present invention relates to a gene therapy plasmid and its construction method, in particular, the present invention relates to a myocardial gene therapy plasmid vector and its construction method. Background technique [0002] Cardiac gene therapy is a novel approach to the treatment of hereditary and congenital heart disease. The exogenous gene is transferred into cardiomyocytes, through gene expression, so as to achieve the purpose of correcting or enhancing the function of cardiomyocytes. [0003] As the key technology of gene therapy, gene transfer is to effectively transfer the gene to enough target cells and make the cells express the target gene. [0004] Gene transfer methods can be divided into four categories according to the different vectors used: viral vector method, non-viral biological vector method, non-biological vector method and non-vector method. Plasmid DNA injection is a non-carrier method, direct intramuscular injection o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K47/00A61K48/00A61P9/10
Inventor 余细勇单志新
Owner GUANGDONG GENERAL HOSPITAL
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