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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 reducing the specificity and effectiveness of gene therapy, and cannot control the specific expression of target genes

Inactive Publication Date: 2004-08-11
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
  • Plasmid for gene therapy of cardiac muscle

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Construction of pEGFP-MLC-VP22 plasmid

[0028] 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 increaser and promoter of the vector were made into pEGFP-MLC vector; the VP22 gene with Bgl II and HindIII 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 The pEGFP-MLC-VP22 vector was constructed between the Bgl II and HindIII sites.

[0029] The specific method is as follows:

[0030] 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 (250 bp). The sequence SEQ ID NO: 2 is a...

Embodiment 2

[0059] Example 2 Plasmid amplification, extraction and purification

[0060] 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

[0061] Example 3 Cytology experiment

[0062] The same amount of pEGFP-N was transferred by liposome method 3 , pEGFP-MLC and pEGFP-MLC-VP22 were transfected with cultured human Hela (human uterine cancer cell line), Ea.hy926 (human embryonic umbilical vein endothelial cells), IMA (human inferior mesenteric artery media 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.

[0063] 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 had GFP expres...

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Abstract

A plasmid for the gene therapy of myocardium is composed of an eucaryotic expression carrier, the myocardium cell specific promotor (CMV-MLC-2V), and single herpesvirus structural protein VP22 gene with cell shuttle function. It can be specifically expressed in myocardium cell, can be fused with object gene for expression, and can be used to conveniently detect the transfection efficiency of object gene and its location in cell.

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 diseases. 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 ...

Claims

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Application Information

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