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Myocardial specific promoter

A specific promoter and promoter technology, applied in the field of promoters

Inactive Publication Date: 2013-06-26
JIANGSU PROVINCE HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far, there is no literature report on which specific domain in the TnIc promoter sequence can specifically regulate the expression of the target gene in the myocardium.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1. Obtaining of TnIc promoters with different sequence lengths

[0070] According to the literature (Identification of cis-acting DNA elements required for expression of the human cardiac troponin I gene promoter. J Mol Cell Cardiol, 2000; 32 (1): 95-108) and GenBank (X90780.1) TNNI3 gene sequence , design primers respectively, and the sequence of each fragment in the primer and gene is shown in the sequence table, and they are respectively named as TnIc858, TnIc491, TnIc301, TnIc168, TnIc129, and the 1107th sequence of the TNNI3 gene is taken as 1, and the 1106th is taken as -1 for calculation, wherein TnIc858 is from -791 to 67 (SEQ ID NO: 7), the size is 858bp, and its amplification primers are the sequences shown in SEQ ID NO: 1 and 2 respectively; TnIc491 is from -463 to 28 (SEQ ID NO: 8 ), the size is 491bp, and its amplification primers are the sequences shown in SEQ ID NO: 3 and 4 respectively; TnIc301 is from -234 to 67 positions (SEQ ID NO: 9), the siz...

Embodiment 2

[0072]Example 2, Construction of an evaluation vector containing a promoter sequence

[0073] The promoters TnIc860, TnIc491, TnIc301, TnIc168, TnIc129 were double digested with HindⅢ and BglⅡ and then cloned into the PGL3-basic-Luciferase vector (purchased from Promega) to obtain PGL3-TnIc-Luciferase (such as figure 1 ). Correctly digested by HindⅢ and BglⅡ (such as figure 2 shown), and was identified correctly by sequencing.

Embodiment 3

[0074] Example 3, Myocardium-specific TnIc promoter identification and screening

[0075] Resuscitated cardiomyocytes (H9C2), fibroblasts (H3T3), and skeletal muscle cells (L6) (Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) and planted them in 96-well plates, and evaluated the sequenced correct promoters The carrier PGL3-TnIc-Luciferase was transfected into cardiomyocytes (H9C2), fibroblasts (H3T3), and skeletal muscle cells (L6). Through the luciferase detection system, the TnIc promoter sequence (such as image 3 ). Specifically: PGL3-TnIc-Luciferase encoded as 1 (TnIc858), 2 (TnIc491), 3 (TnIc301), 4 (TnIc168), 5 (TnIc129) and Renilla luciferase plasmid (Promega Company) in the same amount (2 μg) The liposome method was used to simultaneously transfect myocardial, fibroblast and skeletal muscle cells, using the Promega double reporter luciferase gene detection system, the fluorescence value expressed by Renilla plasmid was...

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Abstract

The invention relates to a myocardial specific promoter, which is a part of a TroponinI promoter and comprises a component (TATA / MEF-2) with rich A / T, two GATA components and an area (comprising CACC box and Sp1 element) with rich cytosine; and preferably, the myocardial specific promoter is a part from the -791st site to the 67th site of a TNNI3 gene, and more preferably, the myocardial specific promoter is a part from the -234th site to the 67th site of the TNNI3 gene. The invention also relates to a nucleic-acid construction body, a carrier and a reconstitution cell comprising the promoter. The invention also relates to an application of the promotor for controlling the specific expression of target genes in myocardium and an application for preparing a medicine for preventing or treating cardiovascular disease. The promoter has the advantages that the target gene can be controlled for high expression to myocardial specificity and a new path for preventing or treating the cardiovascular disease is opened up.

Description

technical field [0001] The invention relates to a promoter for gene expression regulation, in particular to a myocardial specific promoter for gene expression regulation. The present invention also relates to the use of the promoter for regulating intramyocardial gene expression and the use for preparing medicines for preventing or treating cardiovascular diseases. Background technique [0002] Cardiovascular disease is a kind of disease that seriously threatens human health. In my country, the mortality rate of cardiovascular diseases is on the rise. Currently, the treatment methods for cardiovascular diseases include drug therapy, angioplasty, surgical vascular bypass, and therapeutic angiogenesis. Therapeutic angiogenesis, also known as "molecular bypass" or "biological bypass", refers to taking certain measures to promote angiogenesis in the ischemic area to achieve the purpose of treatment. The collateral circulation pathway can complete the self-bridge of the blood ...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/63C12N5/10A61K48/00A61P9/00A61P9/10A61P9/06
Inventor 杨志健陶正贤许智惠陈波王立生杨月峰王华吴祖泽
Owner JIANGSU PROVINCE HOSPITAL
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