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Cardiac sodium channel gene SCN5A novel variant and detection method thereof

A sodium channel and mutant technology, applied in genetic engineering, plant gene improvement, recombinant DNA technology, etc., can solve the problem of high mortality

Inactive Publication Date: 2009-11-18
ZHONGSHAN HOSPITAL FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

About 50% of patients without heart transplantation died within 5 years after diagnosis of dilated cardiomyopathy. Availability of treatments may prolong survival of patients with the disease, but dilated cardiomyopathy remains a disease with high mortality to date

Method used

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  • Cardiac sodium channel gene SCN5A novel variant and detection method thereof
  • Cardiac sodium channel gene SCN5A novel variant and detection method thereof
  • Cardiac sodium channel gene SCN5A novel variant and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0093] Example 2 Functional analysis of a newly discovered SCN5A mutation A1180V

[0094] 1. Experimental materials

[0095] SCN5A normal sequence plasmid and A1180V mutant sequence plasmid were constructed, and human embryonic kidney 293 cells (HEK293 cells) were commercially available. Fluorescence microscope, patch clamp, Axopatch-200B amplifier, pCLAMP v6.03 acquisition software, etc.

[0096] 2. Experimental process

[0097] 1. SCN5A normal sequence plasmid amplification and mutant sequence plasmid recombination: Utilize the constructed human SCN5A normal sequence plasmid, according to the mutation sequence of the pathogenic gene screened in Example 1, recombine the plasmid, and transform and amplify to obtain the SCN5A normal sequence respectively Plasmid and recombinant plasmid containing the mutant sequence.

[0098] 2. Protein expression and electrophysiological detection of wild-type and mutant sodium channels: The wild-type and A1180V mutant plasmids carrying the...

Embodiment 3

[0104] Example 3 Detection of cardiac sodium channel SCN5A mutant A1180V

[0105] 1 Draw peripheral venous blood;

[0106] 2 DNA extraction of white blood cells:

[0107] 2.1 Centrifuge the anticoagulated venous blood with a centrifuge at a speed of 1000 rpm for 5 minutes, take the supernatant and put it into a 1.5ml EP tube, and freeze it at -70°C.

[0108]2.2 The remaining blood cells were first lysed with red blood cell lysate, centrifuged at 4000 rpm for 5 minutes, and the white blood cells were precipitated, and the supernatant was discarded.

[0109] 2.3 Then use 20ml of red blood cell lysate to lyse the remaining red blood cells, and centrifuge at 4000 rpm for 5 minutes to see pure white blood cell precipitates.

[0110] 2.4 Discard the supernatant, shake the precipitated leukocytes gently to disperse, add dropwise 5ml leukocyte lysate, 60ml proteinase K (10mg / ml) to a final concentration of 200ng / ml, shake overnight at 37°C.

[0111] 2.5 Add an equal volume of pheno...

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Abstract

The invention relates to a cardiac sodium channel gene SCN5A novel variant and a detection method thereof, which belong to the biotechnology field. The invention solves the technical problem of providing the SCN5A variant, a plasmid containing the variant, a cell containing the plasmid, and a mutant protein, and a detection method of the variant. The invention discloses the SCN5A novel variant, A positioned at the exon 20 and cardiac sodium channel protein 1180 is changed into V, and the variant is the pathogenesis of the dilated cardiomyopathy. The invention provides a novel method for early diagnosis and new treatment protocol of the dilated cardiomyopathy.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a novel cardiac sodium channel gene SCN5A mutant and a detection method thereof. Background technique [0002] Dilated cardiomyopathy is one of the most common cardiomyopathies, characterized by enlarged ventricular cavity, decreased myocardial systolic function and normal left ventricular wall thickness. It is the third cause of heart failure and the first cause of heart transplantation. It can be caused by coronary heart disease, myocarditis and systemic diseases, but it can also be primary. The incidence of primary dilated cardiomyopathy is about 40 / 100,000, and the cellular and molecular mechanisms of its pathogenesis are still unclear. About 30% of cases are familial, suggesting that genetic factors play an important role in its pathogenesis. [0003] For dilated cardiomyopathy, there is still a lack of effective early diagnosis and treatment methods. About 50% of patients wi...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/63C12N5/10C07K14/47C12Q1/68
Inventor 孙爱军徐磊葛均波范铮王克强邹云增
Owner ZHONGSHAN HOSPITAL FUDAN UNIV
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