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klhl24 gene mutant and its application

A technology of KLHL24 and mutants, applied in the direction of ligase, enzyme, biochemical equipment and methods, etc., can solve the problems of epidermolysis bullosa that need to be further studied

Active Publication Date: 2019-11-08
SHENZHEN HUADA GENE INST +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Therefore, the current research on epidermolysis bullosa still needs to be in-depth

Method used

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  • klhl24 gene mutant and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 Determination of epidermolysis bullosa pathogenic genes and pathogenic mutations

[0055] 1. Sample collection

[0056] The inventor collected a Chinese Han family with three generations of epidermolysis bullosa (Epidermolysis bullosa) and 4 sporadic patient samples. The family of this patient included 6 members, including 2 EB patients. The proband showed extensive exfoliation of the skin on the abdomen and limbs at birth. After receiving good care, the wound showed atrophic scar epithelium, and physical injury induced blisters and erosions. Recurring episodes, most of which were associated with hypo- or hyperpigmentation, but did not heal spontaneously, with mildly affected oral mucosa and toenail dystrophy, with normal hair growth; the affected daughter of the proband suffered from extensive skin detachment shortly after birth and died of secondary infection without obtaining a DNA sample.

[0057] Therefore, the inventor collected samples from the patien...

Embodiment 2

[0073] Example 2 Sanger method sequencing verification

[0074] The KLHL24 gene of 5 patients described in Example 1 (1 family patient, 4 sporadic patients) and normal parents was detected, primers were designed for the sequence of KLHL24, obtained by PCR amplification, product purification and sequencing The relevant sequence of KLHL24, according to whether the sequence determination results belong to the mutant type or the wild type, verifies the correlation between KLHL24 and EB disease. The specific method steps are as follows:

[0075] (1) DNA extraction:

[0076] Genomic DNA was extracted from the peripheral venous blood of 5 patients and normal family members according to the method in Example 1, and the concentration and purity of DNA were measured by a spectrophotometer. The OD260 / OD280 of each sample genomic DNA was between 1.7-2.0. The concentration is not less than 200ng / ul, and the total amount is not less than 30μg.

[0077] (2) Primer design and PCR reaction:...

Embodiment 3

[0084] Example 3 Other biological verification

[0085] 1. Reverse transcription PCR (RT-PCR)

[0086] KLHL24 belongs to the KELCH family of genes, which includes 42 genes. This gene encodes a KELCH protein that shares a highly conserved BTB domain and a BACK domain, and interacts with CUL3 and RBX1 to form a CUL3-RBX1-KELCH ubiquitin ligase complex. The KELCH proteins also share a KELCH domain responsible for the uptake of ubiquitinated substrates, thereby determining the specificity of the ubiquitin ligase E3. Therefore, the inventors determined whether KLH24 is expressed in skin tissue by performing RT-PCR analysis of KLHL24 on cDNA samples of skin and peripheral blood. The specific method is as follows:

[0087] (1) Total RNA was extracted from normal human skin and peripheral blood using Trizol reagent (Invitrogen) and QIAamp RNA BloodMiniKit kit (Qiagen) according to the manufacturer's instructions.

[0088] (2) cDNA was amplified from 50-500 ng of total RNA using Tra...

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Abstract

The invention discloses a KLHL24 gene mutant and application thereof and specifically relates to a separated nucleic acid coding the KLHL24 mutant, a separated polypeptide, a system used for screeningbiological samples susceptible to epidermolysis bullosa, a kit used for screening the biological samples susceptible to epidermolysis bullosa, a construct, and a recombinant cell. Compared with SEQ ID No. 1, the separated nucleic acid coding the KLHL24 mutant has at least one mutation selected from a group consisting of c.1A>G mutation, c.3G>T mutation and c.3G>A mutation. Whether a biological sample is prone to epidermolysis bullosa can be effectively detected by detecting existence of the gene mutant in the biological sample.

Description

technical field [0001] The present invention relates to KLHL24 gene mutant and application thereof. Specifically, the present invention relates to isolated nucleic acids encoding KLHL24 mutants, isolated polypeptides, systems for screening biological samples susceptible to epidermolysis bullosa, methods for screening biological samples susceptible to epidermolysis bullosa Kits, constructs and recombinant cells. Background technique [0002] Epidermolysis bullosa (Epidermolysis bullosa, EB) is a skin disease determined by genes, and the common feature is that the skin and mucous membrane tissue will produce blisters after minor trauma. It has brought great pain to the patient's family, and for the patient himself, it will be accompanied by pain for life. There is currently no treatment for the disease. One in 227 people carries the genetic defect that causes epidermolysis bullosa. Epidermolysis bullosa affects one in every 18,000 newborns. Eighteen pathogenic genes have b...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/00C12N15/52C12Q1/6883
CPCC12N9/93C12Q1/6883C12Q2600/156C12Y603/02019
Inventor 戴兰兰杨勇张建国林志淼刘汉奎冯程谌于蓝汪慧君汪建
Owner SHENZHEN HUADA GENE INST
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