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cyp4v2 gene mutant and its application

A retinal degeneration, primary technology, applied in application, genetic engineering, plant genetic improvement and other directions, can solve problems that need to be deepened

Inactive Publication Date: 2019-10-25
THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the current research on primary BCD, especially its pathogenic genes, still needs to be in-depth

Method used

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  • cyp4v2 gene mutant and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1 Determination of pathogenic mutations in primary crystalline retinal degeneration (BCD)

[0065] 1. Sample collection

[0066] In 2010, the inventor collected a case of BCD patient in Chongqing. There were 4 family members of this patient, including 1 patient and 3 normal members (the father passed away, and his vision and field of vision were normal before his death, without night blindness). A total of 3 people participated in the research of the present invention, including 1 patient, 2 normal members (mother and younger brother), and all family members participating in the research of the present invention signed the informed consent. The patient's pedigree figure 2 As shown, □ indicates a normal male, ■ indicates a male patient, ○ indicates a normal female, Denotes a deceased normal male, M denotes a mutation, and + denotes a wild type.

[0067] In this family, the patient developed night blindness at the age of 49, and the fundus examination showed ...

Embodiment 2

[0078] Example 2: Sanger sequencing verification of candidate CYP4V2 genes

[0079] Primers were designed for the sequence of CYP4V2 gene (exon 1-11), and the related sequence was obtained by PCR amplification, product purification and sequencing. The specific operation is as follows:

[0080] 1. DNA extraction

[0081] The probands in the above-mentioned BCD family were collected ( figure 2 Members represented by middle ■) peripheral blood, using the conventional phenol-chloroform method to extract the genomic DNA in the peripheral blood leukocytes, using a spectrophotometer to measure the concentration and purity of the DNA, and the OD of the genomic DNA of each sample obtained 260 / OD 280 They are all located between 1.7-2.0, the concentration is not less than 200 ng / microliter, and the total amount is not less than 30 micrograms.

[0082] 2. Primer Design

[0083] The PCR reaction primers were designed with reference to the human genome sequence, as shown in Table 1 ...

Embodiment 3

[0096]Example 3: Carry out Sanger method sequencing verification on pathogenic mutations

[0097] The CYP4V2 gene of all family members (including patients and normal people in the family) and 100 normal people outside the family in the BCD patient family described in Example 1 were detected: using the exons of the CYP4V2 gene in Example 2 6 The designed primers were used to obtain the relevant sequence of the mutation site by PCR amplification, product purification and sequencing, and to verify the correlation between the CYP4V2 gene and the mutation and BCD according to whether the sequence determination results belonged to the mutant type or the wild type.

[0098] The specific method steps are as follows:

[0099] 1. DNA extraction

[0100] According to the method for extracting DNA described in Example 2, the genomic DNA in the peripheral venous blood of the subject was extracted and prepared respectively for future use.

[0101] 2. PCR reaction

[0102] Using the prim...

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Abstract

The invention discloses a CYP4V2 gene mutant and an application thereof, and specifically relates to a separate nucleic acid encoding a CYP4V2 mutant, a separate polypeptide, a method for screening a biological sample susceptible to primary crystal-like retinal degeneration, a system for screening a biological sample susceptible to primary crystal-like retinal degeneration and a kit for screening a biological sample susceptible to primary crystal-like retinal degeneration. Compared with SEQ ID No: 1, the separated nucleic acid encoding the CYP4V2 mutant has c.791del T mutation. By detecting the existence of the new mutant in the biological sample or not, whether the biological sample is susceptible to the primary crystal-like retinal degeneration can be effectively detected.

Description

technical field [0001] The present invention relates to CYP4V2 gene mutant and application thereof. Specifically, the present invention relates to isolated nucleic acids encoding CYP4V2 mutants, isolated polypeptides, methods for screening biological samples susceptible to primary crystalline retinal degeneration, systems for screening biological samples susceptible to primary crystalline retinal degeneration , kits, constructs and recombinant cells for screening biological samples susceptible to primary crystalline retinal degeneration. Background technique [0002] Bietti's crystalline corneoretinal dystrophy (BCD) is a rare hereditary blinding disease with an incidence of about 1 / 24,000 in the world. 4000. BCD patients usually develop at the age of 20-30 and become blind at the age of 40-50. At present, it has become a major eye disease that threatens the visual function and quality of young and middle-aged people around the world. It will seriously affect the quality o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12C12N15/63C12N5/10C07K14/47C12Q1/6883C12M1/00C12M1/34
Inventor 孟晓红阴正勤黎其友徐海伟金鑫李世迎
Owner THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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