MAF gene mutant causing congenital cataract, polypeptide, kit, construct, recombinant cell and application

A congenital cataract and recombinant cell technology, applied in the direction of cells modified by introducing foreign genetic material, application, genetic engineering, etc., can solve the problem of hindering the clinical diagnosis of congenital cataract, rebirth, prenatal genetic diagnosis, new mutation data of MAF gene Imperfect and other issues

Active Publication Date: 2022-02-15
AFFILIATED HOSPITAL OF WEIFANG MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current data on new mutations in the MAF gene are not perfect, which hinders the establishm

Method used

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  • MAF gene mutant causing congenital cataract, polypeptide, kit, construct, recombinant cell and application
  • MAF gene mutant causing congenital cataract, polypeptide, kit, construct, recombinant cell and application
  • MAF gene mutant causing congenital cataract, polypeptide, kit, construct, recombinant cell and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Design upstream and downstream amplification primers for the MAF gene mutation site, using the genomic DNA of cataract patients as a template, PCR method to amplify the upstream and downstream 341bp sequence of the MAF gene mutation site, the amplified product was subjected to agarose gel electrophoresis, and the PCR product After separation and purification, Sanger sequencing was performed to analyze the sequence and detect the mutation site of MAF gene in cataract patients.

[0048] Primers used to amplify the c.788A→G mutation site in exon 1 of MAF gene:

[0049] Forward 5'-GGCGGCCTGCACTTC-3' (SEQ ID NO.5);

[0050] Reverse 5'-GGGTTGTCGCTGCTCG-3' (SEQ ID NO. 6)

[0051] The reaction system for amplifying the sequence of the MAF gene mutation site by PCR method is shown in Table 1.

[0052] PCR reaction conditions: denaturation at 98°C for 3 minutes; then 11 cycles, denaturation at 98°C for 20 seconds, annealing temperature from 70°C to 60°C (1°C per cycle) for 20 s...

Embodiment 2

[0060] (1) Construction of MAF constructs with FLAG tag sequences (constructs of MAF wild type and c.788A→G mutants with FLAG tag sequences are shown in Figure 5 Shown): Design the upstream and downstream amplification primers for the full-length open reading frame of the MAF gene, the upstream primer has a Hind III restriction site, the downstream primer has a BamH I restriction site, and the MAF gene exists c.788A→G Genomic DNA of a cataract patient with heterozygous mutation is used as a template. Since one MAF allele of this patient is a wild-type normal sequence, and the other MAF allele contains a c.788A→G mutation sequence, using the patient’s genomic DNA as a template, PCR Methods Amplify the MAF open reading frame sequence, and simultaneously obtain the wild type and c.788A→G mutant MAF open reading frame sequences, and the amplified product is subjected to Hind III and BamH I double enzyme digestion and purification; commercialized pFLAG - After the CMV4 expression ...

Embodiment 3

[0100] The nucleic acid construct containing the wild-type MAF gene and the c.788A→G mutant constructed in Example 2 was used to transfect recipient cells to prepare recombinant cells.

[0101] Recipient cells were derived from Escherichia coli cells (DH5α competent cells, purchased from Tiangen Biochemical Technology Co., Ltd.) or mammalian cells (HEK293T cells, purchased from National Biomedical Experimental Cell Resource Bank).

[0102] The method for transforming DH5α competent cells with MAF constructs: 5 μL FLAG-tagged ligation products were transformed into 30 μL DH5α competent cells, placed on ice for 20 minutes, heat-shocked at 42°C for 1 minute and 30 seconds, incubated on ice for 5 minutes, and inoculated in ampicillin 30 μL of DH5α competent cells were transformed with 5 μL of GFP-labeled ligation products, placed on ice for 20 minutes, heat-shocked at 42°C for 1 minute and 30 seconds, incubated on ice for 5 minutes, and inoculated in kanamycin resistant agarose pl...

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Abstract

The invention relates to an MAF gene mutant causing congenital cataract disease phenotype, a polypeptide, a kit, a construct, a recombinant cell and application, belonging to to the technical field of biological gene engineering. The MAF gene mutant disclosed by the invention is characterized in that nucleotide at the 788 site of a No. 1 exon of an MAF gene is subjected to A-G mutation. The polypeptide has p.D263G mutation. The kit can be used for screening biological samples susceptible to congenital cataract and contains a reagent for detecting the MAF gene mutant. The nucleic acid construct contains the MAF gene mutant. The recombinant cell is obtained by transfecting recipient cells with the nucleic acid construct containing the MAF gene mutant, and can express the MAF polypeptide with the p.D263G mutation. The method can be used for molecular genetic diagnosis of congenital cataract, and provides a basis for prenatal gene diagnosis of patients with cataract caused by MAF gene mutation.

Description

technical field [0001] The invention relates to the technical field of biogenetic engineering, in particular to a MAF gene mutant, polypeptide, kit, construct, recombinant cell and application that cause congenital cataract disease phenotype. Background technique [0002] At present, congenital cataract is a serious blinding lens disease, which is caused by the disorder of lens metabolism in embryonic stage, infection during embryonic development, gene defects and chromosomal abnormalities, which lead to the decrease of its own transparency. Leukocoria in infancy. Clinically, congenital cataract can be manifested as lens opacity with different shapes such as nuclear, anterior pole, and posterior pole. Leukocoria is the most common manifestation of congenital cataract in newborns, while incomplete cataract often presents with abnormalities such as poor vision, strabismus, and nystagmus. The incidence rate of this disease is about 0.01%~0.06%, accounting for 10%~38% of blind...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/85C12N15/65C12N5/10C07K14/47C12Q1/6883G01N33/68
CPCC07K14/47C12N15/85C12N15/65C12Q1/6883G01N33/6893C12Q2600/156G01N2333/47G01N2800/166
Inventor 丰岱荣闫晓娜李金云
Owner AFFILIATED HOSPITAL OF WEIFANG MEDICAL UNIV
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