CN114045292A

CN114045292A - MAF gene mutant, polypeptide, kit, construct, recombinant cell and application for causing congenital cataract

Info

Publication number: CN114045292A
Application number: CN202210034224.9A
Authority: CN
Inventors: 丰岱荣; 闫晓娜; 李金云
Original assignee: Affiliated Hospital of Weifang Medical University
Current assignee: Affiliated Hospital of Weifang Medical University
Priority date: 2022-01-13
Filing date: 2022-01-13
Publication date: 2022-02-15
Anticipated expiration: 2042-01-13
Also published as: CN114045292B

Abstract

The invention relates to a MAF gene mutant, polypeptide, a kit, a construct, a recombinant cell and application thereof, which cause congenital cataract disease phenotype, and belongs to the technical field of biological genetic engineering. The MAF gene mutant is an A → G mutation of 788 nucleotides of the No.1 exon of the MAF gene; the polypeptide has a 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 a MAF gene mutant; the recombinant cell is obtained by transfecting a receptor cell with the nucleic acid construct containing the MAF gene mutant, and can express the MAF polypeptide with the p.D263G mutation. The invention can be used for molecular genetic diagnosis of congenital cataract and provides basis for prenatal gene diagnosis of cataract patients caused by MAF gene mutation.

Description

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

Technical Field

The invention relates to the technical field of biological genetic engineering, in particular to a MAF gene mutant, a polypeptide, a kit, a construct, a recombinant cell and application thereof, wherein the MAF gene mutant causes congenital cataract disease phenotype.

Background

At present, the congenital cataract is a serious blindness-causing lens disease, is a disease of which the self transparency is reduced due to the metabolic disturbance of the lens in the embryonic period, infection in the embryonic development process, gene defect and chromosome abnormality, and is mainly shown as white pupillary disease occurring in infants. Clinically, congenital cataract may be manifested as lenticular opacity with different shapes, such as nuclear, anterior and posterior poles. White pupil is the most common manifestation of congenital cataract in newborn, and incomplete cataract is usually diagnosed by abnormality such as low vision, strabismus, nystagmus, etc. The incidence rate of the disease is about 0.01-0.06%, and accounts for 10-38% of blindness-causing eye diseases of children. Hereditary cataract accounts for 22.3% of the worldwide childhood cataract, and the most common genetic mode is autosomal dominant inheritance.

MAF is mainly used as a transcription factor to directly regulate the expression of downstream crystallin encoding genes, and if the MAF gene is mutated, the combination of the encoded MAF polypeptide and a plurality of crystallin encoding gene promoter regions can be directly influenced, so that the expression of a plurality of crystallins is reduced, and finally cataract expression is caused. MAF is one of the pathogenic genes of the congenital cataract, the pathogenic mutation is rare, so far, 29 pathogenic mutation sites are discovered at home and abroad, the missense mutation and the nonsense mutation caused by point mutation are mainly located in one exon of the MAF gene, and the transcriptional regulation function of the MAF coding protein product is influenced.

The new mutation and pathogenicity of the MAF gene related to the congenital cataract phenotype are determined, and the method has important significance for determining the molecular genetic diagnosis of the patient, guiding the accurate treatment of the patient and avoiding the regeneration risk of the family. However, the existing MAF gene new mutation data is not perfect, and the establishment of the clinical diagnosis of the congenital cataract and the development of the regeneration prenatal gene diagnosis of related families are prevented.

Disclosure of Invention

The invention aims to provide a MAF gene mutant, a polypeptide, a kit, a construct, a recombinant cell and application thereof, which cause the phenotype of the congenital cataract disease. The invention determines the new mutant of the MAF gene, the new mutant is closely related to the occurrence of the congenital cataract, and the new mutant is detected whether to exist in a biological sample, so that whether the biological sample suffers from the congenital cataract can be effectively detected, the molecular genetic diagnosis of the patient is determined, the accurate treatment of the patient is guided, and the regeneration risk of the family is avoided.

The invention provides a MAF gene mutant, and the nucleotide sequence of the MAF gene mutant is shown in SEQ ID No. 3.

The invention also provides the polypeptide coded by the MAF gene mutant in the technical scheme, and the amino acid sequence of the polypeptide is shown in SEQ ID NO. 4.

The invention also provides the application of the reagent for detecting the MAF gene mutant or the polypeptide coded by the MAF gene mutant in the technical scheme in the preparation of an congenital cataract molecular genetics diagnosis kit or a prenatal gene diagnosis kit or a biological sample screening kit susceptible to congenital cataract.

The invention also provides a kit, which comprises a reagent for detecting the MAF gene mutant or the polypeptide coded by the MAF gene mutant; the reagent for detecting the MAF gene mutant contains a reagent for detecting the 788 th nucleotide mutation A → G of the MAF gene, and the nucleotide sequence of the mutated MAF gene mutant is shown as SEQ ID NO. 3.

Preferably, the reagent for detecting the 788 th nucleotide mutation of the MAF gene comprises a primer, and the nucleotide sequence of the primer is shown as SEQ ID NO.5 and SEQ ID NO. 6.

The invention also provides a nucleic acid construct, which contains the MAF gene mutant; the nucleotide sequence of the MAF gene mutant is shown in SEQ ID NO. 3.

Preferably, the nucleic acid construct further comprises a FLAG tag sequence or a GFP tag sequence; when the nucleic acid construct contains a FLAG tag sequence, the skeleton vector of the nucleic acid construct comprises pFLAG-CMV4, and the nucleotide sequences of primers for constructing the nucleic acid construct are shown as SEQ ID NO.7 and SEQ ID NO. 8; when the nucleic acid construct contains a GFP tag sequence, the framework vector of the nucleic acid construct comprises pAcGFP1-C1, and the nucleotide sequences of primers for constructing the nucleic acid construct are shown as SEQ ID NO.9 and SEQ ID NO. 8.

The invention also provides a recombinant cell, wherein the recombinant cell is obtained by transfecting the nucleic acid construct of the technical scheme into a receptor cell.

The invention also provides the application of the construct or the recombinant cell in the technical scheme in the preparation of a kit for researching the function of the polypeptide coded by the MAF gene mutant.

The invention also provides the application of the construct or the recombinant cell in the technical scheme in preparing a kit for defining the molecular genetic diagnosis of the congenital cataract patient, guiding the precise treatment of the patient or avoiding the regeneration risk of the family of the patient.

The invention provides a MAF gene mutant causing congenital cataract disease phenotype. At present, no report that the mutation of the MAF gene c.788A → G (p.D263G) causes congenital cataract exists internationally, and the functional data of the polypeptide coded by the new variation of the MAF gene is lacked, so that the pathogenicity rating of the new variation is influenced, and the establishment of clinical diagnosis of the disease and the development of regenerative prenatal gene diagnosis of related families are hindered. The invention determines the new mutant of the MAF gene, the new mutant is closely related to the disease of the congenital cataract, and whether the biological sample suffers from the congenital cataract or not can be effectively detected by detecting whether the new mutant exists in the biological sample or not. The technical scheme of the invention defines the pathogenicity of new mutation in MAF gene related to the congenital cataract phenotype, and has important significance for defining the molecular genetics diagnosis of the patient, guiding the accurate treatment of the patient and avoiding the regeneration risk of the family.

The invention also provides MAF polypeptides with specific mutations. The nucleic acid construct of the MAF gene with specific mutations of the invention can be used for in vitro functional studies; recombinant cells with specific mutant constructs can be used to determine unambiguously whether a MAF mutant polypeptide affects the transcriptional expression level of downstream crystallin-encoding genes, providing evidence for the pathogenicity rating of new mutations in the MAF gene.

Drawings

FIG. 1 is a pedigree diagram of a family of congenital cataracts provided by the present invention;

FIG. 2 is a schematic diagram of agarose gel electrophoresis of 341bp products at the upstream and downstream of the c.788 locus of MAF gene of patients and normal individuals in cataract family amplified by PCR method provided by the present invention;

FIG. 3 is a schematic diagram of the c.788 positions of wild type MAF and its mutants;

FIG. 4 is a schematic representation of the conservation analysis of the MAF 263 rd amino acid position in different vertebrates according to the present invention;

FIG. 5 is a schematic diagram of the MAF wild-type and c.788A → G mutant constructs provided herein with the FLAG tag sequence;

FIG. 6 is a schematic diagram of the MAF wild-type and c.788A → G mutant constructs provided herein with GFP tag sequences;

FIG. 7 is a schematic diagram of the detection of MAF wild type and p.D263G mutant polypeptide expression levels in recombinant HEK293T cells with a FLAG tag sequence and a C.788A → G mutant construct by the immunoblotting method provided by the present invention; wherein, (a) FLAG-MAF Wild Type (WT) and mutant (D263G) polypeptides in recombinant HEK293T cells; (B) histograms of FLAG-MAF mutant (D263G) polypeptide and Wild Type (WT) polypeptide expression levels in recombinant HEK293T cells: p < 0.05;

FIG. 8 is a schematic diagram showing that in the recombinant HEK293T cells with the MAF wild type and c.788A → G mutant constructs carrying GFP tag sequences, the green fluorescence signals of the MAF wild type and p.D263G mutant polypeptides are localized in the cells detected by the immunofluorescence method provided by the invention.

Detailed Description

The invention provides a MAF gene mutant, and the nucleotide sequence of the MAF gene mutant is shown in SEQ ID No. 3. The MAF gene is localized at 16q23.2, with a total length of 6900bp, consisting of 1 exon, its full-length cDNA comprising 1122 nucleotides, and the encoded product is a 38.5kD transcription factor comprising 373 amino acids. MAF is mainly used as a transcription factor to directly regulate the normal expression of downstream crystallin. The MAF gene mutant of the invention is a wild type MAF gene with A → G mutation at the 788 th nucleotide of the No.1 exon. The nucleotide sequence of the wild type MAF gene is shown in SEQ ID NO. 1. The invention takes an innate cataract family as a research object, and identifies a new heterozygous pathogenic mutation in the MAF gene by using whole exome sequencing, Sanger sequencing and restriction enzyme fragment length polymorphism technology, wherein the 788 coding nucleotide positioned in No.1 exon has A → G mutation. At present, no report that the mutation of the MAF gene c.788A → G (p.D263G) causes congenital cataract exists internationally. The invention determines the new c.788A → G mutation in the congenital cataract pathogenic gene MAF by a method of target region capture sequencing combined with candidate gene mutation verification.

The invention also provides the polypeptide coded by the MAF gene mutant in the technical scheme, and the amino acid sequence of the polypeptide is shown in SEQ ID NO. 4. The polypeptide is characterized in that the 263 th Asp of the polypeptide coded by the wild MAF gene cDNA is mutated into Gly, namely the polypeptide has p.Asp263Gly (p.D263G) mutation, and the mutation is caused by missense mutation of c.A788G. The amino acid sequence of the polypeptide coded by the wild type MAF gene cDNA is shown in SEQ ID NO. 2. Whether the biological sample is susceptible to the congenital cataract or not can be effectively detected by detecting whether the polypeptide is expressed in the biological sample or not, and whether the biological sample is suffered from the congenital cataract or not can be effectively detected by detecting whether the polypeptide exists in an organism or not.

The invention also provides a kit, which comprises a reagent for detecting the MAF gene mutant or the polypeptide coded by the MAF gene mutant; the reagent for detecting the MAF gene mutant contains a reagent for detecting the 788 th nucleotide mutation A → G of the MAF gene, and the nucleotide sequence of the mutated MAF gene mutant is shown as SEQ ID NO. 3. The kit can realize the screening of biological samples susceptible to the congenital cataract, the molecular genetic diagnosis of the congenital cataract and the prenatal gene diagnosis of the congenital cataract. The detection method based on the kit of the invention preferably comprises the following steps: designing an upstream and downstream amplification primer aiming at the MAF gene mutation site, amplifying upstream and downstream 341bp sequences of the MAF gene mutation site by using a PCR method by using genome DNA of a sample to be detected as a template, carrying out agarose gel electrophoresis on an amplification product, separating and purifying the PCR product, and carrying out Sanger sequencing analysis on a nucleotide sequence. The MAF gene mutant is closely related to the onset of the congenital cataract, and whether the biological sample is susceptible to the congenital cataract or not or whether the patient is susceptible to the congenital cataract can be effectively detected by detecting whether the new mutant exists in the biological sample or not.

In the present invention, the reagent for detecting the 788 th nucleotide mutation of the MAF gene preferably comprises primers (upstream and downstream amplification primers for the mutation site of the MAF gene) or probes, the nucleotide sequences of which are shown in SEQ ID NO.5 (forward 5'-GGCGGCCTGCACTTC-3') and SEQ ID NO.6 (reverse 5'-GGGTTGTCGCTGCTCG-3'). The primer can obviously and effectively complete the amplification of the MAF exon 1 in a PCR reaction system, and can efficiently screen biological samples susceptible to the congenital cataract.

In the present invention, the reaction system for PCR amplification is preferably as shown in Table 1:

TABLE 1 reaction System

In the present invention, the reaction procedure for the PCR amplification is preferably: denaturation at 98 ℃ for 3 min; followed by 11 cycles of denaturation at 98 ℃ for 20 seconds, annealing temperature from 70 ℃ to 60 ℃ (1 ℃ reduction per cycle) for 20 seconds, and extension at 72 ℃ for 30 seconds; this was followed by 40 cycles of denaturation at 98 ℃ for 20 seconds, annealing at 59 ℃ for 20 seconds, and extension at 72 ℃ for 30 seconds.

The invention also provides a nucleic acid construct, which contains the MAF gene mutant; the nucleotide sequence of the MAF gene mutant is shown in SEQ ID NO. 3. The nucleic acid of the present invention is any polymer comprising deoxyribonucleotides or ribonucleotides, including but not limited to modified or unmodified DNA, RNA, and the length thereof is not subject to any particular limitation. For constructs used to construct recombinant cells, it is preferred that the nucleic acid be DNA, as DNA is more stable and easier to manipulate than RNA. The term "construct" as used in the present invention refers to a genetic vector comprising a specific nucleic acid sequence and capable of transferring the nucleic acid sequence of interest into a host cell to obtain a recombinant cell. According to an embodiment of the present invention, the form of the construct is not particularly limited. According to an embodiment of the present invention, it is at least one of a plasmid, a virus, preferably a plasmid. The plasmid is used as a genetic carrier, has the characteristics of simple operation, capability of carrying larger fragments and convenience for operation and treatment. The form of the plasmid is also not particularly limited, and may be either a circular plasmid or a linear plasmid. The skilled person can select as desired.

In the present invention, the nucleic acid construct further comprises a FLAG tag sequence or a GFP tag sequence; when the nucleic acid construct contains a FLAG tag sequence, the skeleton vector of the nucleic acid construct comprises pFLAG-CMV4, and the nucleotide sequences of primers for constructing the nucleic acid construct are shown as SEQ ID NO.7 and SEQ ID NO. 8; when the nucleic acid construct contains a GFP tag sequence, the framework vector of the nucleic acid construct comprises pAcGFP1-C1, and the nucleotide sequences of primers for constructing the nucleic acid construct are shown as SEQ ID NO.9 and SEQ ID NO. 8.

The invention also provides a construction method of the nucleic acid construct, which preferably comprises the following steps:

designing an amplification primer with a specific enzyme cutting site according to an open reading frame of the MAF gene, carrying out double enzyme cutting on an amplification product, and connecting the amplification product to an expression vector with a specific tag polypeptide sequence according to research needs to construct a nucleic acid construct with specific site mutation.

Specifically, MAF nucleic acid constructs with FLAG tag sequences were constructed: designing primers with nucleotide sequences of specific enzyme cutting sites as shown in SEQ ID NO.7 and SEQ ID NO.8 aiming at the full-length open reading frame of the MAF gene, amplifying by taking the genomic DNA containing the MAF gene mutant as a template, and connecting the amplified product with an expression vector containing a FLAG tag which is subjected to enzyme cutting by the same enzyme cutting site to obtain the MAF nucleic acid construct with the FLAG tag sequence. In the present invention, the FLAG-tag containing expression vector preferably comprises pFLAG-CMV 4.

Construction of MAF nucleic acid constructs with GFP tag sequences: designing primers with nucleotide sequences of specific enzyme cutting sites as shown in SEQ ID NO.9 and SEQ ID NO.8 aiming at the full-length open reading frame of the MAF gene, amplifying by taking the genomic DNA containing the MAF gene mutant as a template, and connecting the amplified product with an expression vector containing a GFP label and subjected to enzyme cutting at the same enzyme cutting site to obtain the MAF nucleic acid construct with the GFP label sequence. In the present invention, the GFP tag-containing expression vector preferably includes pAcGFP 1-C1.

A MAF nucleic acid construct with a FLAG tag sequence, which can be used for researching and comparing the difference of the expression quantity of the MAF wild-type polypeptide and the p.D263G mutant polypeptide in mammalian cells; MAF nucleic acid constructs with GFP tag sequences can be used to study and compare the difference in expression position of MAF wild-type polypeptide and p.D263G mutant polypeptide in mammalian cells.

The invention also provides a recombinant cell, wherein the recombinant cell is obtained by transfecting the nucleic acid construct of the technical scheme into a receptor cell. The recombinant cell can express the polypeptide coded by the MAF gene mutant. In the present invention, the kind of the recipient cell is not particularly limited, and may be, for example, an escherichia coli cell or a mammalian cell, and the recipient cell is preferably derived from a mammal. In the present invention, the recipient cell is preferably derived from an E.coli cell (DH 5. alpha. competent cell, available from Tiangen Biochemical technology Co., Ltd.) or a mammalian cell (HEK 293T cell, available from national cell resources for biomedical experiments). The recombinant cell obtained by transfecting the receptor cell with the nucleic acid construct can evaluate the protein expression level of the mutant; in addition, the biological hazard of mutants in affecting transcription of the crystallin-encoding gene can be further assessed by quantitative analysis of the level of firefly luciferase gene expression, in conjunction with the firefly and Renilla dual-luciferase reporter systems with specific crystallin-encoding gene promoter sequences.

The MAF gene mutant, the polypeptide, the kit, the construct, the recombinant cell and the application thereof causing the congenital cataract disease phenotype are further described in detail with reference to the following specific examples, and the technical scheme of the invention includes but is not limited to the following examples.

Example 1

Designing an upstream and downstream amplification primer aiming at the MAF gene mutation site, amplifying upstream and downstream 341bp sequences of the MAF gene mutation site by using a PCR (polymerase chain reaction) method by using genome DNA of a cataract patient as a template, carrying out agarose gel electrophoresis on an amplification product, carrying out Sanger sequencing after separation and purification of the PCR product, analyzing the sequence, and detecting the MAF gene mutation site of the cataract patient.

Primers for amplification of the c.788A → G mutation site in exon 1 of the MAF gene:

forward direction 5'-GGCGGCCTGCACTTC-3' (SEQ ID NO. 5);

reverse direction 5'-GGGTTGTCGCTGCTCG-3' (SEQ ID NO. 6)

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

And (3) PCR reaction conditions: denaturation at 98 ℃ for 3 min; followed by 11 cycles of denaturation at 98 ℃ for 20 seconds, annealing temperature from 70 ℃ to 60 ℃ (1 ℃ reduction per cycle) for 20 seconds, and extension at 72 ℃ for 30 seconds; this was followed by 40 cycles of denaturation at 98 ℃ for 20 seconds, annealing at 59 ℃ for 20 seconds, and extension at 72 ℃ for 30 seconds.

Separation and purification of PCR products: after 2% agarose gel separation, the MAF gene PCR amplification product fragment is cut off a gel strip containing a target band in an ultraviolet gel cutting instrument, the gel strip is loaded into a 1.5ml EP tube, the weight of a gel block is weighed by an electronic balance, 100 mul Binding buffer is added into a centrifugal tube containing gel according to 100mg gel, the centrifugal tube is placed in a metal bath and heated at 60 ℃ until the gel is completely melted, and the centrifugal tube is centrifuged for 30 seconds. The gel solution was transferred to a nucleic acid silica gel purification column, centrifuged at 12000 rpm for 1 minute, and the waste solution was discarded. Add 700. mu.l of washing solution (containing 80% ethanol), centrifuge at 12000 rpm for 1 minute, discard the waste solution, centrifuge for 2 minutes again, remove the residual liquid on the wall of the centrifuge tube, and transfer the nucleic acid silica gel purification column to a sterile 1.5ml centrifuge tube. And uniformly dropwise adding 22 mu l of elution buffer solution onto the adsorption membrane, incubating at room temperature for 5-6 minutes, and collecting the purified DNA solution into a centrifuge tube for Sanger sequencing analysis at 12000 r/min of separation center.

As a result, the new mutant has a c.A788G mutation compared with the wild type MAF gene shown in SEQ ID No.1, namely, the 788 th base of the MAF gene mutant of the invention is mutated from A to G relative to the wild type MAF gene. Thus, the encoded product has the p.asp263gly mutation compared to wild-type MAF: amino acid Asp (D) at position 263 of wild type MAF is mutated to amino acid Gly (G).

FIG. 1 is a spectrum chart of the family of congenital cataracts, wherein IV-1 is congenital cataract patients, III-7 is congenital father, III-9 is congenital tertiary patient, all of which are cataract patients. Because a plurality of patients in the family have undergone the operation of replacing the lens, the cataract is not present, but the vision is low, and the vision of both eyes is lower than 0.3.

The method of embodiment 1 is adopted to detect the family of the congenital cataract patient, the agarose gel electrophoresis schematic diagram of 341bp products at the upstream and downstream of the c.788 locus of the patient and the normal individual MAF gene in the cataract family amplified by the PCR method is shown in figure 2, IV-1 is the proband, III-7 proband father and III-9 proband tert-tert of the congenital cataract patient, which are all patients; III-6 proband mothers, III-10 proband girl and IV-2 proband girls are all normal persons.

The c.788 positions of the wild type MAF and the mutant are shown in FIG. 3 by sequencing. After 341bp fragments above and below the mutation site of the cataract patients and the family MAF gene are amplified by PCR, the Sanger sequencing result shows that IV-1 is a heterozygous patient for the proband of the congenital cataract patients, the father of the proband of III-7 and the tertiaryt of the proband of III-9; the sites of III-6 proband mother, III-10 proband girald and IV-2 proband girl are normal and have no mutation. Confirming that the mutation cosegregated with the cataract genotype phenotype.

Subsequently, the polypeptide amino acid coded by the nucleic acid mutant is compared with the wild type polypeptide to obtain a conservative analysis schematic diagram of the amino acid site 263 of the MAF shown in the figure 4 in different vertebrates; the results show that this site is highly conserved among different species (chicken, cattle, orangutan, etc.), indicating that it has important functionality.

Example 2

(1) Construction of MAF constructs with FLAG tag sequence (MAF wild-type with FLAG tag sequence and c.788a → G mutant constructs schematic as shown in figure 5): designing an upstream and downstream amplification primer aiming at a full-length open reading frame of the MAF gene, wherein the upstream primer is provided with a Hind III enzyme cutting site, the downstream primer is provided with a BamH I enzyme cutting site, the genomic DNA of a cataract patient with C.788A → G heterozygous mutation of the MAF gene is taken as a template, one MAF allele of the patient is a wild type normal sequence, the other MAF allele of the patient comprises a c.788A → G mutant sequence, the genomic DNA of the patient is taken as a template, the MAF open reading frame sequence is amplified by a PCR method, the MAF open reading frame sequence comprising a wild type and a c.788A → G mutant MAF open reading frame sequence can be obtained at the same time, and the amplified product is subjected to double enzyme cutting and purification of Hind III and BamH I; after a commercial pFLAG-CMV4 expression vector is subjected to double digestion and purification by Hind III and BamH I, the pFLAG-CMV4 expression vector is connected with a cut and purified MAF open reading frame fragment to form a circular double-stranded DNA by T4 ligase, 5 mu L of a ligation product is taken to transform 30 mu L of DH5 alpha competent cells, the cells are inoculated in an ampicillin resistant agarose plate and cultured overnight at 37 ℃, and a single clone is picked for Sanger sequencing to identify the sequences of a wild type MAF gene construct (pFLAG-CMV 4-MAF-WT) with a FLAG tag and a MAF mutant gene construct (pFLAG-CMV 4-MAF-D263G) with a FLAG tag;

primers used for constructing pFLAG-CMV4-MAF-WT, pFLAG-CMV 4-MAF-D263G:

forward primer Hind III-maforrf-F:

5’-AACGCGAAGCTTGCATCAGAACTGGCAATGAG-3’（SEQ ID NO.7）；

reverse primer BamH I-MAFORF-R:

5’-AACGCGGGATCCTCACATGAAAAACTCGGGAGA-3’（SEQ ID NO.8）。

the reaction system for amplifying the MAF open reading frame sequence with Hind III and BamH I cleavage sites by PCR method is shown in Table 2.

TABLE 2 reaction System

(2) Construction of MAF constructs with GFP tag sequences (constructs of MAF wild type with GFP tag sequence and c.788A → G mutant are schematically shown in FIG. 6): designing an upstream and downstream amplification primer aiming at a full-length open reading frame of a MAF gene, wherein the upstream primer is provided with a Kpn I enzyme cutting site, the downstream primer is provided with a BamH I enzyme cutting site, the genomic DNA of a cataract patient with c.788A → G heterozygous mutation of the MAF gene is taken as a template, one MAF allele of the patient is a wild type normal sequence, the other MAF allele of the patient comprises a c.788A → G mutant sequence, the genomic DNA of the patient is taken as a template, the MAF open reading frame sequence is amplified by a PCR method, the open reading frame sequence comprising the wild type and the c.788A → G mutant MAF is obtained at the same time, and the amplified product is subjected to double enzyme cutting and purification of the Kpn I and the BamH I; after a commercial pAcGFP1-C1 expression vector is subjected to double digestion and purification by Kpn I and BamH I, the expression vector is connected with a cut and purified MAF open reading frame fragment to form circular double-stranded DNA by T4 ligase, 5 mu L of a connection product is transformed into 30 mu L of DH5 alpha competent cells, the competent cells are inoculated into an agarose plate resistant to kanamycin and cultured overnight at 37 ℃, a single clone is selected for Sanger sequencing, and the sequences of a wild type MAF gene construct (pAcGFP 1-C1-MAF-WT) with a GFP label and a mutant MAF gene construct (pAcGFP 1-C1-MAF-D263G) with the GFP label are identified;

primers used for constructing pAcGFP1-C1-MAF-WT, pAcGFP 1-C1-MAF-D263G:

forward primer KpnI-MAFORF-F:

5’-AACGCGGGTACCGCATCAGAACTGGCAATGAG-3’（SEQ ID NO.9），

the reverse primer BamHI-MAFORF-R is the same as SEQ ID NO. 8.

The reaction system for amplifying the MAF open reading frame sequence with KpnI and BamH I cleavage sites is shown in Table 3.

TABLE 3 reaction System

PCR conditions for MAF open reading frame sequence with restriction enzyme sites: denaturation at 98 ℃ for 3 min; then 11 cycles, denaturation at 98 ℃ for 15 seconds, annealing temperature from 70 ℃ to 60 ℃ (1 ℃ reduction per cycle) for 15 seconds, and extension at 72 ℃ for 1 min and 10 seconds; after 40 cycles, denaturation at 98 ℃ for 15 seconds, annealing at 59 ℃ for 15 seconds, and extension at 72 ℃ for 1 min 10 seconds.

Separation and purification of the PCR product of the MAF open reading frame sequence with the enzyme cutting site: after the PCR product was separated by 1% agarose gel, a gel strip containing the target band was cut in an ultraviolet gel cutter, and the cut gel strip was put into a 1.5ml EP tube, the weight of the gel block was weighed by an electronic balance, 100. mu.l Binding buffer was added to each 100mg gel in a centrifuge tube containing the gel, and the gel was heated in a metal bath at 60 ℃ until the gel was completely melted, and centrifuged for 30 seconds. The gel solution was transferred to a nucleic acid silica gel purification column, centrifuged at 12000 rpm for 1 minute, and the waste solution was discarded. Add 700. mu.l of washing solution (containing 80% ethanol), centrifuge at 12000 rpm for 1 minute, discard the waste solution, centrifuge for 2 minutes again, remove the residual liquid on the wall of the centrifuge tube, and transfer the nucleic acid silica gel purification column to a sterile 1.5ml centrifuge tube. And uniformly dropwise adding 22 mu l of elution buffer solution onto the adsorption membrane, incubating at room temperature for 5-6 minutes, and collecting the purified DNA solution into a centrifuge tube for double enzyme digestion reaction at 12000 r/min of centrifugation.

The double cleavage system for the PCR product with the MAF open reading frame sequence with Hind III and BamH I cleavage sites is shown in Table 4.

TABLE 4 double enzyme digestion System

The pFLAG-CMV4 vector Hind III and BamH I double digestion system is shown in Table 5.

TABLE 5 double enzyme digestion System

The double digestion system of the PCR purified product with MAF open reading frame sequence with KpnI and BamH I cleavage sites is shown in Table 6.

TABLE 6 double enzyme digestion System

The double enzyme digestion system of pAcGFP1-C1 vector Kpn I and BamH I is shown in Table 7.

TABLE 7 double enzyme digestion System

The conditions of the 4 enzyme digestion systems are as follows: incubate at 37 ℃ for 59 minutes, inactivate at 80 ℃ for 15 minutes, and cool to room temperature.

The products of PCR purification of the MAF open reading frame sequence digested with Hind III and BamH I were ligated with the recovered products of FLAG tag vector digested with Hind III and BamH I, and the ligation reaction system is shown in Table 8.

TABLE 8 ligation reaction System

The PCR purified product of the open reading frame sequence of MAF digested with KpnI and BamH I was ligated with the recovered product of GFP tag vector digested with KpnI and BamH I, and the ligation reaction system was shown in Table 9.

TABLE 9 ligation reaction System

The four nucleic acid constructs of pFLAG-CMV4-MAF-WT, pFLAG-CMV4-MAF-D263G, pAcGFP1-C1-MAF-WT and pAcGFP1-C1-MAF-D263G constructed according to the technical method prove that the construction is successful.

Example 3

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.

Recipient cells are derived from E.coli cells (DH 5. alpha. competent cells, available from Tiangen Biochemical technology Co., Ltd.) or mammalian cells (HEK 293T cells available from national cell resources for biomedical experiments).

Method for transformation of DH5 α competent cells with MAF constructs: mu.L of FLAG-tagged ligation product was transformed into 30. mu.L of DH 5. alpha. competent cells, placed on ice for 20 minutes, heat-shocked at 42 ℃ for 1 minute and 30 seconds, incubated on ice for 5 minutes, and plated on ampicillin-resistant agarose plates; mu.L of the GFP-tagged ligation product was transformed into 30. mu.L of DH 5. alpha. competent cells, incubated on ice for 20 minutes, heat-shocked at 42 ℃ for 1 minute and 30 seconds, incubated on ice for 5 minutes, and plated on kanamycin-resistant agarose plates. The plates were incubated overnight at 37 ℃ and single colonies were picked for expansion culture of recombinant cells containing the corresponding MAF nucleic acid construct.

Transfection of the MAF constructs HEK293T mammalian cells: HEK293T cells (accession number: 3111C0001CCC000091, purchased from national Biomedicine laboratory cell resources Bank) were transfected when cultured to 60% -70% confluence, specifically, first an Opti-MEM-construct suspension was prepared: adding a proper amount of Opti-MEM (Thermo Fisher) culture medium into an EP tube, adding a p3000 reagent (2 mu L/1 mu g construct, Thermo Fisher), sequentially adding a wild type or mutant MAF construct, and fully mixing; and preparing an Opti-MEM-liposome suspension: add right amount of Opti-MEM medium to EP tube, add Lipofectamine 3000 liposome (ThermoFisher) reagent transfection reagent (3. mu.L/1. mu.g construct), blow gently and beat to mix well; the Opti-MEM-construct suspension is then added to the Opti-MEM-liposome suspension (the order cannot be reversed), mixed well, and incubated at room temperature for 5-10 minutes to form a construct-liposome mixture. And (3) gently dripping the construct-liposome mixture into a culture medium, putting the culture medium into an incubator for continuous culture for 24 hours, detecting the expression and the positioning of the FLAG-labeled or GFP-labeled MAF polypeptide by an immunoblotting method and an immunofluorescence method, and evaluating whether the recombinant cells are successfully constructed.

Immunoblot detection of wild-type and mutant polypeptides with FLAG tag MAF:

cell lysates containing FLAG-MAF wild-type and mutant polypeptides were prepared and tested (HEK 293T cells as an example): HEK293T cells containing constructs pFLAG-CMV4-MAF-WT and pFLAG-CMV4-MAF-D263G (containing the co-transfected pAcGFP1-C1 vector as a control) were collected into EP tubes using a cell scraper, cell pellets were collected by centrifugation, lysate (containing IP lysis buffer, 1 xPIC, 2mM PMSF, 8mM NaF, 2mM Na3VO 4) was added in a volume 5 times the volume of the cell pellets, the cells were blown down to completion of lysis, incubated on ice for 10 minutes and placed in a 4 ℃ centrifuge, centrifuged at 12000 rpm/separation for 10 minutes, the supernatant was transferred to a new EP tube, and FLAG-MAF polypeptide in the supernatant could be detected by denaturing polyacrylamide gel electrophoresis. Further, total protein was separated by 10% SDS-PAGE, and total protein in PAGE gel was transferred to PVDF membrane by wet transfer method, and then PVDF membrane was blocked with 1% TBS skim milk solution at room temperature for 0.5 hour, and then antibody α -FLAG (Sigma, F1804) capable of specifically recognizing the polypeptide sequence of FLAG tag was added. Meanwhile, alpha-GFP (Santa Cruz, sc-9996) was added to identify GFP polypeptides, and the expression level of FLAG-MAF polypeptides was quantitatively analyzed as a control. After overnight incubation, PVDF membrane was incubated with horseradish peroxidase-labeled secondary antibody m-IgGKBP-HRP (Santa Cruz, SC-516102) diluted in blocking solution at room temperature, ECL developed, and film exposure developed to show a protein band between 40 and 50 kD, demonstrating the successful construction of recombinant cells containing the constructs pFLAG-CMV4-MAF-WT and pFLAG-CMV 4-MAF-D263G. ImageJ software was used to analyze the band optical density and further to quantify the expression levels of FLAG-MAF wild-type and mutant polypeptides.

Immunofluorescence detection method of GFP-tagged MAF wild-type and mutant polypeptides:

the constructs pAcGFP1-C1-MAF-WT and pAcGFP1-C1-MAF-D263G were transfected into HEK293T cells (pre-plated on a 0.1mg/mL poly L-lysine-coated cover glass), the cell culture medium was removed after 24 hours of transfection, the cells were rinsed twice with 1% BSA/TBS at room temperature, and 4% paraformaldehyde was used to immobilize the cells for 15 min in 1 mL; after 1% BSA/TBS is rinsed twice, 0.5ml Triton-X100 is dripped on a cover glass to incubate for 10 minutes, and cell punching is carried out; washing cells twice with 1% BSA/TBS, adding DAPI fluorescent dye dropwise, incubating for 1 hour in a dark place, and washing twice with 1% BSA/TBS; the construction of recombinant cells with the constructs pAcGFP1-C1-MAF-WT and pAcGFP1-C1-MAF-D263G was confirmed successfully by dropping 20. mu.L of anti-quencher onto the slide, gently covering the slide with the cell-bearing side, smearing the blocking tablet around the slide, and observing the green signal under an inverted fluorescence microscope. By comparing the intensity and position of the green fluorescent signal, the difference in intracellular localization of the GFP-MAF wild-type and mutant polypeptides was confirmed.

The detection result proves that: the recombinant cells obtained by transfection of four constructs, namely pFLAG-CMV4-MAF-WT, pFLAG-CMV4-MAF-D263G, pAcGFP1-C1-MAF-WT and pAcGFP1-C1-MAF-D263G, can be used for analyzing and comparing the difference of the expression level and the expression position of the MAF wild-type polypeptide and the p.D263G mutant polypeptide in mammalian cells.

FIG. 7 is a graph showing the level of MAF wild-type and p.D263G mutant polypeptide expression in recombinant HEK293T cells with the MAF wild-type and c.788A → G mutant constructs with the FLAG tag sequence. (A) FLAG-MAF Wild Type (WT) and mutant (D263G) polypeptides in recombinant HEK293T cells; (B) histograms show that expression of FLAG-MAF mutant (D263G) polypeptide in recombinant HEK293T cells was significantly increased compared to Wild Type (WT) polypeptide: p < 0.05.

FIG. 8 is a schematic diagram showing the localization of MAF wild-type and p.D263G mutant polypeptide green fluorescence signals in recombinant HEK293T cells with MAF wild-type and c.788A → G mutant constructs with GFP tag sequences detected by immunofluorescence. In most recombinant cells, GFP-MAF wild-type polypeptide is uniformly expressed, and is expressed in such a way that green fluorescence signals are uniformly distributed in the cells, only individual cells have weak green fluorescence point-like aggregation, and the statistical analysis result shows that the average rate is only 0.78 points/cell; the expression of the mutant polypeptide in the recombinant cell is in punctate aggregation and is expressed as an obvious dense punctate green fluorescence signal, and the average is up to 30.2 points/cell. Immunofluorescence detection shows that in the MAF mutant recombinant cells, the position of the MAF p.D263G mutant polypeptide in the cells is changed, and the MAF p.D263G mutant polypeptide is not uniformly distributed in the cells like the MAF wild-type polypeptide, but is changed into a punctate aggregation state.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Weifang medical college subsidiary hospital

<120> MAF gene mutant, polypeptide, kit, construct, recombinant cell and application causing congenital cataract

<160> 9

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1122

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atggcatcag aactggcaat gagcaactcc gacctgccca ccagtcccct ggccatggaa 60

tatgttaatg acttcgatct gatgaagttt gaagtgaaaa aggaaccggt ggagaccgac 120

cgcatcatca gccagtgcgg ccgtctcatc gccgggggct cgctgtcctc cacccccatg 180

agcacgccgt gcagctcggt gcccccttcc cccagcttct cggcgcccag cccgggctcg 240

ggcagcgagc agaaggcgca cctggaagac tactactgga tgaccggcta cccgcagcag 300

ctgaaccccg aggcgctggg cttcagcccc gaggacgcgg tcgaggcgct catcagcaac 360

agccaccagc tccagggcgg cttcgatggc tacgcgcgcg gggcgcagca gctggccgcg 420

gcggccgggg ccggtgccgg cgcctccttg ggcggcagcg gcgaggagat gggccccgcc 480

gccgccgtgg tgtccgccgt gatcgccgcg gccgccgcgc agagcggcgc gggcccgcac 540

taccaccacc accaccacca cgccgccggc caccaccacc acccgacggc cggcgcgccc 600

ggcgccgcgg gcagcgcggc cgcctcggcc ggtggcgctg ggggcgcggg cggcggtggc 660

ccggccagcg ctgggggcgg cggcggcggc ggcggcggcg gaggcggcgg gggcgcggcg 720

ggggcggggg gcgccctgca cccgcaccac gccgccggcg gcctgcactt cgacgaccgc 780

ttctccgacg agcagctggt gaccatgtct gtgcgcgagc tgaaccggca gctgcgcggg 840

gtcagcaagg aggaggtgat ccggctgaag cagaagaggc ggaccctgaa aaaccgcggc 900

tatgcccagt cctgccgctt caagagggtg cagcagagac acgtcctgga gtcggagaag 960

aaccagctgc tgcagcaagt cgaccacctc aagcaggaga tctccaggct ggtgcgcgag 1020

agggacgcgt acaaggagaa atacgagaag ttggtgagca gcggcttccg agaaaacggc 1080

tcgagcagcg acaacccgtc ctctcccgag tttttcatgt ga 1122

<210> 2

<211> 373

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Ala Ser Glu Leu Ala Met Ser Asn Ser Asp Leu Pro Thr Ser Pro

1 5 10 15

Leu Ala Met Glu Tyr Val Asn Asp Phe Asp Leu Met Lys Phe Glu Val

20 25 30

Lys Lys Glu Pro Val Glu Thr Asp Arg Ile Ile Ser Gln Cys Gly Arg

35 40 45

Leu Ile Ala Gly Gly Ser Leu Ser Ser Thr Pro Met Ser Thr Pro Cys

50 55 60

Ser Ser Val Pro Pro Ser Pro Ser Phe Ser Ala Pro Ser Pro Gly Ser

65 70 75 80

Gly Ser Glu Gln Lys Ala His Leu Glu Asp Tyr Tyr Trp Met Thr Gly

85 90 95

Tyr Pro Gln Gln Leu Asn Pro Glu Ala Leu Gly Phe Ser Pro Glu Asp

100 105 110

Ala Val Glu Ala Leu Ile Ser Asn Ser His Gln Leu Gln Gly Gly Phe

115 120 125

Asp Gly Tyr Ala Arg Gly Ala Gln Gln Leu Ala Ala Ala Ala Gly Ala

130 135 140

Gly Ala Gly Ala Ser Leu Gly Gly Ser Gly Glu Glu Met Gly Pro Ala

145 150 155 160

Ala Ala Val Val Ser Ala Val Ile Ala Ala Ala Ala Ala Gln Ser Gly

165 170 175

Ala Gly Pro His Tyr His His His His His His Ala Ala Gly His His

180 185 190

His His Pro Thr Ala Gly Ala Pro Gly Ala Ala Gly Ser Ala Ala Ala

195 200 205

Ser Ala Gly Gly Ala Gly Gly Ala Gly Gly Gly Gly Pro Ala Ser Ala

210 215 220

Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Ala Ala

225 230 235 240

Gly Ala Gly Gly Ala Leu His Pro His His Ala Ala Gly Gly Leu His

245 250 255

Phe Asp Asp Arg Phe Ser Asp Glu Gln Leu Val Thr Met Ser Val Arg

260 265 270

Glu Leu Asn Arg Gln Leu Arg Gly Val Ser Lys Glu Glu Val Ile Arg

275 280 285

Leu Lys Gln Lys Arg Arg Thr Leu Lys Asn Arg Gly Tyr Ala Gln Ser

290 295 300

Cys Arg Phe Lys Arg Val Gln Gln Arg His Val Leu Glu Ser Glu Lys

305 310 315 320

Asn Gln Leu Leu Gln Gln Val Asp His Leu Lys Gln Glu Ile Ser Arg

325 330 335

Leu Val Arg Glu Arg Asp Ala Tyr Lys Glu Lys Tyr Glu Lys Leu Val

340 345 350

Ser Ser Gly Phe Arg Glu Asn Gly Ser Ser Ser Asp Asn Pro Ser Ser

355 360 365

Pro Glu Phe Phe Met

370

<210> 3

<211> 1122

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atggcatcag aactggcaat gagcaactcc gacctgccca ccagtcccct ggccatggaa 60

tatgttaatg acttcgatct gatgaagttt gaagtgaaaa aggaaccggt ggagaccgac 120

cgcatcatca gccagtgcgg ccgtctcatc gccgggggct cgctgtcctc cacccccatg 180

agcacgccgt gcagctcggt gcccccttcc cccagcttct cggcgcccag cccgggctcg 240

ggcagcgagc agaaggcgca cctggaagac tactactgga tgaccggcta cccgcagcag 300

ctgaaccccg aggcgctggg cttcagcccc gaggacgcgg tcgaggcgct catcagcaac 360

agccaccagc tccagggcgg cttcgatggc tacgcgcgcg gggcgcagca gctggccgcg 420

gcggccgggg ccggtgccgg cgcctccttg ggcggcagcg gcgaggagat gggccccgcc 480

gccgccgtgg tgtccgccgt gatcgccgcg gccgccgcgc agagcggcgc gggcccgcac 540

taccaccacc accaccacca cgccgccggc caccaccacc acccgacggc cggcgcgccc 600

ggcgccgcgg gcagcgcggc cgcctcggcc ggtggcgctg ggggcgcggg cggcggtggc 660

ccggccagcg ctgggggcgg cggcggcggc ggcggcggcg gaggcggcgg gggcgcggcg 720

ggggcggggg gcgccctgca cccgcaccac gccgccggcg gcctgcactt cgacgaccgc 780

ttctccggcg agcagctggt gaccatgtct gtgcgcgagc tgaaccggca gctgcgcggg 840

gtcagcaagg aggaggtgat ccggctgaag cagaagaggc ggaccctgaa aaaccgcggc 900

tatgcccagt cctgccgctt caagagggtg cagcagagac acgtcctgga gtcggagaag 960

aaccagctgc tgcagcaagt cgaccacctc aagcaggaga tctccaggct ggtgcgcgag 1020

agggacgcgt acaaggagaa atacgagaag ttggtgagca gcggcttccg agaaaacggc 1080

tcgagcagcg acaacccgtc ctctcccgag tttttcatgt ga 1122

<210> 4

<211> 373

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Met Ala Ser Glu Leu Ala Met Ser Asn Ser Asp Leu Pro Thr Ser Pro

1 5 10 15

Leu Ala Met Glu Tyr Val Asn Asp Phe Asp Leu Met Lys Phe Glu Val

20 25 30

Lys Lys Glu Pro Val Glu Thr Asp Arg Ile Ile Ser Gln Cys Gly Arg

35 40 45

Leu Ile Ala Gly Gly Ser Leu Ser Ser Thr Pro Met Ser Thr Pro Cys

50 55 60

Ser Ser Val Pro Pro Ser Pro Ser Phe Ser Ala Pro Ser Pro Gly Ser

65 70 75 80

Gly Ser Glu Gln Lys Ala His Leu Glu Asp Tyr Tyr Trp Met Thr Gly

85 90 95

Tyr Pro Gln Gln Leu Asn Pro Glu Ala Leu Gly Phe Ser Pro Glu Asp

100 105 110

Ala Val Glu Ala Leu Ile Ser Asn Ser His Gln Leu Gln Gly Gly Phe

115 120 125

Asp Gly Tyr Ala Arg Gly Ala Gln Gln Leu Ala Ala Ala Ala Gly Ala

130 135 140

Gly Ala Gly Ala Ser Leu Gly Gly Ser Gly Glu Glu Met Gly Pro Ala

145 150 155 160

Ala Ala Val Val Ser Ala Val Ile Ala Ala Ala Ala Ala Gln Ser Gly

165 170 175

Ala Gly Pro His Tyr His His His His His His Ala Ala Gly His His

180 185 190

His His Pro Thr Ala Gly Ala Pro Gly Ala Ala Gly Ser Ala Ala Ala

195 200 205

Ser Ala Gly Gly Ala Gly Gly Ala Gly Gly Gly Gly Pro Ala Ser Ala

210 215 220

Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Ala Ala

225 230 235 240

Gly Ala Gly Gly Ala Leu His Pro His His Ala Ala Gly Gly Leu His

245 250 255

Phe Asp Asp Arg Phe Ser Gly Glu Gln Leu Val Thr Met Ser Val Arg

260 265 270

Glu Leu Asn Arg Gln Leu Arg Gly Val Ser Lys Glu Glu Val Ile Arg

275 280 285

Leu Lys Gln Lys Arg Arg Thr Leu Lys Asn Arg Gly Tyr Ala Gln Ser

290 295 300

Cys Arg Phe Lys Arg Val Gln Gln Arg His Val Leu Glu Ser Glu Lys

305 310 315 320

Asn Gln Leu Leu Gln Gln Val Asp His Leu Lys Gln Glu Ile Ser Arg

325 330 335

Leu Val Arg Glu Arg Asp Ala Tyr Lys Glu Lys Tyr Glu Lys Leu Val

340 345 350

Ser Ser Gly Phe Arg Glu Asn Gly Ser Ser Ser Asp Asn Pro Ser Ser

355 360 365

Pro Glu Phe Phe Met

370

<210> 5

<211> 15

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ggcggcctgc acttc 15

<210> 6

<211> 16

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gggttgtcgc tgctcg 16

<210> 7

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

aacgcgaagc ttgcatcaga actggcaatg ag 32

<210> 8

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

aacgcgggat cctcacatga aaaactcggg aga 33

<210> 9

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

aacgcgggta ccgcatcaga actggcaatg ag 32

Claims

1. The MAF gene mutant is characterized in that the nucleotide sequence of the MAF gene mutant is shown in SEQ ID NO. 3.

2. The MAF gene mutant encoded polypeptide of claim 1, wherein the amino acid sequence of said polypeptide is as shown in SEQ ID No. 4.

3. Use of a reagent for detecting the MAF gene mutant as defined in claim 1 or the polypeptide encoded by the MAF gene mutant as defined in claim 2 for the preparation of a kit for molecular genetic diagnosis of congenital cataract or prenatal gene diagnosis kit or screening kit for biological samples susceptible to congenital cataract.

4. A kit comprising reagents for detecting a MAF gene mutant or a polypeptide encoded by a MAF gene mutant; the reagent for detecting the MAF gene mutant contains a reagent for detecting the 788 th nucleotide mutation A → G of the MAF gene, and the nucleotide sequence of the mutated MAF gene mutant is shown as SEQ ID NO. 3.

5. The kit according to claim 4, wherein the reagent for detecting the nucleotide mutation A → G at position 788 in MAF gene comprises primers having nucleotide sequences shown in SEQ ID No.5 and SEQ ID No. 6.

6. A nucleic acid construct comprising a MAF gene mutant; the nucleotide sequence of the MAF gene mutant is shown in SEQ ID NO. 3.

7. The nucleic acid construct of claim 6, further comprising a FLAG tag sequence or a GFP tag sequence; when the nucleic acid construct contains a FLAG tag sequence, the skeleton vector of the nucleic acid construct comprises pFLAG-CMV4, and the nucleotide sequences of primers for constructing the nucleic acid construct are shown as SEQ ID NO.7 and SEQ ID NO. 8; when the nucleic acid construct contains a GFP tag sequence, the framework vector of the nucleic acid construct comprises pAcGFP1-C1, and the nucleotide sequences of primers for constructing the nucleic acid construct are shown as SEQ ID NO.9 and SEQ ID NO. 8.

8. A recombinant cell obtained by transfecting a recipient cell with the nucleic acid construct of claim 6 or 7.

9. Use of the nucleic acid construct according to claim 6 or 7 or the recombinant cell according to claim 8 for the preparation of a kit for studying the function of a polypeptide encoded by a MAF gene mutant.

10. Use of the nucleic acid construct of claim 6 or 7 or the recombinant cell of claim 8 for the preparation of a kit for the unequivocal molecular genetic diagnosis of patients with congenital cataract, for guiding the precise treatment of patients or for avoiding the risk of regeneration in the patient's home.