CN116064618B - ECHS1 gene mutant, protein polypeptide, kit and application thereof - Google Patents
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- CN116064618B CN116064618B CN202211551908.2A CN202211551908A CN116064618B CN 116064618 B CN116064618 B CN 116064618B CN 202211551908 A CN202211551908 A CN 202211551908A CN 116064618 B CN116064618 B CN 116064618B
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/527—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving lyase
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention provides an ECHS1 gene mutant, a protein polypeptide, a kit and application thereof, belonging to the technical field of genetic engineering, wherein the ECHS1 gene mutant is formed by G-A mutation of 724 th nucleotide of a cDNA nucleotide sequence of the ECHS1 gene, and the nucleotide sequence is shown as SEQ ID NO. 3; the protein polypeptide has a p.E242K mutation; the kit can be used for screening biological samples susceptible to Leigh syndrome and contains a reagent for detecting ECHS1 gene mutants. The ECHS1 gene mutant, the protein polypeptide, the kit and the application thereof provided by the invention can be used for molecular genetic diagnosis of the Leigh syndrome, and provide basis for prenatal gene diagnosis of patients with the Leigh syndrome caused by ECHS1 gene mutation.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to an ECHS1 gene mutant, a protein polypeptide, a kit and application thereof.
Background
Leigh Syndrome (LS) is a globally rare, fatal, severe inherited metabolic disease that belongs to mitochondrial disease. Leigh syndrome occurs in infants or childhood with a morbidity of about 1 in live infants: 5000-8000, no effective therapeutic measures exist at present. Patients often suffer from impaired self-care due to the accompanying muscle tension reversal, motor and mental retardation, which presents a heavy burden to the home and society.
Currently, it is widely believed that a defective mitochondrial respiratory chain (including pyruvate dehydrogenase) causes a disorder of body energy metabolism, which is a major cause of the disease. Currently, more than 75 genes are associated with the disease, distributed on mitochondrial genomic DNA (mtDNA) and mitochondrial component related nuclear DNA (nDNA), respectively. Because of the obvious genetic heterogeneity of the disease, it is difficult to diagnose accurately by clinical and general laboratory tests alone, and therefore genetic diagnosis is a gold standard for diagnosing the disease.
ECHS1 consists of 290 amino acids and is responsible for its traction to the mitochondrial matrix by the N-terminal mitochondrial signal peptide. Functionally, first, the ECHS1 protein polypeptide participates in fatty acid beta-oxidation, is responsible for catalyzing intermediate metabolite short acyl-CoA, forms 3-L-hydroxyacyl-CoA (3-L-hydroxyycyl-CoA), and finally forms acetyl CoA to enter TCA cycle; second, the ECHS1 protein polypeptide is involved in the metabolism of branched-chain amino acids. Thus, defects in ECHS1 can lead to abnormalities in fatty acid, amino acid, and energy metabolism. The ECHS1 gene defect is mainly caused by autosomal recessive inheritance.
Since 2014 the first ECHS1 mutation was found, 42 mutations have been reported on this gene, ECHS1 has gradually become a hot spot causative gene of Leigh syndrome. However, almost all ECHS1 pathogenic reports are from western populations, and there are currently only 6 in chinese populations. The reason for this is that the disease is not low in incidence in the Chinese population, but potential patients are found successively due to the vigorous development of accurate medicine in Europe and America, but with the deep depth of large-scale sequencing in China clinical in recent years, it is believed that the patients with Leigh syndrome caused by ECHS1 gene defects in the Chinese population can be found continuously.
In view of the severity of LS and the randomness and the non-directionality of the mutation of the gene, the new mutation and pathogenicity of the ECHS1 gene related to the Leigh syndrome in Chinese people are clear, and the rapid improvement of the mutation spectrum of the gene in Chinese people has very urgent practical significance. For suspected Leigh syndrome patients, the method has important clinical significance in preparing their molecular genetic diagnosis, guiding accurate treatment of the patients and avoiding the regeneration and breeding risks of the families. However, the current tools for detecting ECHS1 gene variation and researching functions are imperfect, which hinders establishment of clinical diagnosis of Leigh syndrome and progress of gene diagnosis before regeneration and birth of related families.
Disclosure of Invention
In order to solve the problems, the invention provides an ECHS1 gene mutant for causing Leigh syndrome, which is found in Chinese people, and the nucleotide sequence of the ECHS1 gene mutant is shown as SEQ ID NO. 3.
In order to solve the problems, the invention also provides a protein polypeptide coded by the ECHS1 gene mutant, and the amino acid sequence of the protein polypeptide is shown as SEQ ID NO. 4.
In order to solve the problems, the invention also provides an application of the reagent for detecting the ECHS1 gene mutant or the protein polypeptide coded by the ECHS1 gene mutant in preparation of a Leigh syndrome molecular genetic diagnosis kit or a prenatal gene diagnosis kit or a Leigh syndrome biological sample screening kit.
In order to solve the above problems, the present invention also provides a kit comprising:
reagents for detecting the ECHS1 gene mutant described above or the protein polypeptide encoded by the ECHS1 gene mutant described above;
wherein the reagent for detecting ECHS1 gene mutant contains the reagent for detecting the 724 th nucleotide mutation G-A of ECHS1 gene cDNA, and the nucleotide sequence of the ECHS1 gene mutant after mutation is shown as SEQ ID NO. 3.
Preferably, the reagent for detecting the mutation G-A of the 724 th nucleotide of the cDNA nucleotide sequence of the ECHS1 gene comprises a primer;
wherein, the nucleotide sequence of the primer is shown as SEQ ID NO.5 and SEQ ID NO. 6.
In order to solve the above problems, the present invention also provides a nucleic acid construct comprising the ECHS1 gene mutant as described above; the nucleotide sequence of the ECHS1 gene mutant is shown as SEQ ID NO. 3.
Preferably, the backbone vector of the nucleic acid construct comprises pcdna3.1;
the nucleotide sequences of the primers used for constructing the nucleic acid construct are shown as SEQ ID NO.11 and SEQ ID NO. 12;
preferably, when the nucleic acid construct contains a FLAG tag sequence, the nucleotide sequence of the primer used to construct the nucleic acid construct is shown in SEQ ID NO.11 and SEQ ID NO. 15.
In order to solve the above problems, the present invention also provides a recombinant cell obtained by transfecting a recipient cell with the nucleic acid construct as described above.
In order to solve the problems, the invention also provides an application of the nucleic acid construct or the recombinant cell in preparation of a kit for researching the functions of the polypeptide coded by the ECHS1 gene mutant.
In order to solve the problems, the invention also provides an application of the nucleic acid construct or the recombinant cell in preparation of a kit for providing molecular genetic diagnosis of a Leigh syndrome patient, guiding accurate treatment of the patient and/or avoiding regeneration and breeding risks of the patient family.
The invention provides an ECHS1 gene mutant, a protein polypeptide and application thereof, wherein the nucleotide sequence of the ECHS1 gene mutant is shown as SEQ ID NO. 3. At present, no report of Leigh syndrome caused by ECHS1 gene c.724G > A mutation is internationally available, and functional data of protein polypeptide coded by the ECHS1 gene mutant is lacking, so that pathogenicity rating of new variation is influenced, and establishment of clinical diagnosis of the disease and progress of regeneration and prenatal gene diagnosis of related families are hindered. The invention determines a novel mutant of ECHS1 gene, which is closely related to the pathogenesis of Leigh syndrome, and can effectively detect whether the biological sample suffers from Leigh syndrome by detecting whether the novel mutant exists in the biological sample. The technical scheme of the invention confirms the pathogenicity of new variation in the ECHS1 gene related to the Leigh syndrome phenotype, and has important significance for defining the molecular genetic diagnosis of patients, guiding the accurate treatment of the patients and avoiding the regeneration and breeding risks of the families.
The invention also provides ECHS1 protein polypeptides with site-specific amino acid mutations. The nucleic acid construct with the ECHS1 gene with specific mutation can be used for in vitro function research; the recombinant cells with the specific mutation constructs can be used for definitely judging whether ECHS1 mutant protein polypeptide influences the enzyme catalytic activity and the expression level of the ECHS1 mutant protein polypeptide, and provide evidence for pathogenicity rating of new mutation of ECHS1 genes.
Drawings
FIG. 1 is a lineage diagram of Leigh syndrome core family provided by the invention;
FIG. 2 is a schematic diagram of agarose gel electrophoresis of a 409bpDNA fragment upstream and downstream of ECHS1 gene mutation site c.724G > A of a patient and a normal individual in a Leigh syndrome family member amplified by the PCR method provided by the invention;
FIG. 3 is a sequence diagram of a mutation site c.724G > A on the family member ECHS1 provided by the present invention;
FIG. 4 shows the conservation analysis of the amino acid (p.Glu242) of the mutation site of ECHS1 gene provided by the invention;
FIG. 5 is a schematic diagram showing the construction of wild type and c.724G > A mutant ECHS1 gene expression constructs provided by the present invention;
FIG. 6 is a schematic diagram of the construction of the wild type and c.724G > A mutant ECHS1 gene expression construct with FLAG tag sequence provided by the present invention;
FIG. 7 is a schematic diagram of immunoblotting method for detecting expression level difference of ECHS1 wild type and mutant p.E242K protein in recombinant HEK293 cells;
FIG. 8 is a schematic representation of subcellular localization of ECHS1 detected by immunofluorescence method of the present invention for wild-type (WT) and mutant (p.E242K, MT 1) protein polypeptides;
FIG. 9 is a schematic representation of the effect of the p.E242K mutation on the three-dimensional structure of ECHS1 protein (PDB code of protein is 2HW 5);
FIG. 10 is a schematic representation of the inhibition of ECHS1 enzyme catalytic activity by the p.E242K mutation.
The achievement of the objects, functional features and advantages of the present invention will be further described with reference to the accompanying drawings, in conjunction with the embodiments.
Detailed Description
The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Unless defined otherwise hereinafter, all technical and scientific terms used in the detailed description of the invention are intended to be identical to what is commonly understood by one of ordinary skill in the art. While the following terms are believed to be well understood by those skilled in the art, the following definitions are set forth to better explain the present invention.
As used herein, the terms "comprising," "including," "having," "containing," or "involving" are inclusive or open-ended and do not exclude additional unrecited elements or method steps. The term "consisting of …" is considered to be a preferred embodiment of the term "comprising". If a certain group is defined below to contain at least a certain number of embodiments, this should also be understood to disclose a group that preferably consists of only these embodiments.
The indefinite or definite article "a" or "an" when used in reference to a singular noun includes a plural of that noun.
The term "about" in the present invention means a range of accuracy that one skilled in the art can understand while still guaranteeing the technical effect of the features in question. The term generally means a deviation of + -10%, preferably + -5%, from the indicated value.
Furthermore, the terms first, second, third, (a), (b), (c), and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.
The following is provided merely to aid in the understanding of the present invention. These definitions should not be construed to have a scope less than understood by those skilled in the art.
The technical solution of the present invention is further described in detail below with reference to specific embodiments, but the present invention is not limited thereto, and any modifications made by anyone within the scope of the claims of the present invention are still within the scope of the claims of the present invention.
The embodiment provides an ECHS1 gene mutant, and the nucleotide sequence of the ECHS1 gene mutant is shown as SEQ ID NO. 3.
ECHS1 is located on human chromosome 10q26.3, has the total length of 10869bp, is composed of 8 exons and codes for a protein composed of 290 amino acids. ECHS1 plays a role in the formation of 3-L-hydroxyester acyl-CoA intermediate mainly in the fatty acid beta-oxidation pathway.
In this example, the ECHS1 gene mutant is generated by missense mutation of the wild ECHS1 gene at c.724G > A (p.E242K), which is located on exon 6. The cDNA nucleotide sequence of the wild ECHS1 gene is shown as SEQ ID NO. 1.
In this example, the Leigh syndrome core family was used as a study object, and a new pathogenic mutation c.724G > A on the ECHS1 gene was identified by combining Sanger sequencing verification with whole exome sequencing screening. The mutation is not found through the international thousand-person genome database, the human whole-exome sequencing project ESP6500 database and the sixty thousand-person exome ExAC integration database, and the Clinvar and HGMD databases are queried, and the report about the mutation is not found through literature retrieval. Thus, this example identified a new Leigh syndrome mutation, c.724G > A, on the ECHS1 gene by target region capture sequencing combined with candidate gene mutation verification.
The embodiment also provides a protein polypeptide encoded by the ECHS1 gene mutant, and the amino acid sequence of the protein polypeptide is shown as SEQ ID NO. 4.
The protein polypeptide of this example is a mutant of the 242 th glutamic acid to lysine of the polypeptide encoded by the wild ECHS1 gene cDNA, i.e. the protein polypeptide has an amino acid change of p.Glu242Lys (p.E242K), which is caused by missense mutation c.724G > A.
It should be noted that, in this embodiment, the cDNA nucleotide sequence of the wild type ECHS1 gene is shown as SEQ ID NO.1, and the encoded polypeptide amino acid sequence is shown as SEQ ID NO. 2. By detecting whether the mutant protein polypeptide is expressed in the biological sample, whether the biological sample is susceptible to the Leigh syndrome can be effectively detected, and whether the biological sample is suffering from the Leigh syndrome can be effectively detected by detecting whether the novel mutant exists in the biological sample.
The embodiment also provides an application of the reagent for detecting the ECHS1 gene mutant or the protein polypeptide coded by the ECHS1 gene mutant in preparation of a Leigh syndrome molecular genetic diagnosis kit or a prenatal gene diagnosis kit or a reagent for screening biological samples susceptible to the Leigh syndrome.
The invention also provides a kit comprising:
reagents for detecting the ECHS1 gene mutant described above or the protein polypeptide encoded by the ECHS1 gene mutant described above; wherein the reagent for detecting ECHS1 gene mutant contains the reagent for detecting mutation G-A of 724 th nucleotide of ECHS1 gene cDNA nucleotide sequence, and the nucleotide sequence of the ECHS1 gene mutant after mutation is shown as SEQ ID NO. 3.
The reagent for detecting ECHS1 gene mutant contains the reagent for detecting ECHS1 gene point mutation c.724G > A.
In the embodiment, the kit can screen biological samples susceptible to the Leigh syndrome, can also realize molecular genetic diagnosis of the Leigh syndrome, and can also realize prenatal gene diagnosis of the Leigh syndrome.
Further, the detection method based on the kit described in this embodiment preferably includes the following steps:
(1) Designing an upstream and downstream amplification primer aiming at ECHS1 gene mutation sites;
(2) Taking genomic DNA of a sample to be detected as a template, and amplifying upstream and downstream 409bp sequences of ECHS1 gene mutation sites by a PCR method;
(3) Performing agarose gel electrophoresis identification on the amplified product;
(4) After purification of the PCR product, sanger sequencing was performed to analyze the nucleotide sequence.
As described above, ECHS1 gene mutants are closely related to the onset of Leigh syndrome, and by detecting the presence or absence of the novel mutants in biological samples, it is possible to effectively detect whether or not the biological samples thus suffer from Leigh syndrome.
Further, the reagent for detecting the mutation G-A of the 724 th nucleotide of the cDNA nucleotide sequence of the ECHS1 gene comprises a primer; wherein, the nucleotide sequence of the primer is shown as SEQ ID NO.5 and SEQ ID NO. 6.
As described above, in this example, the reagent for detecting nucleotide mutation c.724G > A of ECHS1 gene preferably comprises a primer sequence; the nucleotide sequences of the primers are shown as SEQ ID NO.5 and SEQ ID NO. 6. The primer can effectively amplify ECHS1 exon 6 in a PCR system, and can efficiently screen biological samples susceptible to Leigh syndrome.
In the present invention, the PCR gene amplification system is preferably as shown in Table 1:
TABLE 1 PCR preparation System
Reagent(s) | Usage amount |
2×Premix Taq(Ex Taq version 2.0plus dye,TAKARA) | 10μL |
Forward primer (SEQ ID NO.5, 10. Mu.M) | 0.4μL |
Reverse primer (SEQ ID NO.6, 10. Mu.M) | 0.4μL |
Sample genomic DNA | 50ng |
Deionized water | Make up to 20 mu L |
In this embodiment, the reaction procedure for amplifying the PCR method is preferably as follows:
denaturation at 95℃for 3min, denaturation at 98℃for 10s, annealing at 60℃for 30s, extension at 72℃for 30s, 35 cycles, incubation at 72℃for 10min, and storage at 4 ℃.
The present embodiment also provides a nucleic acid construct comprising the ECHS1 gene mutant; the nucleotide sequence of the ECHS1 gene mutant is shown as SEQ ID NO. 3.
As described above, in this embodiment, the reagent for amplifying ECHS1 gene mutant preferably comprises a primer sequence; the nucleotide sequences of the primers are shown as SEQ ID NO.7 and SEQ ID NO.8, the primers can effectively amplify the full length of ECHS1 cDNA in a PCR system, and the cDNA sequence for encoding ECHS1 protein in a biological sample can be obtained efficiently.
In the present invention, the PCR system for amplifying the cDNA sequence of ECHS1 is preferably as shown in Table 2:
TABLE 2 preparation of PCR reaction solution
In this embodiment, the reaction procedure for amplifying the PCR method is preferably as follows:
denaturation at 95℃for 3min, denaturation at 98℃for 10s, annealing at 62℃for 30s, elongation at 68℃for 1min, 33 cycles, incubation at 72℃for 10min, and storage at 4 ℃.
As described above, the nucleic acid of this embodiment is any polymer comprising deoxyribonucleotides or ribonucleotides, including but not limited to modified or unmodified DNA, RNA, and the length thereof is not limited in any way.
As described above, for the construct used to construct the recombinant cell, it is preferable that the nucleic acid is DNA because DNA is more stable than RNA and is easy to handle.
It should be noted that the term "construct" as used in this example refers to a genetic vector having the following characteristics or properties:
(1) Can self-replicate and functionally carry the target nucleic acid sequence into the host cell;
(2) Expressing the carried genetic information to obtain recombinant cells.
In this embodiment, the form of the construct is not particularly limited, and is at least one of a plasmid and a virus, preferably a plasmid. The plasmid, as a genetic carrier, has the advantages of simple operation, convenient insertion of foreign nucleic acid fragments, easy separation from host cells, easy labeling and the like. The form of the plasmid is not particularly limited, and may be a circular plasmid or a linear plasmid. Those skilled in the art can make selections as desired.
Further, when the nucleic acid construct is an expression vector, the backbone vector of the nucleic acid construct is pcDNA3.1;
the nucleotide sequences of the primers used for constructing the nucleic acid expression construct are shown as SEQ ID NO.11 and SEQ ID NO. 12.
Further, when the nucleic acid expression construct contains a FLAG tag sequence, the nucleotide sequence of the primer used to construct the nucleic acid construct is shown in SEQ ID NO.11 and SEQ ID NO. 15.
The invention also provides a construction method of the nucleic acid construct, preferably comprising the following steps:
(1) Extracting total RNA from the blood of a patient;
(2) Reverse transcription into cDNA using a reverse transcription kit (SuperScript III, cat.no.18080-051;Life Technologies);
(3) Designing a primer based on a transcript reference sequence NM_004092 of the ECHS1 gene, and taking cDNA as a template;
(4) The amplified ECHS1 fragment is connected to a T vector, after point mutation, the ECHS1 gene sequence is inserted into an expression vector according to the research requirement by a Gibson homologous connection method, and an ECHS1 nucleic acid expression construct with a specific mutation site is constructed.
Specifically, construction of the ECHS1 nucleic acid expression construct:
primers capable of amplifying the full-length coding region (CDS) of ECHS1 protein are designed according to a transcript reference sequence NM_004092 of ECHS1 gene, and the sequence information is shown as SEQ ID NO.7 and SEQ ID NO. 8. And (3) taking the patient leucocyte cDNA as a template, carrying out PCR amplification, connecting the amplified ECHS1 fragment to a T carrier, constructing ECHS1 gene plasmid with a mutation site, and then designing a primer according to sequence information around the mutation site to restore the mutation site nucleotide to a normal genotype, wherein the nucleotide sequence of the primer is shown as SEQ ID NO.9 and SEQ ID NO. 10.
Specifically, the constructed ECHS1 gene T vector plasmid with mutation sites is used as a template, PCR amplification is carried out, after the product is purified, cyclizing is carried out by a point mutation kit (E0552S; NEB), and the cyclized product is transformed into competent cells, thus obtaining ECHS1 wild genotype plasmid. Finally, two pairs of primers are designed according to the Gibson connection requirement: (1) A pair of primers for amplifying ECHS1 fragment and containing an expression vector (pcDNA3.1) homologous region at the 5' end, wherein the nucleotide sequences of the primers are shown as SEQ ID NO.11 and SEQ ID NO. 12; (2) The other pair of primers is used for amplifying the pcDNA3.1 sequence of the vector, and the nucleotide sequences of the primers are shown as SEQ ID NO.13 and SEQ ID NO. 14.
PCR amplification is carried out by taking ECHS1 gene plasmid with mutation site and ECHS1 wild type T vector plasmid as templates, taking SEQ ID NO.11 and SEQ ID NO.12 as primers, respectively mixing amplified products with pcDNA3.1 vector fragments obtained by amplification by taking SEQ ID NO.13 and SEQ ID NO.14 as primers (pcDNA3.1 plasmid as templates), and connecting DNA fragments by using Gibson reaction kit (E2611S; NEB). When the ligation reaction is completed, the reaction solution transforms competent cells, whereby an ECHS1 gene expression construct with a specific mutation site and an ECHS1 gene expression construct of a normal genotype can be obtained.
In this embodiment, the expression vector preferably comprises pcDNA3.1.
Specifically, construction of the ECHS1 gene expression construct with FLAG tag sequence: two pairs of primers are designed according to the Gibson connection requirement, one pair is used for amplifying ECHS1 fragments, wherein the 5 'end of the forward primer contains pcDNA3.1 carrier homologous region, the 5' end of the reverse primer contains FLAG tag homologous sequence, and the nucleotide sequences of the primers are shown as SEQ ID NO.11 and SEQ ID NO. 15. The other pair is used for amplifying an expression vector pcDNA3.1, wherein a FLAG tag sequence is added to the 5' end of a forward primer, and the nucleotide sequences of the primers are shown as SEQ ID NO.16 and SEQ ID NO. 14. PCR amplification is carried out by taking ECHS1 gene plasmid with mutation site and ECHS1 wild type T vector plasmid as templates, taking SEQ ID NO.11 and SEQ ID NO.15 as primers, respectively mixing amplified products with pcDNA3.1 vector fragments obtained by amplification by taking SEQ ID NO.16 and SEQ ID NO.14 as primers (pcDNA3.1 plasmid as templates), and connecting DNA fragments by using Gibson reaction kit (E2611S; NEB). When the ligation reaction is completed, the reaction solution transforms competent cells, thereby obtaining an ECHS1 gene mutant expression construct with a FLAG tag sequence and an ECHS1 gene expression construct of a normal genotype.
In this embodiment, the expression vector preferably comprises pcDNA3.1.
The ECHS1 gene expression construct with FLAG tag sequence can be used to study and compare differences in expression positions of ECHS1 wild type and mutant (p.E242K) proteins in mammalian cells.
The invention also provides a recombinant cell obtained by transfecting a recipient cell with a nucleic acid construct as described above.
The recombinant cells are capable of expressing the proteins encoded by the ECHS1 gene mutants. In this example, the recipient cells are preferably derived from E.coli cells (DH 5. Alpha. Competent cells, available from Tiangen Biochemical Co., ltd.) or mammalian cells (HEK 293 cells, no. CRL-1573, available from ATCC).
Recombinant cells obtained by transfecting the recipient cells with the nucleic acid construct of the present example can evaluate the protein expression level of the mutant; the catalytic activity of the mutant protein is evaluated by detection of the enzyme activity, thereby comprehensively evaluating the biological harmfulness of the mutant.
The embodiment also provides an application of the nucleic acid construct or the recombinant cell in preparation of a kit for researching the functions of the polypeptide coded by the ECHS1 gene mutant.
The present embodiment also provides the use of a nucleic acid construct as described above, or a recombinant cell as described above, in the preparation of a kit for providing a molecular genetic diagnosis of a patient with Leigh syndrome, guiding the accurate treatment of the patient and/or avoiding the risk of regeneration of the patient's family.
The invention is further illustrated by the following specific examples, but it should be understood that these examples are for the purpose of illustration only and are not to be construed as limiting the invention in any way.
Example 1:
upstream and downstream amplification primers are designed aiming at ECHS1 gene mutation sites.
The genomic DNA of the patient is used as a template, a 409bp sequence containing ECHS1 gene mutation sites is amplified by a PCR method, and Sanger sequencing is carried out after PCR product purification, and sequence comparison analysis is carried out to detect the mutation sites on ECHS1 genes of the Leigh syndrome patient.
Upstream and downstream primers for amplifying a nucleotide fragment containing the ECHS1 gene mutation site c.724G > A:
forward primer 5'-CCACGACTCTACAGACAGCAGAAC-3' (SEQ id No. 5);
reverse primer 5'-TGTGGAAGCCGCCCTTGCTTAG-3' (SEQ ID NO. 6);
the reaction system for PCR amplification is shown in Table 1.
PCR reaction conditions: denaturation at 95℃for 3min, denaturation at 98℃for 10s, annealing at 60℃for 30s, extension at 72℃for 30s, 35 cycles, incubation at 72℃for 10min, and storage at 4 ℃.
Purification of PCR products: 200ul Binding buffer is added into each 100ul of RCR products, all the mixed solution is transferred into a nucleic acid silica gel purification column after uniform mixing, 11000 Xg is centrifuged for 30s, and waste liquid is discarded; adding 700ul of cleaning solution, centrifuging for 30s at 11000 Xg, repeating the cleaning step once, and then carrying out air separation at 11000 Xg for 1min; then add 15-30ul deionized water to the silica gel membrane, incubate for 1min at room temperature, centrifuge for 1min at 11000 Xg last, collect the eluate into new centrifuge tube, purify the product for Sanger sequencing analysis.
Referring to FIG. 1, there is shown a Leigh syndrome core family pattern. II-2 is the first person, and is manifested by inflexible eyes, askew head, inability to prop up, high tension of the limb muscles, inability to take things by hand, and reverse development. The urine examination revealed that the indexes of lactic acid, glycolic acid, 3-hydroxyisobutyric acid and 3-hydroxyisovaleric acid were all increased. Head MRI examination showed bilateral basal ganglia and brainstem abnormalities, midbrain, brain angular symmetry T2 high signals, highly suspected LS. The other family members have normal phenotypes, parents are not matched closely, and no medicine is used or adverse environment is contacted during pregnancy.
The patient family members were detected by the method of example 1, a fragment of 409bp containing exon 6 of the ECHS1 gene was amplified from the genome of the family members by PCR, and the PCR product was detected by agarose gel electrophoresis, as shown in FIG. 2, II-2 was the prover, II-1 was the prover Colgo, I-1 was the prover father, and I-2 was the prover mother.
The purified PCR product was subjected to Sanger sequencing using SEQ ID NO.5 as a sequencing primer. Compared with human GRCh38 genome data, the novel ECHS1 mutant from the patient was found to have a c.724G > A mutation, i.e., the 724 th base of the cDNA nucleotide sequence of ECHS1 mutant of this example was mutated from G to A relative to the wild-type ECHS1 gene, and the encoded protein polypeptide product had a p.Glu242Lys mutation, i.e., the 242 th glutamic acid (E) mutation of wild-type ECHS1 to lysine (K) compared to wild-type ECHS 1. The sequencing of ECHS1 mutation site c.724G > A is shown in FIG. 3, which shows heterozygozygous, and is found in the prover (II-2) and the mother (I-2) thereof, and the prover brother (II-1) and the father (I-1) thereof are all wild homozygously, indicating that the prover mutation of the site is inherited to the mother. Since ECHS1 is a recessive pathogenic gene, the precursor in the examples is ill because another mutation site inherited from the father, namely mutation 2 (the mutation site is not described in the patent) is found on the precursor ECHS1 gene, and the two mutations act together to cause disease in the form of double heterozygotes.
Then, carrying out sequence comparison and analysis on the amino acid sequence of the ECHS1 gene mutation site in different species to obtain a conservation analysis schematic diagram of the ECHS1 242 th amino acid site in different species of animals shown in figure 4; the results indicate that the amino acids at this site are very conserved in vertebrates, including mammals, birds, fish and amphibians, and invertebrates, indicating that the amino acids represented by this site are selected for retention in lengthy animal evolution, that mutants thereof are eliminated in natural selection, and that this site would be detrimental if mutated.
Example 2:
(1) Construction of ECHS1 Gene expression constructs (schematic representation of ECHS1 wild type and c.724G > A mutant nucleic acid constructs is shown in FIG. 5). And designing an upstream primer and a downstream primer which can amplify the full length of ECHS1 cDNA, wherein the nucleotide sequences of the primers are shown as SEQ ID NO.7 and SEQ ID NO. 8.
PCR amplification was performed using patient's leukocyte cDNA as a template, and the amplified ECHS1 fragment was subjected to A addition and purification, and then ligated to a commercial T vector (PGEM-T), to construct a T vector plasmid containing the ECHS1 gene at the mutation site, and 5. Mu.L of the ligation product was transformed into 50. Mu.L of DH 5. Alpha. Competent cells, plated on ampicillin-resistant LB plates, cultured overnight at 37℃and monoclonal was picked for Sanger sequencing to identify the ECHS1 gene construct (pGEMT-ECHS 1-MT 1) with the mutation site c.724G > A. And designing a primer aiming at the mutation site to correct the nucleotide variation into a normal genotype, wherein the nucleotide sequence of the primer is shown as SEQ ID NO.9 and SEQ ID NO. 10.
Taking the constructed pGEMT-ECHS1-MT1 plasmid as a template, carrying out PCR amplification, carrying out agarose gel electrophoresis on an amplified product, recovering and purifying the amplified product by gel, taking 1 mu L of the purified product, carrying out the action (phosphorylation, cyclization and plasmid template removal) of KLD mixed enzyme in a point mutation kit for 15min at room temperature, taking 5 mu L of reaction liquid to convert 50 mu L of DH5 alpha competent cells, and picking up monoclonal to carry out Sanger sequencing, thereby obtaining the ECHS1 gene construct (pGEMT-ECHS 1-WT) of a normal genotype.
Finally, two pairs of primers are designed according to the requirement of Gibson homologous connection, one pair of primers is used for amplifying ECHS1 fragments, a small length of pcDNA3.1 vector sequence with the length of about 23bp is added to the 5' end of each primer, and the nucleotide sequences of the primers are shown as SEQ ID NO.11 and SEQ ID NO. 12. The other pair of primers is used for amplifying the expression vector pcDNA3.1, and the nucleotide sequences of the primers are shown in SEQ ID NO.13 and SEQ ID NO. 14.
Respectively taking a T vector plasmid (pGEMT-ECHS 1-MT 1) of ECHS1 gene with mutation sites and a T vector plasmid (pGEMT-ECHS 1-WT) of ECHS1 gene with normal genotype as templates, taking SEQ ID NO.11 and SEQ ID NO.12 as primers, carrying out PCR amplification, and respectively mixing the amplified products with pcDNA3.1 vector fragments obtained by amplification by taking SEQ ID NO.13 and SEQ ID NO.14 as primers (pcDNA3.1 plasmid as templates), wherein the molar ratio of the vector fragments to the insert fragment is 1:4, wherein 50ng of the vector pcDNA3.1 fragment was used, the fragments were mixed with enzymes in a Gibson ligation kit, gibson ligation was completed by incubating for 1h at 50 ℃, 50. Mu.L of DH 5. Alpha. Competent cells were transformed with 3. Mu.L of the reaction solution, and Sanger sequencing was performed by picking up a monoclonal to obtain an ECHS1 gene expression construct with a specific mutation site (pcDNA3.1-ECHS 1-MT 1) and an ECHS1 gene expression construct of a normal genotype (pcDNA3.1-ECHS 1-WT).
I) Primers used to construct the ECHS1 gene plasmid pGEMT-ECHS1-MT1 with mutation site (c.724G > A):
forward primer:
5’-GCCGGGCGAGGAGTCCAGAGAG-3’(SEQ ID NO.7);
reverse primer:
5’-CACACCACGGACACTGCTCTTG-3’(SEQ ID NO.8).
the reaction system for amplifying the cDNA nucleic acid sequence of ECHS1 by PCR method is shown in Table 2.
PCR reaction conditions: denaturation at 95℃for 3min, denaturation at 98℃for 10s, annealing at 62℃for 30s, elongation at 68℃for 1min, 33 cycles, incubation at 72℃for 10min, and storage at 4 ℃.
Adding A to the 3' -end of the PCR product: the PCR product was purified to 20. Mu.L by a silica gel column, 14.5. Mu.L of the purified product was taken, 2. Mu.L of 10 XEx Taq Buffer (containing Mg2+, TAKARA), 3. Mu.L of dNTP (2.5mM each,TAKARA) and 0.5. Mu.L of Ex Taq DNA polymerase (1.25U/. Mu.L, TAKARA) were added, and reacted at 72℃for 20 minutes, and the reaction product was subjected to agarose gel electrophoresis, followed by separation and purification.
And (3) recycling and purifying PCR product glue: after the PCR amplified product fragments are separated by 1% agarose gel electrophoresis, a band with a target size is cut off on an operation table of an ultraviolet gum machine, a 1.5ml EP tube is filled, a gel block is weighed by an electronic balance, 100ul Binding buffer parts per 100mg of gel are added into a centrifuge tube containing the gel, and the mixture is heated in a water bath at 50 ℃ for 5-10min until complete melting. Transferring the gel solution into a nucleic acid silica gel purification column, centrifuging for 30s at 11000 Xg, and discarding the waste liquid; adding 700ul of cleaning solution, centrifuging for 30s at 11000 Xg, repeating the cleaning step once, and then carrying out air separation at 11000 Xg for 1min; next, 15-30ul of deionized water was added to the silica gel membrane, incubated for 1min at room temperature, centrifuged for 1min at 11000 Xg, and DNA eluted into a new centrifuge tube.
The purified ECHS1 nucleic acid fragment was inserted into T vector, and the reaction system is shown in Table 3.
TABLE 3 configuration of connection reaction System
Reagent(s) | Usage amount |
2×Rapid Ligation Buffer(Promega) | 2.5μL |
pGEM-T(50ng/μL,Promega) | 0.4μL |
Purified PCR products | 1.5μL |
T4 DNA Ligase(3U/μL,Promega) | 0.6μL |
Total volume of | 5.0μL |
Connection reaction conditions: incubate overnight at 4 ℃.
The ligation solution transformed DH 5. Alpha. Competent cells, and the monoclone was picked and sequenced by Sanger to obtain ECHS1 gene construct (pGEMT-ECHS 1-MT 1) with mutation site c.724G > A.
II) primers for constructing the normal ECHS1 genotype plasmid pGEMT-ECHS 1-WT:
forward primer:
5’-GATGGCCAAAaAATCAGTGAATGC-3’(SEQ ID NO.9);
reverse primer:
5’-GCTACTACAATTTTAGAATTGCTGGC-3’(SEQ ID NO.10).
PCR amplification was performed using the constructed pGEMT-ECHS1-MT1 plasmid as a template, and after gel recovery and purification, cyclization reaction was performed with the reaction system shown in Table 4.
TABLE 4 reaction System
Reagent (E0552S; NEB) | Usage amount |
2×KLD Reaction Buffer(NEB) | 2.5μL |
Purified PCR products | 0.5μL |
2 XKLD enzyme mixture (NEB) | 0.5μL |
Deionized water | Make up to 5.0 mu L |
Connection reaction conditions: incubate at 25℃for 15min.
The ligation solution transformed DH 5. Alpha. Competent cells, were selected from the monoclonal, and sequenced by Sanger to obtain the normal ECHS1 gene construct (pGEMT-ECHS 1-WT).
III) primers for constructing ECHS1 gene expression constructs with specific mutation sites (pcDNA3.1-ECHS 1-MT 1) and ECHS1 gene expression constructs of normal genotype (pcDNA3.1-ECHS 1-WT):
A pair of primers for amplifying the ECHS1 gene, wherein:
forward primer:
5’-cccgggatccaccggtcgccacc ATGGCCGCCCTGCGTGTCCTGCTGT-3’(SEQ ID NO.11);
reverse primer:
5’-tctagactcgagcggccgcttta CTGGTCTTTGAAGTTGGCCTTTCTC-3’(SEQ ID NO.12).
another pair of primers is used to amplify the vector fragment, wherein:
forward primer:
5’-TAAAGCGGCCGCTCGAGTCTAG-3’(SEQ ID NO.13);
reverse primer:
5’-GGTGGCGACCGGTGGATCCCG-3’(SEQ ID NO.14).
ECHS1 insert is obtained by PCR amplification using the T vector (pGEMT-ECHS 1-MT 1) of ECHS1 gene with mutation site and ECHS1 gene T vector plasmid (pGEMT-ECHS 1-WT) of normal genotype as templates and SEQ ID NO.11 and SEQ ID NO.12 as primers, respectively. And then using pcDNA3.1 plasmid as a template, using SEQ ID NO.13 and SEQ ID NO.14 as primers, carrying out PCR amplification to obtain pcDNA3.1 vector fragments, carrying out agarose gel electrophoresis on all products, and carrying out Gibson ligation reaction after gel recovery and purification, wherein the reaction system is shown in Table 5.
TABLE 5 reaction System
Reagent (E2611S; NEB) | Usage amount |
2×Gibson Assembly Master Mix(NEB) | 2.5μL |
Purified PCR product (916 bp) | 35ng |
pcDNA3.1 vector fragment (5456 bp) | 50ng |
Deionized water | Make up to 5 mu L |
Connection reaction conditions: incubate at 50℃for 60min.
mu.L of the reaction solution was used to transform DH 5. Alpha. Competent cells, and monoclonals were picked and sequenced by Sanger to obtain ECHS1 gene expression construct with specific mutation sites (pcDNA3.1-ECHS 1-MT 1) and ECHS1 gene expression construct of normal genotype (pcDNA3.1-ECHS 1-WT).
(2) Construction of the FLAG-tagged ECHS1 gene expression construct (schematic representation of ECHS1 mutant c.724G > A and wild-type nucleic acid construct is shown in FIG. 6). According to the Gibson connection requirement, two pairs of primers are designed, one pair is used for amplifying ECHS1 fragments, wherein the 5 'end of the forward primer contains a carrier homologous region, the 5' end of the reverse primer contains a FLAG tag homologous sequence, and the nucleotide sequences of the primers are shown as SEQ ID NO.11 and SEQ ID NO. 15. The other pair is used for amplifying an expression vector pcDNA3.1, wherein a FLAG tag sequence is added to the 5' end of a forward primer, and the nucleotide sequences of the primers are shown as SEQ ID NO.16 and SEQ ID NO. 14. The T vector (pGEMT-ECHS 1-MT 1) of ECHS1 gene with mutation site and ECHS1 gene T vector plasmid (pGEMT-ECHS 1-WT) of normal genotype are used as templates, plasmid and ECHS1 wild type plasmid are used as templates, SEQ ID NO.11 and SEQ ID NO.15 are used as primers, PCR amplification is carried out, and the amplified products are respectively mixed with pcDNA3.1 vector fragments obtained by amplification with SEQ ID NO.16 and SEQ ID NO.14 as primers (pcDNA3.1 plasmid is used as template). The molar ratio of the carrier fragment to the insert fragment is 1:4, wherein 50ng of the vector pcDNA3.1 fragment was used, these fragments were mixed with the enzyme in the Gibson ligation kit, gibson ligation was completed by incubating for 1h at 50℃and 3. Mu.L of reaction solution was used to transform 50. Mu.LDH5α competent cells, and the monoclone was picked for Sanger sequencing to obtain the ECHS1 gene containing the specific mutation site and FLAG-tagged expression construct (pcDNA3.1-ECHS 1-MT 1-FLAG) and the normal ECHS1 genotype carrying FLAG tag expression construct (pcDNA3.1-ECHS 1-WT-FLAG).
Referring to FIG. 6, a schematic of the construction of the wild type and c.724G > A mutant ECHS1 gene expression constructs with FLAG tag sequences is shown.
Primers for construction of the expression construct containing the ECHS1 gene at the specific mutation site and carrying the FLAG tag (pcDNA3.1-ECHS 1-MT 1-FLAG) and the expression construct carrying the FLAG tag for the normal ECHS1 genotype (pcDNA3.1-ECHS 1-WT-FLAG):
a pair of primers for amplifying the ECHS1 gene, wherein:
the forward primer is identical with SEQ ID NO.11;
reverse primer:
5’-cttatcgtcgtcatccttgtaatc CTGGTCTTTGAAGTTGGCCTTTCTC-3’(SEQ ID NO.15)。
another pair of primers is used to amplify the vector fragment, wherein:
forward primer:
5’-gattacaaggatgacgacgataagTAAAGCGGCCGCTCGAGTCTAG-3’(SEQ ID NO.16);
the reverse primer is identical to SEQ ID NO.14.
The ECHS1 insert is obtained by PCR amplification using the T vector (pGEMT-ECHS 1-MT 1) of ECHS1 gene with mutation site and the ECHS1 gene T vector plasmid (pGEMT-ECHS 1-WT) of normal genotype as templates and SEQ ID NO.11 and ID NO.15 as primers, respectively. And then pcDNA3.1 plasmid is used as a template, SEQ ID NO.16 and SEQ ID NO.14 are used as primers, PCR amplification is carried out, pcDNA3.1 vector fragments are obtained, all products are subjected to agarose gel electrophoresis, gel recovery and purification, gibson ligation reaction is carried out, a reaction system is shown in table 5, 3 mu L ligation reaction liquid is used for transforming DH5 alpha competent cells, monoclonal is selected, and Sanger sequencing is carried out, so that the ECHS1 gene expression construct with FLAG tag is successfully obtained: mutant (pcDNA3.1-ECHS 1-MT 1-FLAG) and wild type (pcDNA3.1-ECHS 1-WT-FLAG).
Example 3:
recombinant cells were prepared by transfecting recipient cells with the nucleic acid construct of the ECHS1 gene wild type and its mutant (c.724G > A) constructed in example 2.
The recipient cells were derived from E.coli (DH 5. Alpha. Competent cells, available from Tiangen Biochemical Co., ltd.) or mammalian cells (HEK 293 cells).
Method for transforming DH 5. Alpha. Competent cells with ECHS1 nucleic acid construct: mu.L of the ligation product was gently mixed with 50. Mu.L of DH 5. Alpha. Competent cells, placed on ice for 30min, heat-shocked at 42℃for 90 seconds, transferred to ice for 3min, added with 950. Mu.L of LB liquid medium, incubated at 37℃for 45min (140 rpm) on a shaker, and evenly spread with 100. Mu.L of the medium on the surface of a preheated ampicillin-resistant LB agar plate. The plates were incubated overnight at 37℃in an incubator, and the monoclonal was picked up to obtain recombinant cells containing the corresponding ECHS1 nucleic acid construct.
ECHS1 nucleic acid construct transfected HEK293 mammalian cells: HEK293 cells were plated in 6-well plates and transfected when cultured to 80-90% confluence. Specifically, 2 μg of ECHS1 nucleic acid construct was mixed well with 200 μ L jetPRIME buffer, followed by the addition of 8 μl of jetPRIME transfection reagent (Cat #144-15;Polyplus Transfection) to form a construct-liposome mixture. After incubation for 10min at room temperature, the construct-liposome mixture was gently added dropwise to the cell culture broth, mixed well, incubated in an incubator at 37℃for 4h, and then the fresh medium was replaced and the culture was continued for 24h. And detecting the expression and the positioning of ECHS1 protein by an immunoblotting method and an immunofluorescence method, and evaluating whether the recombinant cell is constructed successfully.
Immunoblotting method for detection of wild type and mutant ECHS1 proteins (exemplified by HEK293 cells): recombinant HEK293 cells containing pcDNA3.1-ECHS1-MT1-FLAG and pcDNA3.1-ECHS1-WT-FLAG constructs were digested with 0.25% pancreatin, the cells were washed with PBS, centrifuged at 500 Xg for 5min to collect pellet into EP tube, and 10 volumes of RIPA lysate (Cat. No.89900; thermo Scientific) and appropriate amount of protease inhibitor (200X, cat. No. 53934; millipore) were added. After sonication until cells were completely lysed and incubation on ice for 10min, centrifugation was performed at 13600rpm for 10min at 4℃and the supernatant was collected and transferred to another EP tube and assayed for protein concentration by the Bradford method. Protein capillary electrophoresis separation was performed by WES system using a 12-230kDa separation kit (SM-W004; proteinSimple) and ECHS1 antibody (ab 174312; abcam) and HRP-labeled secondary antibody (DM-001; proteinSimple) were used for immunostaining, and an action antibody (ab 179467; abcam) was used as an internal reference antibody. The expression levels of wild-type and mutant ECHS1 protein polypeptides were quantitatively analyzed by calculating the band optical density using the Compass analysis software carried by the WES system.
Detection of ECHS1 protein bands by Western immunoblotting method A schematic diagram is shown in FIG. 7, and HEK293 cells are transfected with ECHS1 wild type pcDNA3.1-ECHS1-WT nucleic acid construct; MT1 is an ECHS1 mutant pcDNA3.1-ECHS1-MT1 nucleic acid construct transfected HEK293 cells, and transfected GFP plasmid and HEK293 cells (Control) without any treatment were used as background controls for protein overexpression and intracellular expression, respectively. The detection result shows that the mutation of c.724G > A (p.E242K, MT 1) on ECHS1 leads to significantly lower intracellular residual quantity compared with ECHS1 Wild Type (WT), indicating that the mutation affects the stability of ECHS1 protein, indicating that it has important protective function.
Immunofluorescence method for detecting FLAG-tagged wild-type and mutant ECHS1 proteins: HEK293 cells (pre-seeded on 0.1mg/ml poly L-lysine coated coverslips) were transfected with pcDNA3.1-ECHS1-MT1-FLAG and pcDNA3.1-ECHS1-WT-FLAG constructs, respectively, in 6-well plates. After 24 hours of transfection, the cell culture medium is removed, and 1ml of 4% paraformaldehyde is added to fix the cells for 15min at room temperature; the fixative was aspirated and washed three times with 1 XPBS for 5min each. After immersing the coverslip in a blocking buffer (blocking buffer) and incubating for 1h at room temperature, the cells were incubated with FLAG antibody (F7425; sigma) overnight at 4℃and then washed three times with 1 XPBS for 5min each. Cells were then incubated with a fluorescent secondary antibody (AlexaFluor 48nm, ab150077; abcam) for 1h at room temperature in the absence of light, washed three times with 1 XPBS for 5min each. And finally, lightly covering one surface of the cover glass with cells on the glass slide, smearing sealing tablets around for sealing, and observing green fluorescent signals under a laser confocal microscope, namely, indicating that the recombinant cells of the pcDNA3.1-ECHS1-MT1-FLAG and pcDNA3.1-ECHS1-WT-FLAG constructs with Flag labels are successfully constructed. By comparing the positions of the green fluorescent signals, the difference in the localization of the wild type and mutant ECHS1 proteins in the cell was analyzed.
Referring to FIG. 8, a schematic representation of FLAG-tag immunofluorescence for detecting the intracellular localization of wild-type and mutant ECHS1 proteins (green fluorescent signal). It can be seen that in recombinant cells, the ECHS1 Wild Type (WT) is specifically mitochondrial expressed, co-localized with the mitochondrial specific dye MitroTracker (Red, M7512; life Technologies), and the c.724G > A mutation does not affect the (MT 1) subcellular localization of ECHS1 protein. Immunofluorescence assays also showed that the ECHS1 mutant had no effect on the morphology of mitochondria, which remained filamentous and distributed in a network. This is different from the other type of genetic metabolic disease, hyperlysinemia, which is a disease in which the mitochondrial shape of a patient increases spherically, although it is also associated with disturbed amino acid metabolism, nerve damage and developmental retardation.
The detection result shows that: the successful construction of transfected mammalian cells with the two constructs pcDNA3.1-ECHS1-MT1-FLAG and pcDNA3.1-ECHS1-WT-FLAG can be used to analyze the differences in expression positions of ECHS1 wild type and mutant (p.E242K) in mammalian cells.
Example 4:
three-dimensional structure prediction was performed on ECSH1 mutant protein (p.E242K) by Pymol software, and the prediction results are shown in FIG. 9. The mutation changes the 242 th amino acid residue from glutamic acid to lysine, not only the amino acid residue becomes larger, but also the electrical property changes from an originally negatively charged amino acid to a positively charged amino acid. ECHS1 generally functions in vivo in the form of a hexamer, and this transition interferes with the formation of inter-subunit hydrogen bonds, reducing the five hydrogen bonding actions originally occurring at this site in the amino acid to three. The HOPE analysis also indicated that the amino acid at position 242 was located on the surface of the protein and that mutation at this site would interfere with the action of the ECHS1 enzyme protein on the substrate molecule in addition to other subunits. In addition, given that the 242 amino acid site is in the catalytically active region of the ECHS1 protein (the enoyl hydratase superfamily domain), mutations at this site are highly likely to affect the biological reactions in which ECHS1 participates.
We therefore examined the effect of p.E242K on ECSH1 enzyme activity (exemplified by HEK293 cells). Specifically, HEK293 cells were transfected with pcDNA3.1-ECHS1-MT1-FLAG and pcDNA3.1-ECHS1-WT-FLAG constructs, respectively, in 6-well cell culture plates. Cell culture medium was removed 24 hours after transfection, and cell pellet was collected after 0.25% pancreatin digestion. After adding 250. Mu.L of cell lysate (containing 50mM Tris-HCl,100mM NaCl,pH =8.0) and sonicating the cells, 16000×g was centrifuged at 4℃for 10min, and the lysate supernatant was collected and assayed for protein concentration by the Bradford method. Mu.g of cellular protein was pipetted into 100. Mu.L of reaction solution (configured with 50mM Tris-HCl, pH=8.0 buffer) containing 0.25mM substrate Crotonyl-CoA (Crotonyl-CoA, cat. No.28007; sigma-Aldrich). After fully mixing, placing the mixture into an enzyme-labeled instrument to detect the light absorption value at 263nm, collecting data every 10s, and detecting for 60min.
The results of the ECHS1 enzyme activity assay are shown in FIG. 10. Using crotonyl-CoA as a substrate, the recombinant cell lysate is mixed with the substrate and the intracellular ECHS1 enzyme activity is determined by detecting the residue of the substrate. 293 cells transfected with GFP plasmid and 293 cells (Control) without any treatment were used as controls for protein overexpression and cell background activity. As can be seen, the mutation c.724G > A (E242K, MT 1) resulted in a significant decrease in ECHS1 enzyme activity compared to ECHS1 wild-type recombinant cells (pcDNA3.1-ECHS 1-WT), which was comparable to the cell background (Control) and still had a significant amount of substrate residue (high substrate uptake value) until the end of the detection time. This mutation was shown to affect the enzymatic activity of the ECHS1 protein, indicating that it has important functionality in maintaining ECHS1 biological activity.
The detection result shows that: the successful construction of mammalian cells transfected with the two constructs pcDNA3.1-ECHS1-MT1 and pcDNA3.1-ECHS1-WT can be used to analyze the differences in enzyme activity of ECHS1 wild type and mutant (p.E242K) in mammalian cells.
While the foregoing is directed to the preferred embodiments and examples of the present invention, it will be apparent to those skilled in the art that various modifications and improvements can be made without departing from the inventive concepts, including but not limited to, adjustments in the ratio, flow, amount and reaction vessel, such as the use of a continuous flow reactor, which are within the scope of the present invention.
Claims (8)
1. The ECHS1 gene mutant is characterized in that the nucleotide sequence of the ECHS1 gene mutant is shown as SEQ ID NO. 3.
2. The protein polypeptide encoded by the ECHS1 gene mutant according to claim 1, wherein the amino acid sequence of said protein polypeptide is shown in SEQ ID No. 4.
3. A nucleic acid construct comprising the ECHS1 gene mutant of claim 1; the nucleotide sequence of the ECHS1 gene mutant is shown as SEQ ID NO. 3.
4. The nucleic acid construct of claim 3, wherein the backbone vector of the nucleic acid construct comprises pcdna3.1;
the nucleotide sequences of the primers used for constructing the nucleic acid construct are shown as SEQ ID NO.11 and SEQ ID NO. 12.
5. The nucleic acid construct of claim 3, wherein when the nucleic acid construct comprises a FLAG tag sequence, the nucleic acid construct is constructed using the nucleotide sequences of the primers shown in SEQ ID No.11 and SEQ ID No. 15.
6. A recombinant cell obtained by transfecting a recipient cell with the nucleic acid construct of any one of claims 3-5.
7. Use of a nucleic acid construct according to any one of claims 3 to 5, or a recombinant cell according to claim 6, for the preparation of a kit for studying the function of a polypeptide encoded by an ECHS1 gene mutant.
8. Use of the nucleic acid construct of any one of claims 3-5, or the recombinant cell of claim 6, in the preparation of a kit for providing a molecular genetic diagnosis of a patient with Leigh syndrome, guiding accurate treatment of the patient and/or avoiding risk of regeneration of the patient's family.
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Novel ECHS1 mutations in Leigh syndrome identified by whole-exome sequencing in five Chinese families: case report;Dan Sun等;BMC Medical Genetics;第21卷;摘要,表2,图4 * |
Two novel ECHS1 variants, affecting splicing and reducing enzyme activity, is associated with mitochondrial encephalopathy in infant: a case report;Miaojuan Wu等;BMC Neurology;第20卷;摘要、图3-4、表1 * |
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