CN116496377A - Ush2a基因突变的多肽、核酸及其应用 - Google Patents
Ush2a基因突变的多肽、核酸及其应用 Download PDFInfo
- Publication number
- CN116496377A CN116496377A CN202310223969.4A CN202310223969A CN116496377A CN 116496377 A CN116496377 A CN 116496377A CN 202310223969 A CN202310223969 A CN 202310223969A CN 116496377 A CN116496377 A CN 116496377A
- Authority
- CN
- China
- Prior art keywords
- polypeptide
- nucleic acid
- cell
- mutation
- combination
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 62
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 62
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 62
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 32
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 32
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 32
- 101100428002 Homo sapiens USH2A gene Proteins 0.000 title claims abstract description 23
- 206010064571 Gene mutation Diseases 0.000 title abstract description 7
- 230000035772 mutation Effects 0.000 claims abstract description 48
- 208000011580 syndromic disease Diseases 0.000 claims abstract description 18
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 6
- 210000004027 cell Anatomy 0.000 claims description 35
- 238000001514 detection method Methods 0.000 claims description 28
- 108020004414 DNA Proteins 0.000 claims description 24
- 238000012163 sequencing technique Methods 0.000 claims description 20
- 239000003795 chemical substances by application Substances 0.000 claims description 16
- 208000014769 Usher Syndromes Diseases 0.000 claims description 15
- 239000000523 sample Substances 0.000 claims description 15
- 230000037430 deletion Effects 0.000 claims description 11
- 238000012217 deletion Methods 0.000 claims description 11
- 238000000034 method Methods 0.000 claims description 10
- 239000002773 nucleotide Substances 0.000 claims description 9
- 125000003729 nucleotide group Chemical group 0.000 claims description 9
- 101000805941 Homo sapiens Usherin Proteins 0.000 claims description 8
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 5
- 108020004485 Nonsense Codon Proteins 0.000 claims description 5
- 239000003814 drug Substances 0.000 claims description 5
- 210000000349 chromosome Anatomy 0.000 claims description 3
- 239000013612 plasmid Substances 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 238000012360 testing method Methods 0.000 claims description 3
- 230000001131 transforming effect Effects 0.000 claims description 3
- 241001465754 Metazoa Species 0.000 claims description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 2
- 210000001671 embryonic stem cell Anatomy 0.000 claims description 2
- 210000004602 germ cell Anatomy 0.000 claims description 2
- 230000001225 therapeutic effect Effects 0.000 claims description 2
- 108090000623 proteins and genes Proteins 0.000 abstract description 26
- 230000001717 pathogenic effect Effects 0.000 abstract description 12
- 238000012216 screening Methods 0.000 abstract description 10
- 238000001228 spectrum Methods 0.000 abstract description 3
- 230000004784 molecular pathogenesis Effects 0.000 abstract description 2
- 238000006243 chemical reaction Methods 0.000 description 32
- 239000011324 bead Substances 0.000 description 29
- 201000008579 Usher syndrome type 2A Diseases 0.000 description 27
- 239000013598 vector Substances 0.000 description 21
- 239000000203 mixture Substances 0.000 description 14
- 239000000047 product Substances 0.000 description 12
- 102000004169 proteins and genes Human genes 0.000 description 12
- 239000003153 chemical reaction reagent Substances 0.000 description 11
- 238000003776 cleavage reaction Methods 0.000 description 11
- 230000007017 scission Effects 0.000 description 11
- 239000011534 wash buffer Substances 0.000 description 10
- 239000012634 fragment Substances 0.000 description 9
- 238000009396 hybridization Methods 0.000 description 9
- 239000006228 supernatant Substances 0.000 description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 8
- 238000004458 analytical method Methods 0.000 description 8
- 230000003321 amplification Effects 0.000 description 7
- 238000010828 elution Methods 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 7
- 238000002156 mixing Methods 0.000 description 7
- 238000003199 nucleic acid amplification method Methods 0.000 description 7
- 238000012408 PCR amplification Methods 0.000 description 6
- 230000002159 abnormal effect Effects 0.000 description 6
- 239000002299 complementary DNA Substances 0.000 description 6
- 238000010276 construction Methods 0.000 description 6
- 238000001890 transfection Methods 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- 201000010099 disease Diseases 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 230000006870 function Effects 0.000 description 5
- 238000011534 incubation Methods 0.000 description 5
- 238000010008 shearing Methods 0.000 description 5
- 201000007737 Retinal degeneration Diseases 0.000 description 4
- 238000000246 agarose gel electrophoresis Methods 0.000 description 4
- 239000012472 biological sample Substances 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 230000001720 vestibular Effects 0.000 description 4
- 108091092195 Intron Proteins 0.000 description 3
- 239000013614 RNA sample Substances 0.000 description 3
- 108010090804 Streptavidin Proteins 0.000 description 3
- 201000008614 Usher syndrome type 2 Diseases 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 230000035484 reaction time Effects 0.000 description 3
- 230000008439 repair process Effects 0.000 description 3
- 238000007480 sanger sequencing Methods 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 238000013518 transcription Methods 0.000 description 3
- 230000035897 transcription Effects 0.000 description 3
- 108700024394 Exon Proteins 0.000 description 2
- 208000001140 Night Blindness Diseases 0.000 description 2
- 101710163270 Nuclease Proteins 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 238000007664 blowing Methods 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- PHIQHXFUZVPYII-UHFFFAOYSA-N carnitine Chemical group C[N+](C)(C)CC(O)CC([O-])=O PHIQHXFUZVPYII-UHFFFAOYSA-N 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 239000003480 eluent Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000001976 enzyme digestion Methods 0.000 description 2
- 230000004438 eyesight Effects 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 102000051625 human USH2A Human genes 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 210000005259 peripheral blood Anatomy 0.000 description 2
- 239000011886 peripheral blood Substances 0.000 description 2
- 230000004853 protein function Effects 0.000 description 2
- 239000012264 purified product Substances 0.000 description 2
- 239000002096 quantum dot Substances 0.000 description 2
- 238000003757 reverse transcription PCR Methods 0.000 description 2
- 208000023573 sensorineural hearing loss disease Diseases 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 238000007482 whole exome sequencing Methods 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 102100036799 Adhesion G-protein coupled receptor V1 Human genes 0.000 description 1
- 101100519158 Arabidopsis thaliana PCR2 gene Proteins 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 230000007018 DNA scission Effects 0.000 description 1
- 206010011882 Deafness congenital Diseases 0.000 description 1
- 206010011891 Deafness neurosensory Diseases 0.000 description 1
- 108091092584 GDNA Proteins 0.000 description 1
- 101000928167 Homo sapiens Adhesion G-protein coupled receptor V1 Proteins 0.000 description 1
- 238000012404 In vitro experiment Methods 0.000 description 1
- 208000026350 Inborn Genetic disease Diseases 0.000 description 1
- 101150102573 PCR1 gene Proteins 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 108091036333 Rapid DNA Proteins 0.000 description 1
- 208000007014 Retinitis pigmentosa Diseases 0.000 description 1
- 208000009966 Sensorineural Hearing Loss Diseases 0.000 description 1
- 201000008616 Usher syndrome type 1 Diseases 0.000 description 1
- 201000008612 Usher syndrome type 3 Diseases 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 238000010009 beating Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 238000005352 clarification Methods 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 208000016361 genetic disease Diseases 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 208000016354 hearing loss disease Diseases 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000004379 myopia Effects 0.000 description 1
- 208000001491 myopia Diseases 0.000 description 1
- 238000007857 nested PCR Methods 0.000 description 1
- 239000013610 patient sample Substances 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 210000003370 receptor cell Anatomy 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 102200017861 rs397514703 Human genes 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 238000003260 vortexing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0684—Cells of the urinary tract or kidneys
- C12N5/0686—Kidney cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0693—Tumour cells; Cancer cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2503/00—Use of cells in diagnostics
- C12N2503/02—Drug screening
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2510/00—Genetically modified cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/136—Screening for pharmacological compounds
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/10—Screening for compounds of potential therapeutic value involving cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/14—Disorders of ear, nose or throat
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/16—Ophthalmology
- G01N2800/164—Retinal disorders, e.g. retinopathy
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Urology & Nephrology (AREA)
- Immunology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Cell Biology (AREA)
- General Engineering & Computer Science (AREA)
- Hematology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Pathology (AREA)
- Biophysics (AREA)
- Toxicology (AREA)
- Food Science & Technology (AREA)
- General Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Gastroenterology & Hepatology (AREA)
- Oncology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Peptides Or Proteins (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
本发明公开了一种USH2A基因突变的多肽、核酸及其应用。所述多肽的氨基酸序列如SEQ ID NO:13或SEQ ID NO:14所示。所述的核酸编码所述的多肽。本发明确定了一个导致Usher综合征2A型的USH2A基因的新的致病位点(c.14343+5G>A),确定了其产生的截短体多肽和编码核酸,扩大了USH2A基因的突变谱。更深入阐明了Usher综合征2A型的分子发病机制,为Usher综合征2A型的早期致病基因筛查和干预治疗提供科学依据。
Description
技术领域
本发明属于疾病检测领域,具体涉及USH2A基因突变的多肽、核酸及其应用。
背景技术
Usher综合征又称遗传性耳聋-色素性视网膜炎综合征,是一组以视网膜色素变性及不同程度听力损伤为特征,伴或不伴有前庭功能异常的常染色体隐性遗传病,具有遗传异质性。据统计,Usher综合征在全球范围内的发病率约为3.3~16.6/100000,呈散发性,无明显性别差异。根据患者听力和前庭功能及视野受损情况,将Usher综合征分为3种亚型:USH1、USH2、USH3。
USH2症状较轻,进展较慢,但发病率最高,主要表现为中度-重度感音神经性耳聋(SNHL),视网膜色素变性(RP)出现在10-38岁(平均年龄15±8.5岁),目前多认为USH2患者前庭功能正常,但也有研究表明USH2A突变可能引起前庭功能潜在损伤。目前已定位了4个基因座位(USH2A-D),已明确与该亚型相关的致病基因有3种,分别是USH2A(USH2A)、GPR98(USH2C)、WHRN(USH2D)。
虽然目前已发现一些Usher综合征2A型的致病基因,但仍存在着相当一部分未知致病基因位点。在目前对Usher综合征2A型的致病基因的研究过程中,因USH2A序列本身很长(800558bp),突变的可能性多样,在哪个位点,将核苷酸突变成哪种,能达到的效果都是未知的。而且,并非USH2A基因上任一位点的突变就必然会导致对翻译后蛋白的功能产生影响,更不能确保该基因上任一位点的突变必然会引起Usher综合征2A型的疾病:例如USH2A基因的转录本NM_206933.4上的突变c.15297+3A>G(Chr1:215807798)、c.8559-65T>C(Chr1:216051287)和c.6486-18G>A(Chr1:216172418),都不会影响所翻译的蛋白的正常功能。即便通过高通量测试,试图从数量庞大的突变体库中筛选出能够实现降低原蛋白质功能效果(例如外显子缺失)的突变也是很小概率的,并且这样的方法成本高昂,不具备实践的可能性。
因而,目前对Usher综合征2A型的研究仍有待深入。
发明内容
针对现有技术中Usher综合征2A型的USH2A基因突变谱尚有许多未知的致病突变位点的缺陷,本发明筛选得到了一种多肽、多肽组合、核酸、检测剂、构建体、重组细胞、试剂盒及应用。
本发明是基于下列工作完成的:通过高通量外显子组测序联合候选基因突变验证的方法确定了Usher综合征2A型新的致病基因突变位点。具体地,本发明涉及分离编码USH2A突变体的核酸,对该核酸样本进行二代测序,筛选出USH2A内含子区域的可能影响剪接的位点。通过克隆带有变异位点(适用于在剪切位点附近的内含子、外显子以及深度内含子中的点突变、插入及缺失等变异类型)的目标基因组片段,构建重组表达载体,转染细胞株,RNA抽提及cDNA反转,再利用电泳和测序技术验证该突变影响mRNA剪接(包括外显子缺失、内含子滞留等),从而确定了一个导致Usher综合征2A型的USH2A基因的新的致病位点以及其产生的截短体蛋白。
为解决上述技术问题,本发明提供的一个技术方案为:一种多肽,所述多肽为多肽1,所述多肽1的氨基酸序列如SEQ ID NO:13所示。所述多肽1为野生型编码人USH2A基因中Exon66中产生提前终止密码子PTC的截短体蛋白。
Exon66中产生提前终止密码子PTC的USH2A截短体蛋白(SEQ ID NO:13):
MNCPVLSLGSGFLFQVIEMLIFAYFASISLTESRGLFPRLENVGAFKKVSIVPTQAVCGLPDRSTFCHSSAAAESIQFCTQRFCIQDCPYRSSHPTYTALFSAGLSSCITPDKNDLHPNAHSNSASFIFGNHKSCFSSPPSPKLMASFTLAVWLKPEQQGVMCVIEKTVDGQIVFKLTISEKETMFYYRTVNGLQPPIKVMTLGRILVKKWIHLSVQVHQTKISFFINGVEKDHTPFNARTLSGSITDFASGTVQIGQSLNGLEQFVGRMQDFRLYQVALTNREILEVFSGDLLRLHAQSHCRCPGSHPRVHPLAQRYCIPNDAGDTADNRVSRLNPEAHPLSFVNDNDVGTSWVSNVFTNITQLNQGVTISVDLENGQYQVFYIIIQFFSPQPTEIRIQRKKENSLDWEDWQYFARNCGAFGMKNNGDLEKPDSVNCLQLSNFTPYSRGNVTFSILTPGPNYRPGYNNFYNTPSLQEFVKATQIRFHFHGQYYTTETAVNLRHRYYAVDEITISGRCQCHGHADNCDTTSQPYRCLCSQESFTEGLHCDRCLPLYNDKPFRQGDQVYAFNCKPCQCNSHSKSCHYNISVDPFPFEHFRGGGGVCDDCEHNTTGRNCELCKDYFFRQVGADPSAIDVCKPCDCDTVGTRNGSILCDQIGGQCNCKRHVSGRQCNQCQNGFYNLQELDPDGCSPCNCNTSGTVDGDITCHQNSGQCKCKANVIGLRCDHCNFGFKFLRSFNDVGCEPCQCNLHGSVNKFCNPHSGQCECKKEAKGLQCDTCRENFYGLDVTNCKACDCDTAGSLPGTVCNAKTGQCICKPNVEGRQCNKCLEGNFYLRQNNSFLCLPCNCDKTGTINGSLLCNKSTGQCPCKLGVTGLRCNQCEPHRYNLTIDNFQHCQMCECDSLGTLPGTICDPISGQCLCVPNRQGRRCNQCQPGFYISPGNATGCLPCSCHTTGAVNHICNSLTGQCVCQDASIAGQRCDQCKDHYFGFDPQTGRCQPCNCHLSGALNETCHLVTGQCFCKQFVTGSKCDACVPSASHLDVNNLLGCSKTPFQQPPPRGQVQSSSAINLSWSPPDSPNAHWLTYSLLRDGFEIYTTEDQYPYSIQYFLDTDLLPYTKYSYYIETTNVHGSTRSVAVTYKTKPGVPEGNLTLSYIIPIGSDSVTLTWTTLSNQSGPIEKYILSCAPLAGGQPCVSYEGHETSATIWNLVPFAKYDFSVQACTSGGCLHSLPITVTTAQAPPQRLSPPKMQKISSTELHVEWSPPAELNGIIIRYELYMRRLRSTKETTSEESRVFQSSGWLSPHSFVESANENALKPPQTMTTITGLEPYTKYEFRVLAVNMAGSVSSAWVSERTGESAPVFMIPPSVFPLSSYSLNISWEKPADNVTRGKVVGYDINMLSEQSPQQSIPMAFSQLLHTAKSQELSYTVEGLKPYRIYEFTITLCNSVGCVTSASGAGQTLAAAPAQLRPPLVKGINSTTIHLRWFPPEELNGPSPIYQLERRESSLPALMTTMMKGIRFIGNGYCKFPSSTHPVNTDFTGIKASFRTKVPEGLIVFAASPGNQEEYFALQLKKGRLYFLFDPQGSPVEVTTTNDHGKQYSDGKWHEIIAIRHQAFGQITLDGIYTGSSAILNGSTVIGDNTGVFLGGLPRSYTILRKDPEIIQKGFVGCLKDVHFMKNYNPSAIWEPLDWQSSEEQINVYNSWEGCPASLNEGAQFLGAGFLELHPYMFHGGMNFEISFKFRTDQLNGLLLFVYNKDGPDFLAMELKSGILTFRLNTSLAFTQVDLLLGLSYCNGKWNKVIIKKEGSFISASVNGLMKHASESGDQPLVVNSPVYVGGIPQELLNSYQHLCLEQGFGGCMKDVKFTRGAVVNLASVSSGAVRVNLDGCLSTDSAVNCRGNDSILVYQGKEQSVYEGGLQPFTEYLYRVIASHEGGSVYSDWSRGRTTGAAPQSVPTPSRVRSLNGYSIEVTWDEPVVRGVIEKYILKAYSEDSTRPPRMPSASAEFVNTSNLTGILTGLLPFKNYAVTLTACTLAGCTESSHALNISTPQEAPQEVQPPVAKSLPSSLLLSWNPPKKANGIITQYCLYMDGRLIYSGSEENYIVTDLAVFTPHQFLLSACTHVGCTNSSWVLLYTAQLPPEHVDSPVLTVLDSRTIHIQWKQPRKISGILERYVLYMSNHTHDFTIWSVIYNSTELFQDHMLQYVLPGNKYLIKLGACTGGGCTVSEASEALTDEDIPEGVPAPKAHSYSPDSFNVSWTEPEYPNGVITSYGLYLDGILIHNSSELSYRAYGFAPWSLHSFRVQACTAKGCALGPLVENRTLEAPPEGTVNVFVKTQGSRKAHVRWEAPFRPNGLLTHSVLFTGIFYVDPVGNNYTLLNVTKVMYSGEETNLWVLIDGLVPFTNYTVQVNISNSQGSLITDPITIAMPPGAPDGVLPPRLSSATPTSLQVVWSTPARNNAPGSPRYQLQMRSGDSTHGFLELFSNPSASLSYEVSDLQPYTEYMFRLVASNGFGSAHSSWIPFMTAEDKPGPVVPPILLDVKSRMMLVTWQHPRKSNGVITHYNIYLHGRLYLRTPGNVTNCTVMHLHPYTAYKFQVEACTSKGCSLSPESQTVWTLPGAPEGIPSPELFSDTPTSVIISWQPPTHPNGLVENFTIERRVKGKEEVTTLVTLPRSHSMRFIDKTSALSPWTKYEYRVLMSTLHGGTNSSAWVEVTTRPSRPAGVQPPVVTVLEPDAVQVTWKPPLIQNGDILSYEIHMPDPHITLTNVTSAVLSQKVTHLIPFTNYSVTIVACSGGNGYLGGCTESLPTYVTTHPTVPQNVGPLSVIPLSESYVVISWQPPSKPNGPNLRYELLRRKIQQPLASNPPEDLNRWHNIYSGTQWLYEDKGLSRFTTYEYMLFVHNSVGFTPSREVTVTTLAGLPERGANLTASVLNHTAIDVRWAKPTVQDLQGEVEYYTLFWSSATSNDSLKILPDVNSHVIGHLKPNTEYWIFISVFNGVHSINSAGLHATTCDGEPQGMLPPEVVIINSTAVRVIWTSPSNPNGVVTEYSIYVNNKLYKTGMNVPGSFILRDLSPFTIYDIQVEVCTIYACVKSNGTQITTVEDTPSDIPTPTIRGITSRSLQIDWVSPRKPNGIILGYDLLWKTWYPCAKTQKLVQDQSDELCKAVRCQKPESICGHICYSSEAKVCCNGVLYNPKPGHRCCEEKYIPFVLNSTGVCCGGRIQEAQPNHQCCSGYYARILPGEVCCPDEQHNRVSVGIGDSCCGRMPYSTSGNQICCAGRLHDGHGQKCCGRQIVSNDLECCGGEEGVVYNRLPGMFCCGQDYVNMSDTICCSASSGESKAHIKKNDPVPVKCCETELIPKSQKCCNGVGYNPLKYVCSDKISTGMMMKETKECRILCPASMEATEHCGRCDFNFTSHICTVIRGSHNSTGKASIEEMCSSAEETIHTGSVNTYSYTDVNLKPYMTYEYRISAWNSYGRGLSKAVRARTKEDVPQGVSPPTWTKIDNLEDTIVLNWRKPIQSNGPIIYYILLRNGIERFRGTSLSFSDKEGIQPFQEYSYQLKACTVAGCATSSKVVAATTQGVPESILPPSITALSAVALHLSWSVPEKSNGVIKEYQIRQVGKGLIHTDTTDRRQHTVTGLQPYTNYSFTLTACTSAGCTSSEPFLGQTLQAAPEGVWVTPRHIIINSTTVELYWSLPEKPNGLVSQYQLSRNGNLLFLGGSEEQNFTDKNLEPNSRYTYKLEVKTGGGSSASDDYIVQTPMSTPEEIYPPYNITVIGPYSIFVAWIPPGILIPEIPVEYNVLLNDGSVTPLAFSVGHHQSTLLENLTPFTQYEIRIQACQNGSCGVSSRMFVKTPEAAPMDLNSPVLKALGSACIEIKWMPPEKPNGIIINYFIYRRPAGIEEESVLFVWSEGALEFMDEGDTLRPFTLYEYRVRACNSKGSVESLWSLTQTLEAPPQDFPAPWAQATSAHSVLLNWTKPESPNGIISHYRVVYQERPDDPTFNSPTVHAFTVKGTSHQAHLYGLEPFTTYRIGVVAANHAGEILSPWTLIQTLESSPSGLRNFIVEQKENGRALLLQWSEPMRTNGVIKTYNIFSDGFLEYSGLNRQFLFRRLDPFTLYTLTLEACTRAGCAHSAPQPLWTDEAPPDSQLAPTVHSVKSTSVELSWSEPVNPNGKIIRYEVIRRCFEGKAWGNQTIQADEKIVFTEYNTERNTFMYNDTGLQPWTQCEYKIYTWNSAGHTCSSWNVVRTLQAPPEGLSPPVISYVSMNPQKLLISWIPPEQSNGIIQSYRLQRNEMLYPFSFDPVTFNYTDEELLPFSTYSYALQACTSGGCSTSKPTSITTLEAAPSEVSPPDLWAVSATQMNVCWSPPTVQNGKITKYLVRYDNKESLAGQGLCLLVSHLQPYSQYNFSLVACTNGGCTASVSKSAWTMEALPENMDSPTLQVTGSESIEITWKPPRNPNGQIRSYELRRDGTIVYTGLETRYRDFTLTPGVEYSYTVTASNSQGGILSPLVKDRTSPSAPSGMEPPKLQARGPQEILVNWDPPVRTNGDIINYTLFIRELFERETKIIHINTTHNSFGMQSYIVNQLKPFHRYEIRIQACTTLGCASSDWTFIQTPEIAPLMQPPPHLEVQMAPGGFQPTVSLLWTGPLQPNGKVLYYELYRRQIATQPRKSNPVLIYNGSSTSFIDSELLPFTEYEYQVWAVNSAGKAPSSWTWCRTGPAPPEAIRRHGHPADSPWPSSLH
或者,所述多肽为多肽2,所述多肽2的氨基酸序列如SEQ ID NO:14所示。所述多肽2为野生型编码人USH2A基因中Exon65整体缺失的USH2A截短体蛋白。
野生型编码人USH2A基因中Exon65整体缺失的USH2A截短体蛋白(SEQ ID NO:14):
MNCPVLSLGSGFLFQVIEMLIFAYFASISLTESRGLFPRLENVGAFKKVSIVPTQAVCGLPDRSTFCHSSAAAESIQFCTQRFCIQDCPYRSSHPTYTALFSAGLSSCITPDKNDLHPNAHSNSASFIFGNHKSCFSSPPSPKLMASFTLAVWLKPEQQGVMCVIEKTVDGQIVFKLTISEKETMFYYRTVNGLQPPIKVMTLGRILVKKWIHLSVQVHQTKISFFINGVEKDHTPFNARTLSGSITDFASGTVQIGQSLNGLEQFVGRMQDFRLYQVALTNREILEVFSGDLLRLHAQSHCRCPGSHPRVHPLAQRYCIPNDAGDTADNRVSRLNPEAHPLSFVNDNDVGTSWVSNVFTNITQLNQGVTISVDLENGQYQVFYIIIQFFSPQPTEIRIQRKKENSLDWEDWQYFARNCGAFGMKNNGDLEKPDSVNCLQLSNFTPYSRGNVTFSILTPGPNYRPGYNNFYNTPSLQEFVKATQIRFHFHGQYYTTETAVNLRHRYYAVDEITISGRCQCHGHADNCDTTSQPYRCLCSQESFTEGLHCDRCLPLYNDKPFRQGDQVYAFNCKPCQCNSHSKSCHYNISVDPFPFEHFRGGGGVCDDCEHNTTGRNCELCKDYFFRQVGADPSAIDVCKPCDCDTVGTRNGSILCDQIGGQCNCKRHVSGRQCNQCQNGFYNLQELDPDGCSPCNCNTSGTVDGDITCHQNSGQCKCKANVIGLRCDHCNFGFKFLRSFNDVGCEPCQCNLHGSVNKFCNPHSGQCECKKEAKGLQCDTCRENFYGLDVTNCKACDCDTAGSLPGTVCNAKTGQCICKPNVEGRQCNKCLEGNFYLRQNNSFLCLPCNCDKTGTINGSLLCNKSTGQCPCKLGVTGLRCNQCEPHRYNLTIDNFQHCQMCECDSLGTLPGTICDPISGQCLCVPNRQGRRCNQCQPGFYISPGNATGCLPCSCHTTGAVNHICNSLTGQCVCQDASIAGQRCDQCKDHYFGFDPQTGRCQPCNCHLSGALNETCHLVTGQCFCKQFVTGSKCDACVPSASHLDVNNLLGCSKTPFQQPPPRGQVQSSSAINLSWSPPDSPNAHWLTYSLLRDGFEIYTTEDQYPYSIQYFLDTDLLPYTKYSYYIETTNVHGSTRSVAVTYKTKPGVPEGNLTLSYIIPIGSDSVTLTWTTLSNQSGPIEKYILSCAPLAGGQPCVSYEGHETSATIWNLVPFAKYDFSVQACTSGGCLHSLPITVTTAQAPPQRLSPPKMQKISSTELHVEWSPPAELNGIIIRYELYMRRLRSTKETTSEESRVFQSSGWLSPHSFVESANENALKPPQTMTTITGLEPYTKYEFRVLAVNMAGSVSSAWVSERTGESAPVFMIPPSVFPLSSYSLNISWEKPADNVTRGKVVGYDINMLSEQSPQQSIPMAFSQLLHTAKSQELSYTVEGLKPYRIYEFTITLCNSVGCVTSASGAGQTLAAAPAQLRPPLVKGINSTTIHLRWFPPEELNGPSPIYQLERRESSLPALMTTMMKGIRFIGNGYCKFPSSTHPVNTDFTGIKASFRTKVPEGLIVFAASPGNQEEYFALQLKKGRLYFLFDPQGSPVEVTTTNDHGKQYSDGKWHEIIAIRHQAFGQITLDGIYTGSSAILNGSTVIGDNTGVFLGGLPRSYTILRKDPEIIQKGFVGCLKDVHFMKNYNPSAIWEPLDWQSSEEQINVYNSWEGCPASLNEGAQFLGAGFLELHPYMFHGGMNFEISFKFRTDQLNGLLLFVYNKDGPDFLAMELKSGILTFRLNTSLAFTQVDLLLGLSYCNGKWNKVIIKKEGSFISASVNGLMKHASESGDQPLVVNSPVYVGGIPQELLNSYQHLCLEQGFGGCMKDVKFTRGAVVNLASVSSGAVRVNLDGCLSTDSAVNCRGNDSILVYQGKEQSVYEGGLQPFTEYLYRVIASHEGGSVYSDWSRGRTTGAAPQSVPTPSRVRSLNGYSIEVTWDEPVVRGVIEKYILKAYSEDSTRPPRMPSASAEFVNTSNLTGILTGLLPFKNYAVTLTACTLAGCTESSHALNISTPQEAPQEVQPPVAKSLPSSLLLSWNPPKKANGIITQYCLYMDGRLIYSGSEENYIVTDLAVFTPHQFLLSACTHVGCTNSSWVLLYTAQLPPEHVDSPVLTVLDSRTIHIQWKQPRKISGILERYVLYMSNHTHDFTIWSVIYNSTELFQDHMLQYVLPGNKYLIKLGACTGGGCTVSEASEALTDEDIPEGVPAPKAHSYSPDSFNVSWTEPEYPNGVITSYGLYLDGILIHNSSELSYRAYGFAPWSLHSFRVQACTAKGCALGPLVENRTLEAPPEGTVNVFVKTQGSRKAHVRWEAPFRPNGLLTHSVLFTGIFYVDPVGNNYTLLNVTKVMYSGEETNLWVLIDGLVPFTNYTVQVNISNSQGSLITDPITIAMPPGAPDGVLPPRLSSATPTSLQVVWSTPARNNAPGSPRYQLQMRSGDSTHGFLELFSNPSASLSYEVSDLQPYTEYMFRLVASNGFGSAHSSWIPFMTAEDKPGPVVPPILLDVKSRMMLVTWQHPRKSNGVITHYNIYLHGRLYLRTPGNVTNCTVMHLHPYTAYKFQVEACTSKGCSLSPESQTVWTLPGAPEGIPSPELFSDTPTSVIISWQPPTHPNGLVENFTIERRVKGKEEVTTLVTLPRSHSMRFIDKTSALSPWTKYEYRVLMSTLHGGTNSSAWVEVTTRPSRPAGVQPPVVTVLEPDAVQVTWKPPLIQNGDILSYEIHMPDPHITLTNVTSAVLSQKVTHLIPFTNYSVTIVACSGGNGYLGGCTESLPTYVTTHPTVPQNVGPLSVIPLSESYVVISWQPPSKPNGPNLRYELLRRKIQQPLASNPPEDLNRWHNIYSGTQWLYEDKGLSRFTTYEYMLFVHNSVGFTPSREVTVTTLAGLPERGANLTASVLNHTAIDVRWAKPTVQDLQGEVEYYTLFWSSATSNDSLKILPDVNSHVIGHLKPNTEYWIFISVFNGVHSINSAGLHATTCDGEPQGMLPPEVVIINSTAVRVIWTSPSNPNGVVTEYSIYVNNKLYKTGMNVPGSFILRDLSPFTIYDIQVEVCTIYACVKSNGTQITTVEDTPSDIPTPTIRGITSRSLQIDWVSPRKPNGIILGYDLLWKTWYPCAKTQKLVQDQSDELCKAVRCQKPESICGHICYSSEAKVCCNGVLYNPKPGHRCCEEKYIPFVLNSTGVCCGGRIQEAQPNHQCCSGYYARILPGEVCCPDEQHNRVSVGIGDSCCGRMPYSTSGNQICCAGRLHDGHGQKCCGRQIVSNDLECCGGEEGVVYNRLPGMFCCGQDYVNMSDTICCSASSGESKAHIKKNDPVPVKCCETELIPKSQKCCNGVGYNPLKYVCSDKISTGMMMKETKECRILCPASMEATEHCGRCDFNFTSHICTVIRGSHNSTGKASIEEMCSSAEETIHTGSVNTYSYTDVNLKPYMTYEYRISAWNSYGRGLSKAVRARTKEDVPQGVSPPTWTKIDNLEDTIVLNWRKPIQSNGPIIYYILLRNGIERFRGTSLSFSDKEGIQPFQEYSYQLKACTVAGCATSSKVVAATTQGVPESILPPSITALSAVALHLSWSVPEKSNGVIKEYQIRQVGKGLIHTDTTDRRQHTVTGLQPYTNYSFTLTACTSAGCTSSEPFLGQTLQAAPEGVWVTPRHIIINSTTVELYWSLPEKPNGLVSQYQLSRNGNLLFLGGSEEQNFTDKNLEPNSRYTYKLEVKTGGGSSASDDYIVQTPMSTPEEIYPPYNITVIGPYSIFVAWIPPGILIPEIPVEYNVLLNDGSVTPLAFSVGHHQSTLLENLTPFTQYEIRIQACQNGSCGVSSRMFVKTPEAAPMDLNSPVLKALGSACIEIKWMPPEKPNGIIINYFIYRRPAGIEEESVLFVWSEGALEFMDEGDTLRPFTLYEYRVRACNSKGSVESLWSLTQTLEAPPQDFPAPWAQATSAHSVLLNWTKPESPNGIISHYRVVYQERPDDPTFNSPTVHAFTVKGTSHQAHLYGLEPFTTYRIGVVAANHAGEILSPWTLIQTLESSPSGLRNFIVEQKENGRALLLQWSEPMRTNGVIKTYNIFSDGFLEYSGLNRQFLFRRLDPFTLYTLTLEACTRAGCAHSAPQPLWTDEAPPDSQLAPTVHSVKSTSVELSWSEPVNPNGKIIRYEVIRRCFEGKAWGNQTIQADEKIVFTEYNTERNTFMYNDTGLQPWTQCEYKIYTWNSAGHTCSSWNVVRTLQAPPEGLSPPVISYVSMNPQKLLISWIPPEQSNGIIQSYRLQRNEMLYPFSFDPVTFNYTDEELLPFSTYSYALQACTSGGCSTSKPTSITTLEAAPSEVSPPDLWAVSATQMNVCWSPPTVQNGKITKYLVRYDNKESLAGQGLCLLVSHLQPYSQYNFSLVACTNGGCTASVSKSAWTMEALPENMDSPTLQVTGSESIEITWKPPRNPNGQIRSYELRRDGTIVYTGLETRYRDFTLTPGVEYSYTVTASNSQGGILSPLVKDRTSPSAPSGMEPPKLQARGPQEILVNWDPPVRTNGDIINYTLFIRELFERETKIIHINTTHNSFGMQSYIVNQLKPFHRYEIRIQACTTLGCASSDWTFIQTPEIAPLMQPPPHLEVQMAPGGFQPTVSLLWTGPLQPNGKVLYYELYRRQIATQPRKSNPVLIYNGSSTSFIDSELLPFTEYEYQLSEGMATQQTLHGLQAFTNYSIGVEACTCFNCCSKGPTAELRTHPAPPSGLSSPQIGTLASRTASFRWSPPMFPNGVIHSYELQFHVACPPDSALPCTPSQIETKYTGLGQKASLGGLQPYTTYKLRVVAHNEVGSTASEWISFTTQKELPQYRAPFSVDSNLSVVCVNWSDTFLLNGQLKEYVLTDGGRRVYSGLDTTLYIPRTADKTFFFQVICTTDEGSVKTPLIQYDTSTGLGLVLTTPGKKKGSRSKSTEFYSELWFIVLMAMLGLILLAIFLSLILQRKIHKEPYIRERPPLVPLQKRMSPLNVYPPGENHMGLADTKIPRSGTPVSIRSNRSACVLRIPSQNQTSLTYSQGSLHRSVSQLMDIQDKKVLMDNSLWEAIMGHNSGLYVDEEDLMNAIKDFSSVTKERTTFTDTHL
本发明所述野生型人USH2A蛋白全长氨基酸序列的NCBI参考序列为NP_996816.3,全长为5202个氨基酸。
为解决上述技术问题,本发明提供的一个技术方案为:一种多肽组合,所述多肽组合包含如本发明所述多肽1和所述多肽2。
为解决上述技术问题,本发明提供的一个技术方案为:一种分离的核酸,所述的核酸编码如本发明所述的多肽或如本发明所述的多肽组合。
较佳地,所述核酸的核苷酸序列包括:与人基因组GRCh38上1号染色体NC_000001.11的第215622891~216423448位碱基所对应的人的野生型USH2A基因的序列相比,具有g.215650587G>A突变的核苷酸序列。
本发明采用本领域通用表示法HGVS表示突变。所述g.215650587G>A突变为人基因组GRCh38上基因坐标为chr1:215650587的核苷酸由G突变为A。
在本发明中,c.14343+5G>A突变和g.215650587G>A为同一种突变,可互相替换。c.14343+5G>A突变表示cDNA(在本发明中,对应于转录本编号为NM_206933.4的序列)从5’到3’端的第14343位核苷酸下游的内含子(在本发明中即为Intron65)的第5位核苷酸由G突变为A。
在本发明中,人基因组GRCh38上1号染色体NC_000001.11的第215622891~216423448位碱基所对应的人的野生型USH2A基因序列(800558bp)以及转录本编号为NM_206933.4的序列(18938bp)均可通过常用的生物数据库(例如NCBI,National Center forBiotechnology Information,美国国家生物技术信息中心)检索得到。
发明人惊奇的发现,该突变体与Usher综合征2A型的发病密切相关,从而通过检测该突变体在生物样品中是否存在,可以有效地检测生物样品是否易患Usher综合征2A型。
在本发明一具体实施方案中,所述核酸为DNA或者RNA。
为解决上述技术问题,本发明提供的一个技术方案为:一种用于检测如本发明所述的多肽、如本发明所述的多肽组合或如本发明所述的核酸的检测剂,所述检测剂包括与所述多肽、所述多肽组合或所述核酸特异性杂交的生物分子,所述生物分子例如引物、探针和/或抗体;或,所述检测剂包括用于检测如本发明所述的多肽、如本发明所述的多肽组合或如本发明所述的核酸的基因组、转录组和/或蛋白质组测序的试剂。
在本发明一具体实施方案中,所述检测剂为引物,所述引物具有如SEQ ID NO:15和SEQ ID NO:16所示的核苷酸序列。
c.14343+5G>A正向引物(SEQ ID NO:15):AGTTCACCGTAGAGCAACTGA
c.14343+5G>A反向引物(SEQ ID NO:16):GCAGGAAAAGCCCCCAGTAG
为解决上述技术问题,本发明提供的一个技术方案为:一种构建体,所述构建体包含如本发明所述的核酸。由此,利用所述构建体转化受体细胞获得的重组细胞,能够有效地用于筛选治疗Usher综合征2A型的药物。
在本发明一较佳实施方案中,所述构建体的质粒骨架为pcMINI或pcMINI-C。
所述pcMINI的结构可参考专利文献CN113736812A。所述pcMINI-C相比于专利文献CN113736812A中的pcMINI载体,缺少片段intronB-ExonB。
为解决上述技术问题,本发明提供的一个技术方案为:一种重组细胞,所述重组细胞是通过将本发明所述的构建体转化受体细胞而获得的。利用本发明的重组细胞,能够有效地筛选治疗Usher综合征2A型的药物。
在本发明一较佳实施方案中,所述受体细胞不为动物生殖细胞、受精卵或者胚胎干细胞。
在本发明一具体实施方案中,所述受体细胞为HeLa细胞或293T细胞。
为解决上述技术问题,本发明提供的一个技术方案为:一种试剂盒,所述试剂盒包含如本发明所述的多肽、如本发明所述的多肽组合、如本发明所述的核酸、如本发明所述的检测剂、如本发明所述的构建体和如本发明所述的重组细胞中的一种或多种。由此,利用该试剂盒,可以对USH2A基因突变体进行高精度的检测,从而对Usher综合征2A型进行基因层面的检测,有助于更准确地筛选易患Usher综合征2A型的生物样品或有效地筛选治疗Usher综合征2A型的药物。
为解决上述技术问题,本发明提供的一个技术方案为:如本发明所述的多肽、如本发明所述的多肽组合、如本发明所述的核酸、如本发明所述的检测剂、如本发明所述的构建体、如本发明所述的重组细胞和如本发明所述的试剂盒中的一种或多种在制备诊断和/或预测Usher综合征2A型的药物中的应用。
本发明中,所述Usher综合征2A型优选为USH2A基因突变导致的Usher综合征2A型,进一步优选为USH2A基因的Exon66中产生提前终止密码子和/或USH2A基因Exon65整体缺失导致的Usher综合征2A型。
为解决上述技术问题,本发明提供的一个技术方案为:一种检测人的USH2A基因突变体的方法,所述方法包括使用如本发明所述的多肽、如本发明所述的多肽组合、如本发明所述的核酸、如本发明所述的检测剂、如本发明所述的构建体、如本发明所述的重组细胞和如本发明所述的试剂盒中的一种或多种检测待测样本。
在本发明的优选实施方案中,所述检测为非诊断和/或治疗目的的,例如在实验室科研过程中,将如本发明所述的多肽、如本发明所述的多肽组合、如本发明所述的核酸、如本发明所述的检测剂、如本发明所述的构建体、如本发明所述的重组细胞或如本发明所述的试剂盒作为阳性对照或标准品;也可将如本发明所述的检测剂或如本发明所述的试剂盒用于实验样本中人的USH2A突变体基因片段的特异性杂交或扩增或与之相似的其他应用场景。
在符合本领域常识的基础上,上述各优选条件,可任意组合,即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于:本发明确定了一个导致Usher综合征2A型的USH2A基因的新的致病位点(c.14343+5G>A),确定了其产生的截短体多肽和编码核酸,扩大了USH2A基因的突变谱。更深入阐明了Usher综合征2A型的分子发病机制,为Usher综合征2A型的早期致病基因筛查和干预治疗提供科学依据。
附图说明
图1为Usher综合征2A型患者家系图。
图2为先证者外显子测序实验流程图。
图3为突变在基因组中的位置。
图4为Varseak对c.14343+5G>A变异的预测。
图5为SpliceAI对c.14343+5G>A变异的预测。
图6为pcMINI载体检测结果。(A部分)minigene载体构建策略;(B部分)minigene构建测序图,上为wt,下为mut;(C部分)RT-PCR转录分析琼脂糖凝胶电泳图;在HeLa和293T细胞中的条带标为a、b和c;(D部分)剪切条带对应示意图及测序结果图。*表示突变位置。
图7为pcMINI-C载体检测结果。(A部分)minigene载体构建策略;(B部分)minigene构建测序图,上为wt,下为mut;(C部分)RT-PCR转录分析琼脂糖凝胶电泳图;在HeLa和293T细胞中的条带标为a、b和c;(D部分)剪切条带对应示意图及测序结果图。*表示突变位置。
具体实施方式
下面通过实施例的方式进一步说明本发明,但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法,按照常规方法和条件,或按照商品说明书选择。
实施例1确定USH2A基因内含子变异对USH2A转录产物的影响
1、样本收集
收集到一个Usher综合征2A型患者,其主要临床表型为怀疑RP,听力不好,双眼近视0.5、0.6,夜盲;家族史:母亲的弟弟,晚上视力不好,夜盲。其家系图见图1。
本实验经过良培基因生物科技(武汉)有限公司伦理委员会审核通过,所有病人样本和数据采集符合《医疗卫生机构科研用人类生物样本管理暂行办法(征求意见稿)》规定。且取得了病人个人的知情同意。
2、全外显子组测序
基于MGISEQ-T7测序平台采用双末端测序方法,对图1所示Usher综合征2A型患者家系中的患者的外显子组序列进行了测序。实验流程如图2所示。
2.1DNA提取及检测
采集图1所示Usher综合征2A型患者的外周血,采用磁珠法血液基因组DNA提取试剂盒(北京Tiangen Biotech公司),从该外周血样品中提取该患者的基因组DNA,并采用Qubit 3.0荧光计(Qubit双链DNA检测试剂盒,美国Invitrogen)定量基因组DNA的浓度及总量,所得各基因组DNA的浓度≥50ng/μl,纯度A260/A280均应为1.8~2.0之间,DNA总量≥30μg,并用琼脂糖凝胶电泳检测其纯度及完整性。
2.2酶切打断,末端修复,3‘端加“A”
这一步骤对Input DNA进行片段化的同时将片段化DNA末端补平,并在5’端进行磷酸化和3’端加dA尾。
本步骤中使用的诺维赞DNA酶切建库(货号NDM617-MGI)的产品组分如下表1:
表1诺维赞DNA酶切建库的产品组分
将FEA Buffer、FEA Enzyme Mix取出,解冻并混匀、短暂离心收集至管底,置于冰上备用,以下所有步骤均在冰上操作。于灭菌PCR管中配制如下表2反应:
表2末端修复和加A酶反应体系
反应组分 | 分别加入量(μL) |
Input DNA | X(200ng) |
ddH2O | 35-X |
FEA Buffer | 5 |
FEA Enzyme Mix | 10 |
Total volume | 50 |
冰盒上操作,加样完成立即放入PCR仪反应(条件如表3所示)。PCR提前预冷至4℃。
表3 PCR反应条件
反应温度 | 反应时间 |
105℃ | on |
4℃ | 2min |
37℃ | 15min |
65℃ | 30min |
4℃ | Hold |
2.3连接
向上一步反应完成后的50μL体系中加入5μL 10μM的Adapter。
按照表4配制反应体系:
表4接头连接反应体系
反应组分 | 分别加入量(μL) |
末端修复后DNA | 50 |
Adapter(10μM) | 5 |
Rapid Ligation Buffer 3 | 25 |
Rapid DNA Ligase | 5 |
ddH2O | 15 |
Total volume | 100 |
将100μL反应体系加入已含有Adapter的样品中混匀,封膜离心。将样品置入PCR仪中反应,反应条件如表5:热盖OFF,20℃15min;4℃Hold。
表5 PCR反应条件
反应温度 | 反应时间 |
热盖105℃ | on |
20℃ | 15min |
4℃ | Hold |
2.4连接后纯化
使用VAHTS DNA Clean Beads(诺维赞,Vazyme#N411)对反应产物进行纯化,将纯化产物洗脱至合适体积的洗脱液中,再进行双轮磁珠分选或切胶纯化。
2.4.1磁珠平衡至室温后,涡旋振荡混匀VAHTS DNAClean Beads。
2.4.2吸取60μL VAHTS DNAClean Beads至100μL Adapter Ligation产物中,涡旋振荡或使用移液器轻轻吹打10次充分混匀。
2.4.3室温孵育5min。
2.4.4将PCR管短暂离心并置于磁力架中分离磁珠和液体,待溶液澄清后(约5min),小心移除上清。
2.4.5保持PCR管始终置于磁力架中,加入200μL新鲜配制的80%乙醇漂洗磁珠,室温孵育30sec,小心移除上清。
2.4.6重复步骤5,总计漂洗两次。
2.4.7保持PCR管始终置于磁力架中,开盖空气干燥磁珠5-10min至无乙醇残留。
2.4.8将PCR管从磁力架中取出,进行洗脱:加入22.5μL洗脱液(10mM Tris-HCl,pH8.0-pH 8.5)洗脱,涡旋振荡或使用移液器轻轻吹打充分混匀,于室温放置2min,将PCR管短暂离心并置于磁力架中静置,待溶液澄清后(约5min),小心移取20μL上清至新EP管中,切勿触碰磁珠。
2.5 Pre-PCR反应
这一步骤将对纯化或长度分选后的Adapter Ligation产物进行PCR扩增。
按照表6配制PCR反应体系:
表6 Pre-PCR反应体系
反应组分 | 分别加入量(μL) |
连接后DNA | 20 |
Index primer for MGI | 2 |
VAHTS HiFi Amplification Mix | 25 |
Total volume | 50 |
将样品置入PCR仪中按表7反应条件进行PCR反应:
表7 PCR反应条件
2.6 Pre-PCR产物纯化
样本补水至100μL,使用磁珠VAHTS DNA Clean Beads(诺维赞,Vazyme#N411,室温平衡30min),0.6×(60μL磁珠),0.2×(20μL磁珠)双选,80%乙醇漂洗2遍,22μL水洗脱。
2.7杂交
纳昂达DNA文库杂交捕获试剂组分如表8所示:
表8纳昂达DNA文库杂交捕获试剂组分
将上述纯化产物,按下表9比例配置,为杂交做准备:
表9预杂交体系
反应组分 | 分别加入量(μL) |
上步所得DNA样本 | 500ng |
Human Cot DNA | 5μL |
NadPrep NanoBlockers(for Illumina,DI) | 2μL |
其中,NadPrep NanoBlockers(for Illumina,DI)品牌Nanodigmbio,NanoBlockers(for/>),16rxn,货号1006101;/>NanoBlockers(for),96rxn,货号1006102。
60℃浓缩(大概15min)(注意:Hyb#1必须65℃孵育直到结晶完全融解),杂交体系如表10所示。
表10杂交体系
反应组分 | 分别加入量(μL) |
Hyb#1 | 8.5μL |
Hyb#2 | 2.7μL |
Nuclease Free Water | 1.8μL |
Exome.Plus Panel v2.0 | 4μL |
注意:Hyb#1、Hyb#2和Nuclease Free Water先混合,静置5min,使13μL溶液充分溶解,然后再加入探针4μL,见表11。
表11反应条件
反应温度 | 反应时间 |
热盖100℃ | on |
95℃ | 30s |
65℃ | Hold |
2.8洗脱
Wash Buffer 1如果无法融解,可于65℃水浴孵育至完全融解。提前拿出磁珠室温平衡30min。洗脱试剂配制如表12所示。
表12洗脱试剂配制
组分名称 | 无水酶(μL) | 备注 |
Bead Wash Buffer | 320 | 室温 |
Wash Buffer 1 | 280 | 孵育 |
Wash Buffer 2 | 320 | 孵育 |
Wash Buffer 3 | 160 | 室温 |
Wash Buffer 4 | 160 | 室温 |
洗脱试剂供应商及货号:纳昂达科技Hybrid Capture Reagents试剂盒,货号1005102;产品/>Hybrid Capture Reagents,16rxn,货号1005101。
其中,Wash Buffer 1组分分出1管110μL,放置于65℃PCR仪孵育1h后使用,剩余170μL用于后续实验。Wash Buffer 2组分分装2份160μL,放置于65℃PCR仪孵育1h后使用。注意:Wash Buffer 1和Wash Buffer 2两种热洗脱buffer至少需要在65℃孵育15min。磁珠悬浮液配置如表13所示。
表13磁珠悬浮液配置
/>
将DynabeadsTMM-270Streptavidin Beads室温平衡30min。
链霉亲和素磁珠清洗:吸取50μL DynabeadsTMM-270Streptavidin Beads至1个1.5ml低吸附离心管中,加入100μL Bead Wash Buffer,轻柔吹吸混匀10次,瞬时离心,置于磁力架上数分钟,待澄清,弃上清。将离心管从磁力架上移出。重复步骤2两次。加入17μL磁珠悬浮液。转移至1个新的0.2ml低吸附PCR管中。
链霉亲和素磁珠捕获:提前打开另外一台PCR仪运行洗脱程序,将杂交管转移至新的PCR仪上。将17μL杂交体系加入到17μL重悬的链霉亲和素磁珠中,用移液器轻柔吹吸混匀或涡旋混匀。65℃孵育45min,每10-12min吹吸混匀一次。
2.8.1热洗脱
取下PCR管,并加入100μL 65℃Wash Buffer 1,吹吸混匀。
置于磁力架上1min,待澄清后弃上清。
从磁力架上移出,加入150μL 65℃Wash Buffer 2,轻柔混合均匀,放进PCR仪中65℃孵育5min。重复步骤2和3一次。
2.8.2室温洗脱
将PCR管瞬时离心后置于磁力架上1min,弃上清,加入150μL室温Wash Buffer1,涡旋混匀,室温孵育2min,期间涡旋混匀30s后静置30s,交替进行,确保充分混匀。
将PCR管瞬时离心后置于磁力架上1min,弃上清,加入150μL室温Wash Buffer3,同上步。
将PCR管瞬时离心后置于磁力架上1min,弃上清,加入150μL室温Wash Buffer4,同上步,离心后置于磁力架上1min,弃上清,去少量残余Buffer。
用22.5μL Nuclease-free water重悬(注意:不要丢弃磁珠。)
2.9 Post-PCR反应
对上步重悬的液体进行Post-PCR(反应体系和反应条件分别见表14和表15)。
表14 Post-PCR反应体系
反应组分 | 分别加入量(μL) |
待捕获DNA的磁珠 | 20μL |
VAHTS HiFi Amplification Mix | 25μL |
POST PCR1 | 2.5μL |
POST PCR2 | 2.5μL |
Total volume | 50μL |
表15反应条件
2.10 PCR扩增后纯化
VAHTS DNA Clean Beads(诺维赞,Vazyme#N411,室温平衡30min),1×磁珠(50μL),80%乙醇漂洗2遍,25μL水洗脱。
2.11测序
将捕获的DNA文库通过MGISEQ-T7测序平台(深圳华大智造科技股份有限公司)对人类全基因外显子组序列进行2×150bp的双末端测序,获得原始测序数据。
3、变异检测、注释及数据库比较
测序仪下机reads利用BWA(版本:0.7.16a)软件与人类参考基因组GRCh38比对,将生成的bam文件采用GATK软件(版本:4.0.8.1)进行变异检测分析,主要包括:标记PCR重复序列、SNVs和InDels变异检测和分型。使用Annovar(版本:20200608)软件对变异文件vcf进行注释。注释数据库主要有ExAC、dbSNP、1000genome、gnomAD、InterPro、ClinVar、OMIM、HGMD等。变异位点筛选原则:(1)筛选出外显子区域变异、剪切区域变异、非同义突变位点;(2)ExAC_EAS、ExAC_ALL、1000Genome、gnomAD等人群频率数据库中正常人变异携带率小于1%;(3)使用多种预测软件如SIFT、Polyphen2、LRT、MutationTaster进行基因位点变异对其蛋白功能影响预测;(4)参考OMIM、HGMD、ClinVar等多种疾病数据库对致病变异位点进行评估。变异解析和临床意义均根据ACMG变异分类指南(2015)以及受检者临床信息进行致病变异筛选,检出与可解释受检者临床表型的变异位点会采用一代Sanger平台进行位点测序验证。先证者检出的变异如表16所示。
表16先证者检出的变异
基因 | 亚区 | 基因组坐标 | 转录本 | 核苷酸变化 | 氨基酸变化 |
USH2A | Intron65 | chr1:215650587 | NM_206933.4 | c.14343+5G>A | - |
3.1软件预测
突变在基因外显子或内含子中对应的位置,如图3所示。
进一步,使用剪接预测软件Varseak(https://varseak.bio/index.php),SpliceAI(https://spliceailookup.broadinstitute.org/),对该位点进行功能预测,结果见图4和图5。预测结果一致认为该位点会导致蛋白功能改变,与疾病强相关。
预测1——Varseak:该算法显示突变后原有的Donor位点被破坏,提示突变影响剪切。
预测2——SpliceAI:该算法显示突变后原有的Donor位点分值丢失0.79,提示突变影响剪切。
4、Minigene实验方案
所用试剂供应商及货号如表17所示。
表17 Minigene实验所用试剂供应商及货号
4.1minigene钓取及酶切位点引入,引物信息见表18。
表18 minigene钓取引物
4.1.1引物设计:设计两对巢式引物770481-USH2A-F和774389-USH2A-R,770800-USH2A-F和774007-USH2A-R,以正常人DNA为模板进行巢式PCR,得到的PCR产物命名为①。
4.1.2片段扩增:
4.1.2.1以产物①为模板,以pcMINI-USH2A-KpnI-F和pcMINI-USH2A-BamHI-R为引物扩增得到pcMINI-USH2A-wt的片段1620bp;
4.1.2.2以产物①为模板,以pcMINI-USH2A-KpnI-F和USH2A-mut-R为引物扩增得到pcMINI-USH2A-mut的左半段;USH2A-mut-F和pcMINI-USH2A-BamHI-R为引物扩增得到pcMINI-USH2A-mut的右半段;左右半段1:1混合作为模板,以pcMINI-USH2A-KpnI-F和pcMINI-USH2A-BamHI-R为引物扩增得到pcMINI-USH2A-mut片段1620bp;
4.1.2.3 pcMINI-C-wt/mut的扩增同理。
4.1.3连接转化及鉴定:
4.1.3.1载体pcMINI(结构参考专利文献CN113736812A图1)和片段做酶切、回收、连接、转化、菌落PCR鉴定以及测序。
4.1.3.2载体pcMINI-C(结构参考专利文献CN113736812A图1,相比于该pcMINI载体,本实验所用载体pcMINI-C缺少片段intronB-ExonB)和片段做酶切、回收、连接、转化、菌落PCR鉴定以及测序。
4.1.4引物的退火温度为57℃,PCR扩增体系如下表19:
表19 PCR扩增体系
2×Mix | 15μL |
Primer-F | 1μL |
Primer-R | 1μL |
gDNA(wt/mut) | 1μL(0.5μg) |
Total | 补足至30μL |
4.1.5电泳及胶回收PCR产物
4.2构建重组载体
4.2.1重组载体构建包括酶切、酶连、转化及重组克隆验证。
4.2.2酶切体系如下表20:
表20酶切体系
37℃酶切反应2小时后,电泳检测并胶回收目标条带。
连接体系如下表21:
表21连接体系
4℃过夜连接后,转化DH5α感受态,37℃培养过夜后随机选取若干单克隆菌落进行鉴定。鉴定方式包括菌落/菌液PCR及Sanger测序。
4.3细胞转染
将重组载体分别瞬时转染到HeLa和293T细胞系中,转染步骤按照脂质体说明书进行,48小时后收样。
4.4minigene转录分析
提取细胞样品的总RNA,提取方法按试剂盒说明书进行。测定浓度后,用等量RNA进行逆转录合成cDNA。
利用minigene载体上的侧翼引物pcMINI-HindIII-F/pcDNA3.1-R(序列见表18)进行PCR扩增,琼脂糖凝胶电泳检测扩增产生的基因转录条带,分别回收各条带进行Sanger测序。
5、实验结果
5.1重组载体测序结果
Sanger测序结果显示,野生型和突变型的minigene均成功插入到相应载体中,测序结果见图6的A部分和图7的A部分。
5.2RNA浓度及纯度测量结果
质粒转染细胞48hr后,进行RNA样品提取和cDNA制备。下表22显示RNA样品浓度和纯度合格。
表22RNA样品浓度和纯度
5.3转录结果分析
进行minigene构建,将野生型和突变型minigene分别插入到pcMINI与pcMINI-C载体上,共4个重组载体分别转染入HeLa和293T细胞系,转染48hr后共收取8个样本提取RNA。
5.3.1pcMINI系列结果分析
5.3.1.1pcMINI-USH2A-wt/mut的minigene构建策略:Intron64(495bp)-Exon65(210bp)-部分Intron65(895bp)插入到pcMINI载体中,该载体含有通用ExonA-IntronA-MCS-IntronB-ExonB,转染细胞后观察ExonA-Exon65-ExonB的剪切方式是否存在异常,结果如图6所示。
5.3.1.2RT-PCR检测结果显示:在HeLa和293T细胞中野生型为单一条带,其与预期大小(521bp)一致,命名为条带a;突变型有两条带,均比野生型条带小,其中大条带命名为条带b,小条带命名为条带c。将两个细胞系中产生的野生型条带a及突变型条带b和c分别进行Sanger测序。
5.3.1.3测序结果显示:野生型条带a为正常剪切条带,剪切方式为ExonA(192bp)-Exon65(210bp)-ExonB(57bp);突变型条带b为异常剪切条带,Exon65右侧缺失134bp,剪切方式为ExonA(192bp)-ΔExon65(76bp)-ExonB(57bp);突变型条带c为异常剪切条带,Exon65整体缺失,剪切方式为ExonA(192bp)-ExonB(57bp)。
5.3.2pcMINI-C系列结果分析
5.3.2.1pcMINI-C-USH2A-wt/mut的minigene构建策略:将部分Intron64(175bp)-Exon65(210bp)-Intron65(1825bp)-Exon66(239bp)插入到pcMINI-C载体中。该载体含有通用ExonA-IntronA-MCS,转染细胞后观察ExonA-Exon65-Exon66剪切方式是否存在异常,结果如图7所示。
5.3.2.2RT-PCR检测结果显示:在HeLa和293T细胞中野生型为单一条带,其与预期大小(717bp)一致,命名为条带a;突变型有两条带,均比野生型条带小,其中大条带命名为条带b,小条带命名为条带c。将两个细胞系中产生的野生型条带a及突变型条带b和c分别进行Sanger测序。
5.3.2.3测序结果显示:野生型条带a为正常剪切条带,剪切方式为ExonA(192bp)-Exon65(210bp)-Exon66(239bp);突变型条带b为异常剪切条带,Exon65右侧缺失134bp,剪切方式为ExonA(192bp)-ΔExon65(76bp)-Exon66(239bp);突变型条带c为异常剪切条带,Exon65整体缺失,剪切方式为ExonA(192bp)-Exon66(239bp)。
6、结论
Minigene体外实验检测结果表明,突变c.14343+5G>A影响基因mRNA的正常剪接,且pcMINI和pcMINI-C两套载体的检测结果一致。突变后存在两种异常的转录本:①突变后导致Exon65右侧缺失134bp。②突变后导致Exon65整体缺失。
突变后导致Exon65右侧缺失134bp。在cDNA及蛋白水平的表示方式为:c.14210_14343del p.Gly4737Alafs*19,会在Exon66中产生提前终止密码子PTC,会产生一条截短体蛋白,长度为4754aa(见SEQ ID NO:13)。
突变后导致Exon65整体缺失。在cDNA及蛋白水平的表示方式为:c.14134_14343del p.Val4712_Val4781del,Exon65整体缺失并不会导致后续读码框的改变,仅导致蛋白内部缺失70aa,总长为5132aa(见SEQ ID NO:14)。
针对突变c.14343+5G>A,可设计特异性引物c.14343+5G>A正向引物USH2A-65-F(SEQ ID NO:15):AGTTCACCGTAGAGCAACTGA和c.14343+5G>A反向引物USH2A-65-R(SEQ IDNO:16):GCAGGAAAAGCCCCCAGTAG,对待测的DNA样本进行PCR扩增,并对扩增产物进行测序,以判断待测的DNA样本是否携带c.14343+5G>A突变。
Claims (10)
1.一种多肽,其特征在于,所述多肽为多肽1,所述多肽1的氨基酸序列如SEQ IDNO:13所示;或者,所述多肽为多肽2,所述多肽2的氨基酸序列如SEQ ID NO:14所示。
2.一种多肽组合,其特征在于,所述多肽组合包含如权利要求1所述多肽中的多肽1和多肽2。
3.一种分离的核酸,其特征在于,所述的核酸编码如权利要求1所述的多肽或如权利要求2所述的多肽组合。
4.如权利要求3所述的核酸,其特征在于,所述核酸的核苷酸序列包括:与人基因组GRCh38上1号染色体NC_000001.11的第215622891~216423448位碱基所对应的人的野生型USH2A基因的序列相比,具有g.215650587G>A突变的核苷酸序列;和/或,所述核酸为DNA或者RNA。
5.一种用于检测如权利要求1所述的多肽、如权利要求2所述的多肽组合或如权利要求3或4所述的核酸的检测剂,其特征在于,所述检测剂包括与所述多肽、所述多肽组合或所述核酸特异性杂交的生物分子,所述生物分子例如引物、探针和/或抗体;或,所述检测剂包括用于检测如权利要求1所述的多肽、如权利要求2所述的多肽组合或如权利要求3或4所述的核酸的基因组、转录组和/或蛋白质组测序的试剂;
较佳地,所述检测剂为引物,所述引物具有如SEQ ID NO:15和SEQ ID NO:16所示的核苷酸序列。
6.一种构建体,其特征在于,所述构建体包含如权利要求3或4所述的核酸;
较佳地,所述构建体的质粒骨架为pcMINI或pcMINI-C。
7.一种重组细胞,其特征在于,所述重组细胞是通过将如权利要求6所述的构建体转化受体细胞而获得的;
较佳地,所述受体细胞不为动物生殖细胞、受精卵或者胚胎干细胞;
更佳地,所述受体细胞为HeLa细胞或293T细胞。
8.一种试剂盒,其特征在于,所述试剂盒包含如权利要求1所述的多肽、如权利要求2所述的多肽组合、如权利要求3或4所述的核酸、如权利要求5所述的检测剂、如权利要求6所述的构建体和如权利要求7所述的重组细胞中的一种或多种。
9.如权利要求1所述的多肽、如权利要求2所述的多肽组合、如权利要求3或4所述的核酸、如权利要求5所述的检测剂、如权利要求6所述的构建体、如权利要求7所述的重组细胞和如权利要求8所述的试剂盒中的一种或多种在制备诊断和/或预测Usher综合征2A型的药物中的应用;
所述Usher综合征2A型优选为USH2A基因突变导致的Usher综合征2A型,进一步优选为USH2A基因的Exon66中产生提前终止密码子和/或USH2A基因Exon65整体缺失导致的Usher综合征2A型。
10.一种检测人的USH2A基因突变体的方法,其特征在于,所述方法包括使用如权利要求1所述的多肽、如权利要求2所述的多肽组合、如权利要求3或4所述的核酸、如权利要求5所述的检测剂、如权利要求6所述的构建体、如权利要求7所述的重组细胞和如权利要求8所述的试剂盒中的一种或多种检测待测样本;
优选地,所述检测为非诊断和/或治疗目的的。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310223969.4A CN116496377A (zh) | 2023-03-09 | 2023-03-09 | Ush2a基因突变的多肽、核酸及其应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310223969.4A CN116496377A (zh) | 2023-03-09 | 2023-03-09 | Ush2a基因突变的多肽、核酸及其应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN116496377A true CN116496377A (zh) | 2023-07-28 |
Family
ID=87317330
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202310223969.4A Pending CN116496377A (zh) | 2023-03-09 | 2023-03-09 | Ush2a基因突变的多肽、核酸及其应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN116496377A (zh) |
-
2023
- 2023-03-09 CN CN202310223969.4A patent/CN116496377A/zh active Pending
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20180346981A1 (en) | Panel-based Genetic Diagnostic Testing for Inherited Eye Diseases | |
CN104745592B (zh) | Cyp4v2基因突变体及其应用 | |
CN116083560B (zh) | 一种用于着床前胚胎nf1基因检测的试剂盒、系统及其应用方法 | |
CN116926185A (zh) | 常染色体显性多囊肾病致病基因突变检测 | |
CN115725721A (zh) | 用于检测长qt综合征致病基因kcnq1和kcnh2的试剂及其应用 | |
CN110541028A (zh) | 一种检测fus基因突变和tardbp基因突变的方法 | |
CN116496377A (zh) | Ush2a基因突变的多肽、核酸及其应用 | |
CN114774549A (zh) | 一种靶向检测cah候选基因的二代测序试剂盒 | |
CN104745591B (zh) | Cyp4v2基因突变体及其应用 | |
CN116496378A (zh) | Poc1b基因突变的多肽、核酸及其应用 | |
CN108486230B (zh) | 用于无创检测mitf基因突变的试剂盒及其制备方法 | |
CN116217695A (zh) | 多肽、多肽组合、核酸、核酸组合、检测剂及应用 | |
CN116042635A (zh) | 一种分离的编码abca4突变体的核酸、检测剂及应用 | |
CN115820654A (zh) | Loxhd1基因突变体及其应用 | |
CN114381513B (zh) | 一种与x连锁淋巴增生症相关的生物标志物及其应用 | |
CN117487907B (zh) | Kcnh2基因突变体、突变体蛋白、试剂、试剂盒及应用 | |
CN117471107B (zh) | 检测先天性无毛症的hr突变基因、蛋白、试剂、试剂盒及应用 | |
CN115851749A (zh) | 马凡综合征变异基因fbn1及其应用 | |
CN110452910B (zh) | 一种fus突变基因、检测引物和试剂盒 | |
CN115851899B (zh) | 一种3m综合征致病基因cul7复合杂合突变位点的应用及其诊断试剂 | |
CN117487923B (zh) | Habp2基因突变体作为检测靶点的应用、具有其的检测试剂和/或检测试剂盒 | |
CN117535402B (zh) | Frmpd4基因突变体作为检测靶点的应用、具有其的检测试剂及检测试剂盒 | |
CN108531583B (zh) | 用于无创检测mitf基因突变的引物组合及检测方法 | |
CN117757798A (zh) | 一种遗传性小头畸形致病突变基因及其应用 | |
CN115806998A (zh) | 肺动脉高压变异基因notch3及其应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination |