CN109943569A - The nucleic acid of the coding IFNLR1 mutant of separation and its application - Google Patents

Abstract

The present invention relates to gene mutation body and its applications.In particular it relates to detect kit, construct and the recombinant cell of the coding IFNLR1 mutant nucleic acid of purposes and separation in the biological sample that preparation screening is susceptible to suffer from nonsyndromic autosomal dominant deafness disease of the reagent in mutational site, the polypeptide of separation, the system of the biological sample of screening nonsyndromic autosomal dominant deafness, biological sample for screening nonsyndromic autosomal dominant deafness.Wherein, for the isolated coding IFNLR1 mutant nucleic acid compared with wild type IFNLR1 gene, the nucleic acid has c.296G > A mutation.It whether there is by detecting the new mutant in the biological sample, can be effectively detected whether biological sample is susceptible to suffer from ADNSHL disease.

Description

The nucleic acid of the coding IFNLR1 mutant of separation and its application

Technical field

The present invention relates to gene mutation body and its applications.In particular it relates to isolated coding IFNLR1 mutant Nucleic acid and its application, be susceptible to suffer from more particularly, to the nucleic acid of the coding IFNLR1 mutant of separation, the polypeptide of separation, screening The system of the biological sample of nonsyndromic autosomal dominant deafness is susceptible to suffer from nonsyndromic and often dyes for screening Kit, construct, recombinant cell and the method for constructing medicaments sifting model of the biological sample of body dominant heredity deafness.

Background technique

Non-syndromic cleft lip and palate is one of hereditary hearing impairment, refers to that deaf is the unique symptom of Affected individuals, without it Its genetic damage sexual organ dysfunction, accounts for 70% or so in hereditary hearing impairment.According to the difference of mode of inheritance, usually by it Be divided into autosomal dominant (autosomal dominant, DFNA), autosomal recessive (autosomal recessive, DFNB), sex chromosome chain (sex-linked) and mitochondrial inheritance (mitochondrial；maternally Inherited) deaf.

It is identical as other genetic diseases, nonsyndromic autosomal dominant deafness (autosomal Dominant non-syndromic hearing loss, ADNSHL) it is mostly familial disease.In terms of clinical phenotypes, multilist It is now tardy onset, but patient's onset age is different, age of onset focuses mostly in some year age grade in the same large family Section, deaf degree are in gradual exacerbation, final up to severe or pole profound sensorineural hearing loss or even complete deafness.Due to learning Just there is deafness in the language later period, so patient's speech function multipotency normally forms, but as auditory function declines, may result in Part speech function is lost.The characteristics of hearing loss, is also less identical, and most ADNSHL patients, which first appear as, to be originated high frequency and listen Power loss, small number of patients have oneself special phenotype.Since phenotype and genotype have heterogeneity to a certain extent, so Characteristic phenotype often facilitates us and makes reasonable anticipation, thus it is speculated that the genotype of patient.

There are 36 genes to be reported at present related to ADNSHL, but there are still quite a few unknown Disease-causing gene positions Point.The early diagnosis in relation to ADNSHL is up for further studying as a result,.

Summary of the invention

The present invention is directed at least solve one of the technical problems existing in the prior art.For this purpose, one object of the present invention It is to propose that one kind is capable of the biological sample of Effective selection nonsyndromic autosomal dominant deaf (ADNSHL) Method.

It should be noted that the present invention has been determined by means of the method for exon sequencing joint candidate gene mutation verifying The new mutation of nonsyndromic autosomal dominant deaf gene, specifically the present invention is based on the following of inventor Work and complete:

The present invention is collected into the Chinese han population hereditary hearing impairment family of a continuous 3 generation morbidity, has chosen 6 in family A sample obtains the variation data of sample by full exon sequencing technologies.Meanwhile using genetic marker to 15 in family A sample carries out Linkage mapping and haplotype analysis, and the Disease-causing gene of the family is located in the area chr1:p34.1-1p36.12 Between.In conjunction with the sequencing of full exon and linkage analysis as a result, having found on gene IFNLR1 a heterozygosis in the localization region Missense mutation: c.296G > A (p.Arg99His).Then it is verified by PCR-Sanger PCR sequencing PCR, IFNLR1c.296G > A (p.Arg99His) there is the phenomenon that phenotype-genotype isolates in all members of family studied, illustrate the variation It is possible for the pathogenic variation of this research.

Due to IFNLR1 express interferon lambda receptor 1 (Interferon lambda receptor 1), with cell factor and Interferon is related in the identification of extracellular environment and Cellular Signaling Transduction Mediated etc..In rodent, IFNLR1 is revolved in interior ear snail Device (organ of Corti), cochlea nerve, spiral ganglion and inner ear vestibular epithelium in wide expression.So inventor is logical It crosses to the zebra fish for knocking out IFNLR1 gene the study found that the normal development of IFNLR1 and hair cell and air bladder and function have emphatically The effect wanted.Therefore, IFNLR1 gene may be related to auditory nerve system, and the mutation on the gene has with ADNSHL It closes.Full exon sequencing technologies are proven the rare single-gene disorder candidate gene of reduction or even find having for its Disease-causing gene Power, effective means are only sequenced to sieve by the full exon to several seldom individuals (including patient and normal control) Variation relevant to disease is selected, success rate significantly increases.

Thus, according to the first aspect of the invention, the present invention provides a kind of cores of the coding IFNLR1 mutant of separation Acid.According to an embodiment of the invention, the nucleic acid at least has c.296G > A mutation compared with wild type IFNLR1 gene.Hair Bright people has surprisingly found that the morbidity of the mutant and ADNSHL are closely related, thus by detecting the mutant in biological sample In whether there is, can be effectively detected whether biological sample is susceptible to suffer from ADNSHL, according to an embodiment of the invention, the mutant Nucleic acid further enriches the pathogenic mutation map of IFNLR1 gene, deeper into the Molecular pathogenesis of ADNSHL is illustrated, is The early stage Disease-causing gene screening of ADNSHL and therapeutic intervention provide scientific basis.

According to the second aspect of the invention, the present invention also provides a kind of isolated polypeptides.According to an embodiment of the invention, The isolated polypeptide at least has p.Arg99His mutation.In the present invention, it indicates to be mutated using representation generally in the art. P.Arg99His indicates that the 99th amino acids Arg of protein level becomes His.It is more by whether expressing this in detection biological sample Peptide, can be effectively detected whether biological sample is susceptible to suffer from ADNSHL disease.

According to the third aspect of the invention we, the present invention provides a kind of reagents for detecting mutational site is susceptible to suffer from preparation screening Purposes in the biological sample of nonsyndromic autosomal dominant deafness disease.According to an embodiment of the invention, described Mutational site includes c.296G > A mutation on IFNLR1 gene.In the present invention, it indicates to be mutated using representation generally in the art. C.296G > A mutation indicates that the 296th nucleotide of cDNA becomes A by G.Inventor has surprisingly found that, the 296th nucleotide of cDNA A is become by G, the morbidity of the mutational site and ADNSHL are closely related, thus by detecting the mutational site in the biological sample It whether there is, can be effectively detected whether biological sample is susceptible to suffer from ADNSHL, according to an embodiment of the invention, the core of the mutant Acid further enriches the pathogenic mutation map of IFNLR1 gene The early stage Disease-causing gene screening of ADNSHL and therapeutic intervention provide scientific basis.

According to a particular embodiment of the invention, the reagent in the above detection mutational site is susceptible to suffer from nonsyndromic in preparation screening Purposes in the biological sample of autosomal dominant deafness disease, further comprises following additional technical feature:

According to some embodiments of the present invention, the mutational site includes in the polypeptide of IFNLR1 gene coding P.Arg99His mutation.

According to some embodiments of the present invention, the reagent includes nucleic acid probe or primer.

According to some embodiments of the present invention, the nucleic acid probe or primer have SEQ ID NO:3 and SEQ ID NO:4 Shown in nucleotide sequence.

According to some embodiments of the present invention, the reagent includes the nucleic acid of the coding IFNLR1 mutant of separation, with open country Raw type IFNLR1 gene is compared, and the nucleic acid at least has c.296G > A mutation.

According to some embodiments of the present invention, the nucleic acid is DNA.

According to some embodiments of the present invention, the reagent includes isolated polypeptide, and the isolated polypeptide at least has P.Arg99His mutation.

According to some embodiments of the present invention, the polypeptide by separate coding IFNLR1 mutant nucleic acid encode.

According to the fourth aspect of the invention, it is easy in preparation screening that the present invention also provides a kind of reagents for detecting mutational site Suffer from the purposes in the system of the biological sample of nonsyndromic autosomal dominant deafness disease.Implementation according to the present invention Example, the mutational site include c.296G > A mutation on IFNLR1 gene, the system comprises: nucleic acid sequence determining device, institute It states nucleic acid sequence determining device to be connected with the nucleic acid-extracting apparatus, for analyzing the sample of nucleic acid, to determine The nucleic acid sequence of the sample of nucleic acid；Judgment means, the judgment means are connected with the nucleic acid sequence determining device, so as to base In the nucleic acid sequence or its complementary series of the sample of nucleic acid, there is c.296G > A mutation compared with wild type IFNLR1 gene. It is surprisingly found by the inventors that ADNSHL disease can be carried out the detection of gene level using the system, help more accurately to sieve Choosing is susceptible to suffer from the biological sample of ADNSHL disease.

According to the fifth aspect of the invention, the present invention also provides one kind is susceptible to suffer from nonsyndromic autosome for screening The kit of the biological sample of dominant heredity deafness disease.According to an embodiment of the invention, the kit contains: being adapted to detect for The reagent of IFNLR1 gene mutation body, wherein the IFNLR1 gene mutation body has compared with wild type IFNLR1 gene C.296G > A mutation.The kit is utilized as a result, high-precision detection can be carried out to IFNLR1 gene mutation body, thus right ADNSHL disease carries out the detection of gene level, helps more accurately to screen the biological sample for being susceptible to suffer from ADNSHL disease.

According to the sixth aspect of the invention, the present invention also provides a kind of constructs.According to an embodiment of the invention, the structure Build the nucleic acid that body includes the coding IFNLR1 mutant of separation above-mentioned.It is thin using construct transformation receptor of the invention as a result, The recombinant cell that born of the same parents obtain can be efficiently used for the drug of screening treatment ADNSHL disease.

According to the seventh aspect of the invention, the present invention also provides a kind of recombinant cells.According to an embodiment of the invention, should Recombinant cell is obtained by expressing construct transformed acceptor cell above-mentioned.According to some embodiments of the present invention, sharp With recombinant cell of the invention, the drug for the treatment of ADNSHL disease can be effectively screened.

The beneficial effect that the present invention obtains is: the present invention passes through full sequencing of extron group and Linkage mapping and biological information point May there is c.296G > A mutation with non-comprehensive autosomal dominant deaf (ADNSHL) on analysis method discovery IFNLR1 gene It closes, and is confirmed by the science of heredity that phenotype-genotype in family isolates experiment and large sample size normal person group This variation of causing a disease on IFNLR1 gene, while IFNLR1 gene and sense of hearing system are found by mouse model and zebra fish model The association of system.This discovery is by the clinical diagnosis of Widening genetic deafness and detection/screening range, it will is ADNSHL pathogenesis Research establish important foundation, it is also possible to for ADNSHL patient treatment completely new theoretical foundation is provided, thus to ADNSHL suffer from The clinical diagnosis of person provides more supports and reference.In addition, the clone of new ADNSHL Disease-causing gene, in conjunction with heredity or apparently The adjusting gene expression of genetic modification factor and the corresponding animal model of building will provide important for the pathogenesis of announcement ADNSHL Clue furthers elucidate the pathomechanism of human auditory system's exception.

Additional aspect and advantage of the invention will be set forth in part in the description, and will partially become from the following description Obviously, or practice through the invention is recognized.

Detailed description of the invention

Above-mentioned and/or additional aspect of the invention and advantage will become from the description of the embodiment in conjunction with the following figures Obviously and it is readily appreciated that, in which:

Fig. 1: show it is according to an embodiment of the invention screen be susceptible to suffer from ADNSHL disease biological sample system and The schematic diagram of its component part, in which:

A is the schematic diagram that the system of biological sample of ADNSHL disease is susceptible to suffer from according to the screening of the embodiment of the present invention,

B is the schematic diagram according to the nucleic acid-extracting apparatus of the embodiment of the present invention,

C is the schematic diagram according to the nucleic acid sequence determining device of the embodiment of the present invention.

Fig. 2: it shows according to one embodiment of present invention, the pedigree chart of 3 familys containing ADNSHL disease patient.

Fig. 3 shows IFNLR1 genetic homology comparative analysis figure according to an embodiment of the invention.

Fig. 4 shows Sanger verifying peak figure according to an embodiment of the invention.

Fig. 5 shows the pure tone test figure of ADNSHL disease patient according to an embodiment of the invention.

Specific embodiment

The embodiment of the present invention is described below in detail.The embodiments described below is exemplary, it is intended to for explaining this Invention, and be not considered as limiting the invention.

IFNLR1 mutant

According to an aspect of the present invention, the invention proposes a kind of nucleic acid of the coding IFNLR1 mutant of separation.According to The embodiment of the present invention, compared with wild type IFNLR1 gene, the nucleic acid at least has c.296G > A mutation.

It should be noted that isolated nucleic acid encode IFNLR1 mutant of the invention, is referred to as " coding IFNLR1 The nucleic acid of mutant ", the i.e. nucleic acid can be understood as nucleic acid substances corresponding with the coding gene of IFNLR1 mutant, i.e. core The type of acid is not particularly limited, and can be any comprising deoxyribonucleotide corresponding with the encoding gene of IFNLR1 And/or the polymer of ribonucleotide, including but not limited to DNA, RNA or cDNA.A specific example according to the present invention, it is preceding The nucleic acid that IFNLR1 mutant is encoded described in face is DNA.According to an embodiment of the invention, inventor has determined IFNLR1 gene New mutant, these mutant and the morbidity of ADNSHL disease are closely related, thus by detect the mutant in biological sample In whether there is, the whether susceptible ADNSHL disease of biological sample can be effectively detected, can also be existed by detecting these mutant It whether there is in organism, can effectively predict whether organism is susceptible to suffer from ADNSHL disease.

For referring to nucleic acid in description of the invention and claims, it will be appreciated by those skilled in the art that practical packet Include any one or two of complementary double-strand.For convenience, in the present specification and claims, although most cases Under only give a chain, but actually also disclose another chain complementary to it.For example, refer to SEQ ID NO:1, it is practical Including its complementary series.Those skilled in the art, which are further appreciated that, can detecte another chain using a chain, and vice versa.

The nucleic acid of these coding IFNLR1 gene mutation bodies is that present inventor passes through high-throughput sequencing of extron group The method of joint candidate gene mutation verifying, the new mutation on the Disease-causing gene of determining ADNSHL disease, and in the prior art In and have not seen above-mentioned pathogenic mutation report relevant to ADNSHL disease.

The IFNLR1 gene mutation body of inventor's discovery has c.296G > A mutation compared with wild type IFNLR1 gene, I.e. the 296th bit base G sports A.

The network address of the IFNLR1 gene nucleic acid sequence are as follows:http://grch37.ensembl.org/Homo_ Sapiens/Transcript/Summary? db=core；G=ENSG00000185436；R=1:24483620- 24513738；T=ENST00000374421,

IFNLR1 gene has 7 exons, and mutation c.296G > A is located on third exon, leads to three exons 99 arginine sport histidine.

The IFNLR1 gene nucleic acid sequence of corresponding wild type is SEQ ID NO:1:

ATGGCGGGGCCCGAGCGCTGGGGCCCCCTGCTCCTGTGCCTGCTGCAGGCCGCTCCAGGGAGGCCCCGT CTGGCCCCTCCCCAGAATGTGACGCTGCTCTCCCAGAACTTCAGCGTGTACCTGACATGGCTCCCAGGGCTTGGCAA CCCCCAGGATGTGACCTATTTTGTGGCCTATCAGAGCTCTCCCACCCGTAGACGGTGGCGCGAAGTGGAAGAGTGTG CGGGAACCAAGGAGCTGCTATGTTCTATGATGTGCCTGAAGAAACAGGACCTGTACAACAAGTTCAAGGGACGCGTG CGGACGGTTTCTCCCAGCTCCAAGTCCCCCTGGGTGGAGTCCGAATACCTGGATTACCTTTTTGAAGTGGAGCCGGC CCCACCTGTCCTGGTGCTCACCCAGACGGAGGAGATCCTGAGTGCCAATGCCACGTACCAGCTGCCCCCCTGCATGC CCCCACTGGATCTGAAGTATGAGGTGGCATTCTGGAAGGAGGGGGCCGGAAACAAGACCCTATTTCCAGTCACTCCC CATGGCCAGCCAGTCCAGATCACTCTCCAGCCAGCTGCCAGCGAACACCACTGCCTCAGTGCCAGAACCATCTACAC GTTCAGTGTCCCGAAATACAGCAAGTTCTCTAAGCCCACCTGCTTCTTGCTGGAGGTCCCAGAAGCCAACTGGGCTT TCCTGGTGCTGCCATCGCTTCTGATACTGCTGTTAGTAATTGCCGCAGGGGGTGTGATCTGGAAGACCCTCATGGGG AACCCCTGGTTTCAGCGGGCAAAGATGCCACGGGCCCTGGAACTGACCAGAGGGGTCAGGCCGACGCCTCGAGTCAG GGCCCCAGCCACCCAACAGACAAGATGGAAGAAGGACCTTGCAGAGGACGAAGAGGAGGAGGATGAGGAGGACACAG AAGATGGCGTCAGCTTCCAGCCCTACATTGAACCACCTTCTTTCCTGGGGCAAGAGCACCAGGCTCCAGGGCACTCG GAGGCTGGTGGGGTGGACTCAGGGAGGCCCAGGGCTCCTCTGGTCCCAAGCGAAGGCTCCTCTGCTTGGGATTCTTC AGACAGAAGCTGGGCCAGCACTGTGGACTCCTCCTGGGACAGGGCTGGGTCCTCTGGCTATTTGGCTGAGAAGGGGC CAGGCCAAGGGCCGGGTGGGGATGGGCACCAAGAATCTCTCCCACCACCTGAATTCTCCAAGGACTCGGGTTTCCTG GAAGAGCTCCCAGAAGATAACCTCTCCTCCTGGGCCACCTGGGGCACCTTACCACCGGAGCCGAATCTGGTCCCTGG GGGACCCCCAGTTTCTCTTCAGACACTGACCTTCTGCTGGGAAAGCAGCCCTGAGGAGGAAGAGGAGGCGAGGGAAT CAGAAATTGAGGACAGCGATGCGGGCAGCTGGGGGGCTGAGAGCACCCAGAGGACCGAGGACAGGGGCCGGACATTG GGGCATTACATGGCCAGGTGA

The polypeptide of the wild type encoded accordingly is that SEQ ID NO:2, SEQ ID NO:2 sequence are as follows:

MAGPERWGPLLLCLLQAAPGRPRLAPPQNVTLLSQNFSVYLTWLPGLGNPQDVTYFVAYQSSPTRRRWR EVEECAGTKELLCSMMCLKKQDLYNKFKGRVRTVSPSSKSPWVESEYLDYLFEVEPAPPVLVLTQTEEILSANATYQ LPPCMPPLDLKYEVAFWKEGAGNKTLFPVTPHGQPVQITLQPAASEHHCLSARTIYTFSVPKYSKFSKPTCFLLEVP EANWAFLVLPSLLILLLVIAAGGVIWKTLMGNPWFQRAKMPRALELTRGVRPTPRVRAPATQQTRWKKDLAEDEEEE DEEDTEDGVSFQPYIEPPSFLGQEHQAPGHSEAGGVDSGRPRAPLVPSEGSSAWDSSDRSWASTVDSSWDRAGSSGY LAEKGPGQGPGGDGHQESLPPPEFSKDSGFLEELPEDNLSSWATWGTLPPEPNLVPGGPPVSLQTLTFCWESSPEEE EEARESEIEDSDAGSWGAESTQRTEDRGRTLGHYMAR

The reagent for detecting mutational site is susceptible to suffer from nonsyndromic autosomal dominant deafness disease in preparation screening Purposes in biological sample

According to another aspect of the invention, the invention proposes a kind of reagents for detecting mutational site is susceptible to suffer from preparation screening Purposes in the biological sample of nonsyndromic autosomal dominant deafness disease, the mutational site include IFNLR1 base Because it is upper c.296G > A mutation.Inventor has found the close phase of morbidity in c.296G > mutational site A and ADNSHL disease on IFNLR1 gene It closes, to whether there is in the biological sample by detecting the mutational site, it is whether susceptible that biological sample can be effectively detected ADNSHL disease can also be whether there is in organism by detecting these mutant, effectively whether can predict organism It is susceptible to suffer from ADNSHL disease.

According to some embodiments of the present invention, mutational site include in the polypeptide of IFNLR1 gene coding p.Arg99His it is prominent Become.

According to some embodiments of the present invention, the reagent includes nucleic acid probe or primer, the nucleic acid probe or primer With nucleotide sequence shown in SEQ ID NO:3 and SEQ ID NO:4.

According to some embodiments of the present invention, the reagent includes the nucleic acid of the coding IFNLR1 mutant of separation, with open country Raw type IFNLR1 gene is compared, and the nucleic acid at least has c.296G > A mutation.Wherein in a preferred embodiment, described Nucleic acid is DNA.

According to some embodiments of the present invention, the reagent includes isolated polypeptide, and the isolated polypeptide at least has P.Arg99His mutation；Wherein in the preferred embodiment of the present invention, the polypeptide is dashed forward by isolated coding IFNLR1 The nucleic acid encode of variant.

Screen the system and kit of the biological sample of susceptible nonsyndromic autosomal dominant deafness

According to another aspect of the present invention, it can effectively implement above-mentioned screening the invention proposes one kind and be susceptible to suffer from ADNSHL disease Biological sample method system.

With reference to Fig. 1, according to an embodiment of the invention, the system 1000 that the screening is susceptible to suffer from the biological sample of ADNSHL disease is wrapped It includes: nucleic acid-extracting apparatus 100, nucleic acid sequence determining device 200 and judgment means 300.

According to an embodiment of the invention, nucleic acid-extracting apparatus 100 is used for from extraction from biological material sample of nucleic acid.Such as preceding institute It states, according to an embodiment of the invention, the type of sample of nucleic acid is not particularly restricted, for using RNA as sample of nucleic acid, then Nucleic acid-extracting apparatus further comprises RNA extraction unit 101 and reverse transcription unit 102, wherein extraction unit 101 is used for from life Object sample extraction RNA sample, reverse transcription unit 102 are connected with RNA extraction unit 101, anti-for carrying out reverse transcription to RNA sample It answers, to obtain cDNA sample, obtained cDNA sample constitutes sample of nucleic acid.

According to an embodiment of the invention, nucleic acid sequence determining device 200 is connected with nucleic acid-extracting apparatus 100, for core Acid sample is analyzed, to determine the nucleic acid sequence of sample of nucleic acid.As previously shown, nucleic acid can be determined using the method for sequencing The nucleic acid sequence of sample.As a result, according to one embodiment of present invention, the nucleic acid sequence determining device 200 can be further It include: library construction unit 201 and sequencing unit 202.Library construction unit 201 is used to be directed to sample of nucleic acid, constructs nucleic acid Sequencing library；Sequencing unit 202 is connected with library construction unit 201, for nucleic acid sequencing library to be sequenced, to obtain The sequencing result being made of multiple sequencing datas.As previously mentioned, IFNLR1 gene extron can be enriched with by PCR amplification, into One step improves the efficiency of the biological sample of screening nonsyndromic autosomal dominant deafness.Library construction list as a result, Member 201 may further include PCR amplification module (not shown), be provided in the PCR amplification module selected from IFNLR1 At least one of gene extron specific primer, to utilize at least one of IFNLR1 gene extron specific primer, PCR amplification is carried out to the sample of nucleic acid.According to an embodiment of the invention, the sequence of IFNLR1 gene extron specific primer It is not particularly limited, such as human gene data unit sequence library GRCh37.1/hg19 can be referred to, it is online using Primer3.0 Design obtains.Preferred embodiment in accordance with the present invention, the IFNLR1 gene extron specific primer have such as SEQ ID Nucleotide sequence shown in NO:3 and SEQ ID NO:4.According to an embodiment of the invention, sequencing unit 202 may include being selected from HISEQ2000, SOLiD, 454 and single-molecule sequencing device at least one.Newest sequencing technologies are combined as a result, for list A site can achieve higher sequencing depth, and detection sensitivity and accuracy greatly improve, it is thus possible to utilize these sequencings The characteristics of high throughput of device, deep sequencing, further increase the efficiency tested and analyzed to sample of nucleic acid.To improve Subsequent accuracy and accuracy when analyzing sequencing data.

According to an embodiment of the invention, judgment means 300 are connected with nucleic acid sequence determining device 200, it is suitable for nucleic acid sample This nucleic acid sequence is compared, so that the nucleic acid sequence based on sample of nucleic acid is sentenced with the difference of corresponding wildtype gene sequence Whether disconnected biological sample is susceptible to suffer from ADNSHL disease.Specifically, nucleic acid sequence or its complementary series based on the sample of nucleic acid, with open country Raw type IFNLR1 gene, which is compared, has c.296G > A mutation, for judging whether the biological sample is susceptible to suffer from ADNSHL disease.As before It is described, according to an embodiment of the invention, the equipment that nucleic acid sequence is compared with corresponding wildtype gene sequence not by Especially limitation can be operated using the software of any conventional, such as specific example according to the present invention, can be used SOAPALIGNER/SOAP2 is compared.

The system is utilized as a result, can effectively implement the method for the biological sample of aforementioned screening ADNSHL disease, so as to Effectively to screen the biological sample for being susceptible to suffer from ADNSHL disease.

According to another aspect of the invention, the invention proposes a kind of for screening the biological sample for being susceptible to suffer from ADNSHL disease Kit.According to an embodiment of the invention, the kit for being used to screen the biological sample for being susceptible to suffer from ADNSHL disease includes: to be suitable for inspection The reagent for surveying IFNLR1 gene mutation body, wherein the IFNLR1 gene mutation body has compared with wild type IFNLR1 gene C.296G > A mutation.Using the kit of embodiment according to the present invention, the biology for being susceptible to suffer from ADNSHL disease can be effectively screened Sample.Herein, used term " reagent for being adapted to detect for IFNLR1 gene mutation body " shall be understood in a broad sense, it can It is the reagent for detecting IFNLR1 mutant code gene, is also possible to detect at least one examination of IFNLR1 protein mutant Agent, such as can be using the antibody of identification specific position.According to one embodiment of present invention, which is nucleic acid probe. Thus, it is possible to which efficiently screening is susceptible to suffer from the biological sample of ADNSHL disease.

Construct and recombinant cell

According to another aspect of the invention, the invention also provides a kind of constructs.According to an embodiment of the invention, the structure Building body includes mentioned-above isolated nucleic acid.It should be noted that " construct includes mentioned-above isolated nucleic acid " table Show, construct of the invention includes to have c.296G > A mutation IFNLR1 gene mutation body compared with wild type IFNLR1 gene Nucleic acid sequence.The recombinant cell that construct transformed acceptor cell of the invention obtains as a result, can effectively serve as non-synthesis The model of sign type autosomal dominant deafness correlative study.Wherein, the type of the recipient cell is not particularly limited, Such as can be Bacillus coli cells, mammalian cell, preferably this receptor cell origin is in mammal.

Term " construct " as used in the present invention refers to a kind of such genetic carrier, and it includes specific nucleic acid sequences Column, and purpose nucleic acid sequence can be transferred in host cell, to obtain recombinant cell.According to an embodiment of the invention, structure The form for building body is not particularly limited.According to an embodiment of the invention, it can be plasmid, bacteriophage, artificial chromosome, clay (Cosmid), viral at least one, preferred plasmid.Plasmid has easy to operate as genetic carrier, can carry larger piece The property of section, convenient for operating and handling.The form of plasmid is also not particularly limited, either circular plasmids, are also possible to line Property grain, it can be it is single-stranded, be also possible to double-strand.Those skilled in the art, which can according to need, to be selected.At this Term used in invention " nucleic acid " can be any polymer comprising deoxyribonucleotide or ribonucleotide, packet It includes but is not limited to by modification or unmodified DNA, RNA, length is not any particular limitation.For for constructing The construct of recombinant cell, the preferably described nucleic acid is DNA, more stable because DNA is for RNA, and is easy to grasp Make.

According to another aspect of the present invention, the invention also provides a kind of recombinant cells.According to an embodiment of the invention, should Recombinant cell is obtained by mentioned-above construct transformed acceptor cell.To which recombinant cell of the invention can IFNLR1 gene mutation body entrained by effective expression construct.According to some embodiments of the present invention, recombination of the invention is thin Born of the same parents can effectively serve as the model of ADNSHL disease correlative study.According to an embodiment of the invention, the type of recipient cell not by Especially limitation, such as can be Bacillus coli cells, mammalian cell, the preferably described recipient cell is dynamic from inhuman lactation Object.

It should be noted that described in the method part for the biological sample that screening is susceptible to suffer from ADNSHL disease herein above Feature and advantage are equally applicable to system or kit that screening is susceptible to suffer from the biological sample of ADNSHL disease, and details are not described herein.

The method for constructing medicaments sifting model

According to another aspect of the invention, the present invention also provides a kind of methods for constructing medicaments sifting model.According to this The embodiment of invention, this method comprises: at least part cell of animal is made to express polypeptide above-mentioned.Implementation according to the present invention Example, the animal are mouse, pig, dog, primate, zebra fish.According to some embodiments of the present invention, using of the invention Animal model can effectively screen the drug for the treatment of ADNSHL disease.

Wherein, it should be noted that the method that the present invention constructs medicaments sifting model is not particularly limited, as long as making At least part cell of object expresses polypeptide above-mentioned.For example, the method using genetic transformation can be used, it will be noted earlier Construct of the invention be transferred to receptor (inhuman), thus make up to animal at least part cell expression it is above-mentioned more Peptide.

The method for screening the biological sample of nonsyndromic autosomal dominant deafness

According to another aspect of the present invention, the side of the biological sample of ADNSHL disease is susceptible to suffer from the invention proposes a kind of screening Method.According to an embodiment of the invention, the method that the screening is susceptible to suffer from the biological sample of ADNSHL disease may comprise steps of:

Firstly, from extraction from biological material sample of nucleic acid.According to an embodiment of the invention, the type of biological sample is not by spy It does not limit, as long as reflection biological sample IFNLR1 gene can be extracted from the biological sample with the presence or absence of the nucleic acid sample of mutation This.According to an embodiment of the invention, biological sample can be at least one selected from blood of human body, skin, subcutaneous tissue. Thus, it is possible to easily be sampled and detect, so as to further increase the efficiency for the biological sample for screening ADNSHL disease. According to an embodiment of the invention, term " sample of nucleic acid " used herein above shall be understood in a broad sense, can be it is any can be anti- Reflecting IFNLR1 gene in biological sample whether there is the sample of mutation, such as can be the full base directly extracted from biological sample Because of a group DNA, it is also possible to a part in the full-length genome comprising IFNLR1 gene coded sequence, can be from biological sample The total serum IgE of extraction is also possible to the mRNA extracted from biological sample.According to one embodiment of present invention, the nucleic acid sample This is complete genome DNA.Thus, it is possible to expand the source range that comes of biological sample, and can be simultaneously to a variety of of biological sample Information is determined, so as to improve the efficiency for the biological sample for screening ADNSHL disease.In addition, implementation according to the present invention Example, as sample of nucleic acid, may further include from extraction from biological material sample of nucleic acid: mention from biological sample for using RNA RNA sample is taken, preferably RNA sample is mRNA；And it is based on obtained RNA sample, by reverse transcription reaction, obtain cDNA sample This, obtained cDNA sample constitutes sample of nucleic acid.It is screened thus, it is possible to further increase using RNA as sample of nucleic acid The efficiency of the biological sample of ADNSHL disease.

Next, can be analyzed sample of nucleic acid after obtaining sample of nucleic acid, so as to determine acquired core The nucleic acid sequence of acid sample.According to an embodiment of the invention, the method and apparatus of the nucleic acid sequence of sample of nucleic acid obtained by determining It is not particularly restricted.According to a particular embodiment of the invention, the nucleic acid sequence of sample of nucleic acid by sequencing approach, can be determined Column.According to an embodiment of the invention, the method and apparatus that can be used for being sequenced is not particularly restricted.It is according to the present invention Embodiment can use second generation sequencing technologies, can also be using the third generation and forth generation or more advanced sequencing technologies. Specific example according to the present invention can use at least one selected from Hiseq2000, SOLiD, 454 and single-molecule sequencing device Nucleic acid sequence is sequenced in kind.Newest sequencing technologies are combined as a result, and it is deep to can achieve higher sequencing for single locus Degree, detection sensitivity and accuracy greatly improve, it is thus possible to utilize the spy of the high throughput of these sequencing devices, deep sequencing Point further increases the efficiency tested and analyzed to sample of nucleic acid.Subsequent sequencing data is analyzed thus, it is possible to improve When accuracy and accuracy.As a result, according to an embodiment of the invention, determining that the nucleic acid sequence of sample of nucleic acid can be wrapped further It includes: firstly, constructing nucleic acid sequencing library for obtained sample of nucleic acid；And obtained nucleic acid sequencing library is carried out Sequencing, to obtain the sequencing result being made of multiple sequencing datas.According to some embodiments of the present invention, it can use and be selected from Obtained nucleic acid sequencing library is sequenced in Hiseq2000, SOLiD, 454 and at least one of single-molecule sequencing device. In addition, the screening is rich according to an embodiment of the invention, can screen to sample of nucleic acid, enrichment IFNLR1 gene extron Collection can carry out before constructing sequencing library, during constructing sequencing library, or after building sequencing library.According to this hair Bright one embodiment, for sample of nucleic acid, constructing nucleic acid sequencing library further comprises: special using IFNLR1 gene extron Specific primer carries out PCR amplification to sample of nucleic acid；And it is directed to obtained amplified production, construct nucleic acid sequencing library.By This, can be enriched with IFNLR1 gene extron by PCR amplification, so as to further increase the susceptible cone bar nutrition of screening not The efficiency of the biological sample of good disease.According to an embodiment of the invention, the sequence of IFNLR1 gene extron specific primer not by Especially limitation, such as human gene data unit sequence library GRCh37.1/hg19 can be referred to, using Primer3.0 Photographing On-line It obtains.Preferred embodiment in accordance with the present invention, the IFNLR1 gene extron specific primer have such as SEQ ID NO:3 and Nucleotide sequence shown in SEQ ID NO:4.It is surprisingly found by the inventors that by using SEQ ID NO:3 and SEQ ID NO:4 Shown in primer, can significantly be effectively completed in PCR reaction system to corresponding gene mutation where exon sequence expansion Increase.It should be noted that nucleotide sequence shown in SEQ ID NO:3 and SEQ ID NO:4 is that the present inventor is paying After having gone out arduous labor, surprisingly obtain.

About sample of nucleic acid is directed to, the method and process of sequencing library are constructed, those skilled in the art can be according to difference Sequencing technologies suitably selected, about the details of process, may refer to such as Illumina company, manufacturer of sequencing instrument Provided regulation, for example, see Illumina company Multiplexing Sample Preparation Guide (Part# 1005361；) or Paired-End SamplePrep Guide (Part#1005063 Feb2010；Feb 2010), by referring to It is incorporated into herein.According to an embodiment of the invention, from the method and apparatus of extraction from biological material sample of nucleic acid, also not by special Limitation can be carried out using the nucleic acid extraction kit of commercialization.

It should be noted that term " nucleic acid sequence " used herein should broadly understood, can be to core After the sequencing data that acid sample is sequenced is assembled, obtained complete nucleic acid sequence information is also possible to directly Using by be sequenced obtained sequencing data (reads) as nucleic acid sequence, as long as these nucleic acid sequences to sample of nucleic acid Coded sequence containing corresponding IFNLR1 gene in column.

Finally, after determining the nucleic acid sequence of sample of nucleic acid, the nucleic acid sequence of obtained sample of nucleic acid is corresponding Reference sequences are compared, and when having at least one of aforementioned each mutation in obtained nucleic acid sequence, that is, indicate biological sample Product are susceptible to suffer from ADNSHL disease.The method for being susceptible to suffer from the biological sample of ADNSHL disease is screened by according to an embodiment of the present invention as a result, it can Effectively to screen the biological sample for being susceptible to suffer from ADNSHL disease.According to an embodiment of the invention, to nucleic acid sequence and corresponding wild type The method and apparatus that gene order is compared is not particularly restricted, and can be operated using the software of any conventional, root According to specific example of the invention, can be compared using SOAPALIGNER/SOAP2.

It should be noted that the purposes of " method of the biological sample of screening ADNSHL disease " according to an embodiment of the present invention is not It is particularly limited, such as may be used as the screening technique of non-diagnostic purpose.

Below with reference to specific embodiment, the present invention will be described, it should be noted that these embodiments are only explanation Property, and be not considered as limiting the invention.Unless otherwise specified, technological means employed in embodiment is this field Conventional means known to technical staff, are referred to " Molecular Cloning:A Laboratory guide " third edition or Related product carries out, institute The reagent and product of use are also available commercial.The various processes and method being not described in detail are as known in the art Conventional method, the source of agents useful for same, trade name and it is necessary to list its constituent person are indicated on the first occurrence, Thereafter same reagents used unless otherwise specified, is the same as indicated for the first time.

The full sequencing of extron group of embodiment 1 determines Disease-causing gene and mutational site

1, sample collection

Inventor is collected into the Chinese han population hereditary hearing impairment family (pedigree chart is shown in Fig. 2) of a continuous 3 generation morbidity, should Family is by 24 member compositions, wherein patient diagnosed 10, existing cases 9 (diagnostic graph is shown in Fig. 3), collect obtain the family at Member peripheral blood sample, wherein the blood samples of patients sample being collected into be 9 (II:2, II:3, II:4, II:5, II:6, III:3, III:5, III:6, III:7)；Be collected into phenotype normal human blood proper manners sheet 14 (I:2, II:1, II:7, II:8, II:9, II:10, II:11, II:12, III:1, III:2, III:4, III:8, III:9, III:10).Each sample collection peripheral blood sample Product 2mL, addition EDTA is anticoagulant, -80 degrees Celsius of preservations.

Wherein, Fig. 2 shows family maps, wherein zero indicates that normal female, indicate normal male, ● indicate that women suffers from Person, ■ indicate male patient,Indicate dead male patient.

Pure tone test is the test auditory acuity, standardized subjective behavior reaction audiometry, it includes air conduction hearing threshold and bone conduction Threshold of audibility test.It is able to reflect the hearing level of the minimum sound for each frequency that subject can hear under quiet environment, is understood Hearing normally whether and hearing loss degree and property, and as diagnose and processing foundation.Some patientss are older, are Do pure tone test diagnosis.

The embodiment of the present invention have detected under different hertz for 9 patients in the family (II:2, II:3, II:4, II: 5, II:6, III:3, III:5, III:6, III:7) carry out pure tone test.Test result shows that these patient's Hearings are universal 10-20 years old is appeared in, and as the phenomenon that aggravating is presented in the increase at age, high-frequency balance is manifested initially by, with year The increase in age is eventually exhibited as spelling severe and pole severe deafness.With patient (II:3, II:4, II:5, II:6, III:5, III:6, III:7) for, test results are shown in figure 5.What wherein the abscissa of Fig. 5 represented is frequency, and unit is hertz (HZ), ordinate is to listen force value, and unit is (dB), from Fig. 5 test result it can be concluded that, under same frequency, same individual year Age is bigger, listens force value lower.

2, full exon trapping sequencing and analysis of variance

Inventor is based on 2000 high-flux sequence platform of Illumina Hiseq, using NimbleGen SeqCap EZ Exome Library V3.0 chip chooses the sample in family and carries out full exon sequencing and data analysis.

Because this is for autosomal dominant family, the selective rules of sample are as follows:

1) select the farther away diseased individuals of affiliation as case (research case)；

2) the normal former generation of Case is as control (control)；

3) preferential selection is with all akin control of two or more case or choosing and one of them The nearest control of case affiliation；

4) it avoids the normal brother and sister of the phenotype of case as control, avoids the normal filial generation conduct of the phenotype of case control；

5) 4-5 case (case number is more, in conjunction with combinatory analysis, can reduce false positive and false negative rate) or 3- is selected 4case+1control。

Based on the above standard, had chosen in experimentation in family 6 samples (II:1, II:4, III:4, III:5, III:6, III:7) carry out full exon sequencing and data analysis, specific practice are as follows:

2.1 sample preparation

The peripheral blood sample of above-mentioned 6 parts of samples (II:1, II:4, III:4, III:5, III:6, III:7) is taken respectively, is used OMEGA Blood DNA Midi Kit whole blood DNA extracts kit extracts genomic DNA, and utilizes spectrophotometer and gel Electrophoresis measure DNA concentration and purity, the OD260/OD280 of resulting each sample genomic DNA be respectively positioned on 1.7-2.0 it Between, concentration is no less than 200ng/ μ l, and total amount is no less than 30 μ g, spare.

2.2 library constructions and sequencing

Each genomic DNA sample is broken into 150-200bp at random using adaptive high focusing ultrasonic technique (Covaris) The segment of left and right, the operational manual then provided according to manufacturer, being separately connected top connection preparation library at segment both ends (can Build library specification referring to the Illumina/Solexa standard that: http://www.illumina.com/ is provided, by referring to by its It is incorporated by herein).It is tried after library is purified by the linear amplification of Ligation-mediated PCR (LM-PCR) and capture Agent NimbleGen SeqCap EZ Exome (64M) kit carries out hybridization enrichment, using the linear amplification of LM-PCR, library inspection Be available on the machine sequencing after survey is qualified, to obtain raw sequencing data.Wherein, referring to the cluster of Illumina standard and sequencing Protocol is sequenced, and microarray dataset is Illumina Genome Analyzer II, and reading length is 90bp, sample Average sequencing depth be 100 ×.

2.3 variation detections and screening

By above-mentioned sequencing output data successively carry out rough estimates analysis, SNP detection and annotation and amino acid substitution it is pre- It surveys, key step is as follows:

2.3.1 master data analysis statistics

The data that output is sequenced are subjected to master data analysis statistics: the sequence reads length analysis that measures, statistics The yield of reads quantity and data, reads sequence and the comparison with reference to genome sequence, statistics target area are compared to being wanted The coverage (Coverage) of the genome reads of reference and sequencing depth (Depth) etc..According to the statistics of the above master data As a result, obtaining the sample essential information by exon trapping, and judge whether data meet the requirements.

2.3.2SNP detection and Indel detection

The original reads of the high quality of each sample is passed through into SOAPaligner (v2.21) and Burrows-Wheeler Aligner (BWA, v0.7.10) compares software and compares on reference genome (hg19), then utilizes Genome Analysis Toolkit (GATK, v3.3-0) software detection SNP and Indel, using Variant Effect Predictor (VEP) to inspection The variation measured carries out variation annotation, at the same add the frequency databases such as 1KG/ESP/HapMap/ExAC and OMIM, GO, KEGG, The information such as harmfulness prediction.According to the illness rate of nonsyndromic autosomal dominant deaf (ADNSHL), filter out High frequency (1KG/ESP/HapMap/ExAC > 0.005) and the variation for including sub-district.Then, according to linkage analysis and family Hereditary Modules Formula filters out that patient in Linkage mapping region is shared and normal person does not have in family variation, finally filters out 10 candidates Variation.

2.3.3Sanger sequencing

This 10 candidate variations are verified using Sanger sequencing, the PCR primer of amplification mutation region uses Primer3 (http://frodo.wi.mit.edu/primer3/) design.The segment of amplification uses ABI3100 (Applied Biosystems, Foster City, CA) genetic analyzer, using ABI BigDye Terminator cycle Sequencing kit v3.1 (Applied Biosystems, Foster City, CA) is sequenced.

It is verified, on IFNLR1 gene a missense mutation c.296G > A (p.Arg99His) in the family genotype with Phenotype isolates.

And blast is carried out to the amino acid fragment of different material and compares analysis, result is analyzed as shown in figure 3, from this The comparative analysis of IFNLR1 genetic homology is it can be found that IFNLR1p.Arg99His is highly conserved in multiple species, including people Class (H.sapiens), chimpanzee (P.troglodytes), macaque (M.Mulatta), wolf (C.lupus), family ox (B.taurus), house mouse (M.musculus), Rattus noruegicsu (Berkenhout) (R.norvegicus) etc. are highly conserved.

The pathogenic mutation of embodiment 2Sanger method sequence verification ADNSHL disease

The detected IFNLR1 gene of embodiment 1 is detected respectively, the mutational site being related to for said gene Then place primers obtain the related sequence of above-mentioned mutation by PCR amplification, product purification and the method for sequencing, Belong to saltant type or wild type according to determining sequencing results, is heterozygous mutant or homozygous mutation and sequence and table Whether type is verifying the correlation between said gene and ADNSHL disease in isolating in family.

1, DNA is extracted

Peripheric venous blood 10ml is adopted, base is extracted using OMEGA Blood DNA Midi Kit whole blood DNA extracts kit Because of a group DNA, and using the concentration and purity of spectrophotometer and gel electrophoresis measurement DNA and it is diluted to 200ng/ μ l；

2, design of primers and PCR reaction

Candidate gene Genomic DNA standard sequence is from UCSC (http://genome.ucsc.edu/), application Primer3 (version 0.4.0, http://primer3.ut.ee/) designs the primer of candidate gene and synthesis (raw work biology Engineering company's synthesis), utilize Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer- Blast/ primer specificity) is verified.

Primer sequence is as follows:

Forward primer (SEQ ID NO:3): 5'-AGTGGCTGCCTGTCTGGA-3'

Reverse primer (SEQ ID NO:4): 5'-TGGTGCTCAACTGAAAATAATGA-3'

Candidate nucleotide diversity abrupt climatic change is carried out to member in family and normal control population:

PCR amplification: it is expanded using the DNA sample that designed primer pair is extracted；

1) PCR reaction system

2) PCR reaction condition:

3, it is sequenced

PCR product analysis: PCR product (is selected into suitable electricity according to amplified fragments size in 1.5% agarose gel electrophoresis Pressure), analytical electrophoresis map；

Select that banding pattern is single and the higher sample of concentration through PCR product after purification, using American AB I company's automatic gene point 3500 sequencing analysis pcr amplification product of analyzer is analyzed sequencer map, is compared with standard sequence, analysis mutation.Identify the base that causes a disease Cause and pathogenic mutation show that the 296th bit base of the gene coding region IFNLR1 occurs G → A and changes (Fig. 4).

Embodiment 3

IFNLR1 gene expression interferon lambda receptor 1 (Interferon lambda receptor 1) albumen, with cell because Son and interferon are related in the identification of extracellular environment and Cellular Signaling Transduction Mediated etc., and in rodent, IFNLR1 is in inner ear Spiral organ, cochlea nerve, spiral ganglion and inner ear vestibular epithelium in wide expression.Its function includes antiviral activity, resists Proliferation function, anticancer activity and MHC (major histocompatibility complex) composite I/II developed by molecule With immune response etc..So far, not yet effect of the discovery IFNLR1 in auditory system.

In order to further study IFNLR1 in the function of auditory system, the present invention utilizes fluorescence mark by means of mouse model The means of note, research IFNLR1 have found IFNLR1 gene equal table in cochlea and spiral organ in the expression and distribution of murine inner ear Reveal wide expression.

Using zebra fish model, comparative study, hair are carried out to IFNLR1 genic mutation type zebra fish and wild-type zebrafish The normal development of existing IFNLR1 and hair cell and air bladder and function have important role, and knocking out IFNLR1 gene delection will lead to The missing of zebra fish hair cell.Therefore, it is aobvious to will lead to nonsyndromic autosome by IFNLR1c.296G > A (p.Arg99His) The generation of property hereditary hearing impairment (ADNSHL).

4 detection kit of embodiment

A detection kit is prepared, it includes the primers for being adapted to detect for IFNLR1 gene mutation body, for use in screening It is susceptible to suffer from the biological sample of nonsyndromic autosomal dominant deafness, wherein the particular sequence of these primers is shown in embodiment 1。

The tool of the biological sample of nonsyndromic autosomal dominant deafness is susceptible to suffer from using the screening of mentioned reagent box Body step are as follows: method described in the step 1 according to embodiment 2 extract person under test DNA, using extracted DNA as template with it is above-mentioned Exon specific primer carry out PCR reaction, and according to conventional method in that art to PCR product purify, by the product of purifying into Then row sequencing is sequenced whether obtained sequence has at least one mutation selected from said gene by observation, to have Whether detection person under test in effect ground is susceptible to suffer from nonsyndromic autosomal dominant deafness, further, can be from person under test Filter out the biological sample for being susceptible to suffer from nonsyndromic autosomal dominant deafness.

In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show The description of example " or " some examples " etc. means specific features, structure, material or spy described in conjunction with this embodiment or example Point is included at least one embodiment or example of the invention.In the present specification, schematic expression of the above terms are not It must be directed to identical embodiment or example.Moreover, particular features, structures, materials, or characteristics described can be in office It can be combined in any suitable manner in one or more embodiment or examples.In addition, without conflicting with each other, the skill of this field Art personnel can tie the feature of different embodiments or examples described in this specification and different embodiments or examples It closes and combines.

Although the embodiments of the present invention has been shown and described above, it is to be understood that above-described embodiment is example Property, it is not considered as limiting the invention, those skilled in the art within the scope of the invention can be to above-mentioned Embodiment is changed, modifies, replacement and variant.

SEQUENCE LISTING

<110>Chinese People's Liberation Army General Hospital, Shenzhen Hua Da life science institute

<120>nucleic acid of the coding IFNLR1 mutant of separation and its application

<130> PIDC3174935

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 1476

<212> DNA

<213>artificial sequence

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atggcggggc ccgagcgctg gggccccctg ctcctgtgcc tgctgcaggc cgctccaggg 60

aggccccgtc tggcccctcc ccagaatgtg acgctgctct cccagaactt cagcgtgtac 120

ctgacatggc tcccagggct tggcaacccc caggatgtga cctattttgt ggcctatcag 180

agctctccca cccgtagacg gtggcgcgaa gtggaagagt gtgcgggaac caaggagctg 240

ctatgttcta tgatgtgcct gaagaaacag gacctgtaca acaagttcaa gggacgcgtg 300

cggacggttt ctcccagctc caagtccccc tgggtggagt ccgaatacct ggattacctt 360

tttgaagtgg agccggcccc acctgtcctg gtgctcaccc agacggagga gatcctgagt 420

gccaatgcca cgtaccagct gcccccctgc atgcccccac tggatctgaa gtatgaggtg 480

gcattctgga aggagggggc cggaaacaag accctatttc cagtcactcc ccatggccag 540

ccagtccaga tcactctcca gccagctgcc agcgaacacc actgcctcag tgccagaacc 600

atctacacgt tcagtgtccc gaaatacagc aagttctcta agcccacctg cttcttgctg 660

gaggtcccag aagccaactg ggctttcctg gtgctgccat cgcttctgat actgctgtta 720

gtaattgccg cagggggtgt gatctggaag accctcatgg ggaacccctg gtttcagcgg 780

gcaaagatgc cacgggccct ggaactgacc agaggggtca ggccgacgcc tcgagtcagg 840

gccccagcca cccaacagac aagatggaag aaggaccttg cagaggacga agaggaggag 900

gatgaggagg acacagaaga tggcgtcagc ttccagccct acattgaacc accttctttc 960

ctggggcaag agcaccaggc tccagggcac tcggaggctg gtggggtgga ctcagggagg 1020

cccagggctc ctctggtccc aagcgaaggc tcctctgctt gggattcttc agacagaagc 1080

tgggccagca ctgtggactc ctcctgggac agggctgggt cctctggcta tttggctgag 1140

aaggggccag gccaagggcc gggtggggat gggcaccaag aatctctccc accacctgaa 1200

ttctccaagg actcgggttt cctggaagag ctcccagaag ataacctctc ctcctgggcc 1260

acctggggca ccttaccacc ggagccgaat ctggtccctg ggggaccccc agtttctctt 1320

cagacactga ccttctgctg ggaaagcagc cctgaggagg aagaggaggc gagggaatca 1380

gaaattgagg acagcgatgc gggcagctgg ggggctgaga gcacccagag gaccgaggac 1440

aggggccgga cattggggca ttacatggcc aggtga 1476

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Met Ala Gly Pro Glu Arg Trp Gly Pro Leu Leu Leu Cys Leu Leu Gln

1 5 10 15

Ala Ala Pro Gly Arg Pro Arg Leu Ala Pro Pro Gln Asn Val Thr Leu

20 25 30

Leu Ser Gln Asn Phe Ser Val Tyr Leu Thr Trp Leu Pro Gly Leu Gly

35 40 45

Asn Pro Gln Asp Val Thr Tyr Phe Val Ala Tyr Gln Ser Ser Pro Thr

50 55 60

Arg Arg Arg Trp Arg Glu Val Glu Glu Cys Ala Gly Thr Lys Glu Leu

65 70 75 80

Leu Cys Ser Met Met Cys Leu Lys Lys Gln Asp Leu Tyr Asn Lys Phe

85 90 95

Lys Gly Arg Val Arg Thr Val Ser Pro Ser Ser Lys Ser Pro Trp Val

100 105 110

Glu Ser Glu Tyr Leu Asp Tyr Leu Phe Glu Val Glu Pro Ala Pro Pro

115 120 125

Val Leu Val Leu Thr Gln Thr Glu Glu Ile Leu Ser Ala Asn Ala Thr

130 135 140

Tyr Gln Leu Pro Pro Cys Met Pro Pro Leu Asp Leu Lys Tyr Glu Val

145 150 155 160

Ala Phe Trp Lys Glu Gly Ala Gly Asn Lys Thr Leu Phe Pro Val Thr

165 170 175

Pro His Gly Gln Pro Val Gln Ile Thr Leu Gln Pro Ala Ala Ser Glu

180 185 190

His His Cys Leu Ser Ala Arg Thr Ile Tyr Thr Phe Ser Val Pro Lys

195 200 205

Tyr Ser Lys Phe Ser Lys Pro Thr Cys Phe Leu Leu Glu Val Pro Glu

210 215 220

Ala Asn Trp Ala Phe Leu Val Leu Pro Ser Leu Leu Ile Leu Leu Leu

225 230 235 240

Val Ile Ala Ala Gly Gly Val Ile Trp Lys Thr Leu Met Gly Asn Pro

245 250 255

Trp Phe Gln Arg Ala Lys Met Pro Arg Ala Leu Glu Leu Thr Arg Gly

260 265 270

Val Arg Pro Thr Pro Arg Val Arg Ala Pro Ala Thr Gln Gln Thr Arg

275 280 285

Trp Lys Lys Asp Leu Ala Glu Asp Glu Glu Glu Glu Asp Glu Glu Asp

290 295 300

Thr Glu Asp Gly Val Ser Phe Gln Pro Tyr Ile Glu Pro Pro Ser Phe

305 310 315 320

Leu Gly Gln Glu His Gln Ala Pro Gly His Ser Glu Ala Gly Gly Val

325 330 335

Asp Ser Gly Arg Pro Arg Ala Pro Leu Val Pro Ser Glu Gly Ser Ser

340 345 350

Ala Trp Asp Ser Ser Asp Arg Ser Trp Ala Ser Thr Val Asp Ser Ser

355 360 365

Trp Asp Arg Ala Gly Ser Ser Gly Tyr Leu Ala Glu Lys Gly Pro Gly

370 375 380

Gln Gly Pro Gly Gly Asp Gly His Gln Glu Ser Leu Pro Pro Pro Glu

385 390 395 400

Phe Ser Lys Asp Ser Gly Phe Leu Glu Glu Leu Pro Glu Asp Asn Leu

405 410 415

Ser Ser Trp Ala Thr Trp Gly Thr Leu Pro Pro Glu Pro Asn Leu Val

420 425 430

Pro Gly Gly Pro Pro Val Ser Leu Gln Thr Leu Thr Phe Cys Trp Glu

435 440 445

Ser Ser Pro Glu Glu Glu Glu Glu Ala Arg Glu Ser Glu Ile Glu Asp

450 455 460

Ser Asp Ala Gly Ser Trp Gly Ala Glu Ser Thr Gln Arg Thr Glu Asp

465 470 475 480

Arg Gly Arg Thr Leu Gly His Tyr Met Ala Arg

485 490

<210> 3

<211> 18

<212> DNA

<213>artificial sequence

<400> 3

agtggctgcc tgtctgga 18

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tggtgctcaa ctgaaaataa tga 23

Claims (10)

1. a kind of nucleic acid of the coding IFNLR1 mutant of separation, which is characterized in that described compared with wild type IFNLR1 gene Nucleic acid at least has c.296G > A mutation；

Optionally, the nucleic acid is DNA.

2. a kind of isolated polypeptide, which is characterized in that the isolated polypeptide at least has p.Arg99His mutation；

Optionally, the polypeptide is by nucleic acid encode described in claim 1.

3. a kind of reagent for detecting mutational site is susceptible to suffer from nonsyndromic autosomal dominant deafness disease in preparation screening Biological sample in purposes, the mutational site include on IFNLR1 gene c.296G > A mutation；

Optionally, the mutational site includes p.Arg99His mutation in the polypeptide of IFNLR1 gene coding.

4. purposes according to claim 3, which is characterized in that the reagent includes nucleic acid probe or primer,

Optionally, the nucleic acid probe or primer have nucleotide sequence shown in SEQ ID NO:3 and SEQ ID NO:4；

Optionally, the reagent includes the nucleic acid of the coding IFNLR1 mutant of separation, compared with wild type IFNLR1 gene, institute Stating nucleic acid at least has c.296G > A mutation；

Optionally, the nucleic acid is DNA；

Optionally, the reagent includes isolated polypeptide, and the isolated polypeptide at least has p.Arg99His mutation；

Optionally, the polypeptide by separate coding IFNLR1 mutant nucleic acid encode.

5. a kind of reagent for detecting mutational site is susceptible to suffer from nonsyndromic autosomal dominant deafness disease in preparation screening Biological sample system in purposes, the mutational site include on IFNLR1 gene c.296G > A mutation.

6. purposes according to claim 5, which is characterized in that the system comprises:

Nucleic acid-extracting apparatus, the nucleic acid-extracting apparatus are used for from the extraction from biological material sample of nucleic acid；

Nucleic acid sequence determining device, the nucleic acid sequence determining device are connected with the nucleic acid-extracting apparatus, for the core Acid sample is analyzed, to determine the nucleic acid sequence of the sample of nucleic acid；

Judgment means, the judgment means are connected with the nucleic acid sequence determining device, so as to the core based on the sample of nucleic acid Acid sequence or its complementary series have c.296G > A mutation compared with wild type IFNLR1 gene.

7. purposes according to claim 6, which is characterized in that the nucleic acid-extracting apparatus further comprises:

RNA extraction unit, the RNA extraction unit are used for from the extraction from biological material RNA sample；And

Reverse transcription unit, the reverse transcription unit are connected with the RNA extraction unit, for inverting to the RNA sample Record reaction, to obtain cDNA sample, the cDNA sample constitutes the sample of nucleic acid；

Optionally, the nucleic acid sequence determining device further comprises:

Library construction unit, the library construction unit are used to be directed to the sample of nucleic acid, construct nucleic acid sequencing library；And

Unit is sequenced, the sequencing unit is connected with the library construction unit, for surveying to the nucleic acid sequencing library Sequence, to obtain the sequencing result being made of multiple sequencing datas；

Optionally, the library construction unit further comprises:

PCR amplification module is provided with IFNLR1 gene extron specific primer in the PCR amplification module, to utilize institute It states specific primer and PCR amplification is carried out to the sample of nucleic acid；

Optionally, the specific primer has the nucleotide sequence as shown in SEQ ID NO:3 and SEQ ID NO:4；

Optionally, the sequencing unit includes at least one selected from HISEQ2000, SOLiD, 454 and single-molecule sequencing device.

8. it is a kind of for screening the kit for being susceptible to suffer from the biological sample of nonsyndromic autosomal dominant deafness disease, It is characterized in that, contains:

It is adapted to detect for the reagent of IFNLR1 gene mutation body, wherein compared with wild type IFNLR1 gene, the IFNLR1 gene Mutant has c.296G > A mutation；

Optionally, the reagent is nucleic acid probe or primer；

Optionally, the nucleic acid probe or primer have nucleotide sequence shown in SEQ ID NO:3 and SEQ ID NO:4.

9. a kind of construct, which is characterized in that the nucleic acid of the coding IFNLR1 mutant comprising separation described in claim 1.

10. a kind of recombinant cell, which is characterized in that the recombinant cell be by construct as claimed in claim 9 conversion by Body cell and obtain.