CN105985963B - CRX gene mutation bodies and its application - Google Patents

Abstract

The invention discloses CRX gene mutation bodies and its application, the nucleic acid of the coding CRX mutant specifically related to separated, the polypeptide of separation, the method that screening is susceptible to suffer from the biological sample of cone rod cell muscular dystrophy, the system that screening is susceptible to suffer from the biological sample of cone rod cell muscular dystrophy, and for screening the kit for the biological sample for being susceptible to suffer from cone rod cell muscular dystrophy.Wherein, the nucleic acid of the coding CRX mutant of the separation, with SEQ ID NO：1 compares, with c.766C>T is mutated.By detecting that the new mutant whether there is in biological sample, it can effectively detect whether biological sample is susceptible to suffer from cone rod cell muscular dystrophy.

Description

CRX gene mutation bodies and its application

Technical field

The present invention relates to CRX gene mutation bodies and its application.In particular it relates to separate coding CRX mutant Nucleic acid, separation polypeptide, screening be susceptible to suffer from cone rod cell muscular dystrophy biological sample system, for screen be susceptible to suffer from regarding Bore kit, construct, recombinant cell and the structure medicaments sifting model of the biological sample of rod cell muscular dystrophy Method.

Background technology

Cone rod cell malnutrition (cone-rod dystrophies, CORDs) is one group genetic gradual Retinal disease, the incidence of disease is 1/40000.There is dysfunction in morbidity early stage in the CORDs cone cell that is mainly characterized by, There is functional deterioration in subsequent rod cell.The clinical manifestation of such disease includes photophobia, and visual impairment, color defect and center are dark Point.There is nystagmus symptom in some patients.Cone cell afunction or to be badly damaged be CORDs on electroretinogram Typical characteristics.Generally, cone cell function occurs just to observe that rod cell is damaged after obstacle soon.Under extreme case, these Gradual symptom is calm with extensive, serious retinal pigment, and center and peripheral retina atrophy occurs.After arriving Phase, may be difficult to distinguish such disease and retinitis pigmentosa based on clinical symptoms.

CORDs genetic heterogeneity and phenotype heterogeneity is very high.Such disease is not only because its symptom is overlapping and diagnoses Difficulty, and the different mutation of same gene also result in phenotype difference.CORDs can autosomal dominant inheritance (adCORD), also can autosomal recessive (arCORD) or X- linkage inheritances (xlCORD).So far, at least 26 genes Mutation is reported related to various forms of CORD, but still has many diseases because unknown.Thus, it is thin to cone retinal rod at present The research of born of the same parents' muscular dystrophy especially its Disease-causing gene still needs to be goed deep into.

The content of the invention

It is contemplated that at least solving one of technical problem present in prior art.Therefore, one object of the present invention Be propose it is a kind of can Effective selection be susceptible to suffer from cone rod cell muscular dystrophy biological sample means.

The present invention is that the following work based on inventor is completed：

Inventor has found, in the research that current Disease-causing gene and pathogenic mutation are excavated, more using full sequencing of extron group Method.Full sequencing of extron group is to be captured the exon region in full-length genome using special DNA sequence dna probe, then The technology of deep sequencing is carried out for each extron.The case of the sick Disease-causing gene of Mendelian inheritance is found out using sequencing of extron group Example is a lot, such as 2009 U.S. Sarah B Ng et al. successfully orient the base of Miller syndromes using sequencing of extron group Because of DHODH (Ng SB, et al.Exome sequencing identifies the cause of a mendelian disorder.Nat Genet,2009,42(1):30–35).China Wang in 2010 et al. is found that using sequencing of extron group New mutator TGM6 (Wang JL, the et al.TGM6identified as a novel of cerebellar ataxia causative gene of spinocerebellar ataxias using exome sequencing.Brain,2010, 133(Pt 12):3510–3518.).With the extensive use of sequencing of extron group technology and increasingly ripe, a collection of new causes a disease Gene (mutation) is found in succession, has greatly promoted the progress of relevant disease diagnosis and treatment.

Because, human exonic's group sequence only accounts for 1%, about 30Mb of mankind's whole gene group sequence, including 18 The extron of ten thousand or so, it is estimated that 85% human disease's mutation is all located on this 1% protein coding sequence.Cause This, sequencing analysis are carried out to the extron groups of various Diseases, targeted be with maximally related " exome " region of disease, What is caught is most of pathogenic mutation information of disease.Extron sequencing technologies are as a kind of impartial detection method, at present In the detection for being applied successfully to various pathogenic mutations.

Thus, inventor is surveyed for the ill family of the adCORD for occuping southern china collected by extron group The method of sequence joint Sanger sequence verifications carries out pathogenic mutation excavation and checking, and the nutrition of cone rod cell is finally determined not Good disease a new pathogenic mutation --- CRX genes are c.766C>T is mutated.

And then, according to the first aspect of the invention, the present invention proposes a kind of nucleic acid of the coding CRX mutant of separation. Embodiments in accordance with the present invention, the nucleic acid and SEQ ID NO：1 compares, with c.766C>T is mutated, i.e., relative to wild type CRX genes, the bit base C of cDNA sequence the 766th of CRX gene mutation bodies of the invention sports T.According to the implementation of the present invention Example, inventor determines the mutant of CRX genes, the close phase of morbidity of the mutant and cone rod cell muscular dystrophy Close, so as to by detecting that the mutant whether there is in biological sample, can effectively detect whether biological sample is susceptible to suffer from regarding Bore rod cell muscular dystrophy.

According to the second aspect of the invention, the present invention proposes a kind of polypeptide of separation.Embodiments in accordance with the present invention, with SEQID NO：2 compare, and the polypeptide of the separation has p.Q256X mutation, i.e. c.766C the mutation is due to>T is mutated and caused , specifically, relative to wild type CRX, the amino acid sequence the 256th of the polypeptide (i.e. CRX mutant) of separation of the invention Amino acid Q-spoiling is X.By detecting the polypeptide whether is expressed in biological sample, it can effectively detect whether biological sample is easy Suffer from cone rod cell muscular dystrophy.

According to the third aspect of the invention we, the present invention proposes a kind of screening and is susceptible to suffer from cone rod cell muscular dystrophy The system of biological sample.Embodiments in accordance with the present invention, the system includes：Nucleic acid-extracting apparatus, the nucleic acid-extracting apparatus is used In from the extraction from biological material sample of nucleic acid；Nucleotide sequence determining device, the nucleotide sequence determining device and the nucleic acid Extraction element is connected, for analyzing the sample of nucleic acid, to determine the nucleotide sequence of the sample of nucleic acid；Judge dress Put, the judgment means are connected with the nucleotide sequence determining device, so as to the nucleotide sequence based on the sample of nucleic acid or its Complementary series, with SEQ ID NO：1 compares, if with c.766C>T is mutated, and judges whether the biological sample is susceptible to suffer from the cone Rod cell muscular dystrophy.Using the system, the biology for being susceptible to suffer from cone rod cell muscular dystrophy can be effectively screened Sample.

According to the fourth aspect of the invention, the present invention proposes one kind and is susceptible to suffer from cone rod cell malnutrition for screening The kit of the biological sample of disease.Embodiments in accordance with the present invention, the kit contains：It is adapted to detect for CRX gene mutation bodies Reagent, wherein with SEQ ID NO：1 compares, and c.766C the CRX gene mutation bodies have>T is mutated.Utilize the reality according to the present invention The kit of example is applied, the biological sample for being susceptible to suffer from cone rod cell muscular dystrophy can be effectively screened.

According to the fifth aspect of the invention, the invention also provides a kind of construct.Embodiments in accordance with the present invention, the structure Build the nucleic acid of coding CRX mutant of the body comprising foregoing separation.Thus, the construct transformation receptor using the present invention is thin The recombinant cell that born of the same parents obtain, can be efficiently used for the medicine of screening treatment cone rod cell muscular dystrophy.

According to the sixth aspect of the invention, the invention also provides a kind of recombinant cell.Embodiments in accordance with the present invention, should Recombinant cell is obtained by foregoing construct transformed acceptor cell.According to some embodiments of the present invention, profit With the recombinant cell of the present invention, the medicine for the treatment of cone rod cell muscular dystrophy can be effectively screened.

According to the seventh aspect of the invention, the invention also provides a kind of method for building medicaments sifting model.According to this The embodiment of invention, this method includes：At least a portion cell of animal is set to express the core of foregoing coding CRX mutant Acid.Thus, using the medicaments sifting model of the present invention, the medicine for the treatment of cone rod cell muscular dystrophy can effectively be screened Thing.

It should be noted that present invention discover that adCORD a new Disease-causing gene CRX pathogenic mutation c.766C>T, It can be used for early screening adCORD pathogenic mutation carrier, and then the progress early intervention treatment before carrier falls ill； Molecule diagnosis available for adCORD and antidiastole with relevant disease, and quickly, accurately, efficiently, it is easy, examine in early days Disconnected rate is high, and testing result can be the early diagnosis of cone rod cell muscular dystrophy, antidiastole and exploitation cone retinal rod The bad disease medicine of cytotrophy provides scientific basis.

The additional aspect and advantage of the present invention will be set forth in part in the description, and will partly become from the following description Obtain substantially, or recognized by the practice of the present invention.

Brief description of the drawings

The above-mentioned and/or additional aspect and advantage of the present invention will become from description of the accompanying drawings below to embodiment is combined Substantially and be readily appreciated that, wherein：

Fig. 1 shows that screening according to embodiments of the present invention is susceptible to suffer from the biological sample of cone rod cell muscular dystrophy The schematic diagram of system and its part, wherein,

Fig. 1 I are that the biological sample for being susceptible to suffer from cone rod cell muscular dystrophy according to the screening of the embodiment of the present invention is The schematic diagram of system,

Fig. 1 II are the schematic diagram of the nucleic acid-extracting apparatus according to the embodiment of the present invention,

Fig. 1 III are the schematic diagram of the nucleotide sequence determining device according to the embodiment of the present invention；

Fig. 2 shows the family of cone rod cell muscular dystrophy patient's family according to an embodiment of the invention Figure；

Fig. 3 shown according to one embodiment of the invention, the eye examination result of propositus in patient's family shown in Fig. 2, Wherein,

Fig. 3 A- Fig. 3 B are eye-ground photography result,

Fig. 3 C- Fig. 3 D are optical coherence tomography result,

Fig. 3 E- Fig. 3 G are fundus autofluorescence result；

Fig. 4 shows according to one embodiment of present invention, the muscular dystrophy patient's family of cone rod cell shown in Fig. 2 Middle healthy member and propositus, and the outer normal person of family CRX genes c.766C>The representative Sanger in T mutational sites is surveyed Sequence verifies peak figure.

Embodiment

Embodiments of the invention are described below in detail, the example of the embodiment is shown in the drawings, wherein from beginning to end Same or similar label represents same or similar element or the element with same or like function.Below with reference to attached The embodiment of figure description is exemplary, is only used for explaining the present invention, and is not considered as limiting the invention.

CRX gene mutation bodies

According to the first aspect of the invention, the present invention proposes a kind of nucleic acid of the coding CRX mutant of separation.According to this The embodiment of invention, the nucleic acid and SEQ ID NO：1 compares, with c.766C>T is mutated.

Expression way used in herein " nucleic acid of coding CRX mutant ", refers to the base with coding CRX mutant Can be any coding base comprising with CRX mutant because the type of corresponding nucleic acid substances, i.e. nucleic acid is not particularly limited Because of the polymer of corresponding deoxyribonucleotide and/or ribonucleotide, including but not limited to DNA, RNA or cDNA.Root According to the specific example of the present invention, the nucleic acid of foregoing coding CRX mutant is DNA.Embodiments in accordance with the present invention, Inventor determines the mutant of CRX genes, and the morbidity of the mutant and cone rod cell muscular dystrophy is closely related, from And whether there is by detecting the mutant in biological sample, it can effectively detect whether biological sample is susceptible to suffer from cone retinal rod The bad disease of cytotrophy, can also effectively predict organism by detecting that the mutant whether there is in organism Whether cone rod cell muscular dystrophy is susceptible to suffer from.

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

The nucleic acid of coding CRX mutant, is that present inventor is combined by sequencing of extron group, linkage analysis The Disease-causing gene CRX for the cone rod cell muscular dystrophy that the method for Sanger sequence verifications is determined new pathogenic mutation. The pathogenic mutation site is not mentioned in the prior art.

Wherein, the cDNA of wild type CRX genes has nucleotide sequence as follows：

ATGATGGCGTATATGAACCCGGGGCCCCACTATTCTGTCAACGCCTTGGCCCTAAGTGGCCCCAGTGTGGATCTGAT GCACCAGGCTGTGCCCTACCCAAGCGCCCCCAGGAAGCAGCGGCGGGAGCGCACCACCTTCACCCGGAGCCAACTGG AGGAGCTGGAGGCACTGTTTGCCAAGACCCAGTACCCAGACGTCTATGCCCGTGAGGAGGTGGCTCTGAAGATCAAT CTGCCTGAGTCCAGGGTTCAGGTTTGGTTCAAGAACCGGAGGGCTAAATGCAGGCAGCAGCGACAGCAGCAGAAACA GCAGCAGCAGCCCCCAGGGGGCCAGGCCAAGGCCCGGCCTGCCAAGAGGAAGGCGGGCACGTCCCCAAGACCCTCCA CAGATGTGTGTCCAGACCCTCTGGGCATCTCAGATTCCTACAGTCCCCCTCTGCCCGGCCCCTCAGGCTCCCCAACC ACGGCAGTGGCCACTGTGTCCATCTGGAGCCCAGCCTCAGAGTCCCCTTTGCCTGAGGCGCAGCGGGCTGGGCTGGT GGCCTCAGGGCCGTCTCTGACCTCCGCCCCCTATGCCATGACCTACGCCCCGGCCTCCGCTTTCTGCTCTTCCCCCT CCGCCTATGGGTCTCCGAGCTCCTATTTCAGCGGCCTAGACCCCTACCTTTCTCCCATGGTGCCCCAGCTAGGGGGC CCGGCTCTTAGCCCCCTCTCTGGCCCCTCCGTGGGACCTTCCCTGGCCCAGTCCCCCACCTCCCTATCAGGCCAGAG CTATGGCGCCTACAGCCCCGTGGATAGCTTGGAATTCAAGGACCCCACGGGCACCTGGAAATTCACCTACAATCCCA TGGACCCTCTGGACTACAAGGATCAGAGTGCCTGGAAGTTTCAGATCTTGTAG(SEQ ID NO：1),

Its protein encoded has amino acid sequence as follows：

MMAYMNPGPHYSVNALALSGPSVDLMHQAVPYPSAPRKQRRERTTFTRSQLEELEALFAKTQYPDVYAREEVALKIN LPESRVQVWFKNRRAKCRQQRQQQKQQQQPPGGQAKARPAKRKAGTSPRPSTDVCPDPLGISDSYSPPLPGPSGSPT TAVATVSIWSPASESPLPEAQRAGLVASGPSLTSAPYAMTYAPASAFCSSPSAYGSPSSYFSGLDPYLSPMVPQLGG PALSPLSGPSVGPSLAQSPTSLSGQSYGAYSPVDSLEFKDPTGTWKFTYNPMDPLDYKDQSAWKFQIL(SEQID NO：2).

The CRX gene mutation bodies that inventor has found and SEQ ID NO：1 compares, with c.766C>T be mutated, i.e., relative to Wild type CRX genes, the bit base C of cDNA sequence the 766th of CRX gene mutation bodies of the invention sports T.Thus, it is compiled The product of code is compared with the CRX of wild type, and with p.Q256X mutation, i.e. c.766C the mutation is due to>Caused by T mutation, Specifically, relative to wild type CRX, the bit amino of amino acid sequence the 256th of the polypeptide (i.e. CRX mutant) of separation of the invention Sour Q-spoiling is X.

At present, there is not yet CRX genes c.766C>T sports the pathogenic mutation of cone rod cell muscular dystrophy Relevant report.

According to the second aspect of the invention, the present invention proposes a kind of polypeptide of separation.Embodiments in accordance with the present invention, with Wild type CRX is compared, and the polypeptide of the separation has p.Q256X mutation, i.e. c.766C the mutation is due to>Caused by T mutation, Specifically, relative to wild type CRX, the bit amino of amino acid sequence the 256th of the polypeptide (i.e. CRX mutant) of separation of the invention Sour Q-spoiling is X.According to some specific examples of the present invention, the polypeptide is the nucleic acid volume by the coding CRX mutant of foregoing separation Code.By detecting the polypeptide whether is expressed in biological sample, it can effectively detect whether biological sample is susceptible to suffer from cone retinal rod The bad disease of cytotrophy, can also effectively predict organism by detecting that these polypeptides whether there is in organism Whether cone rod cell muscular dystrophy is susceptible to suffer from.

Screening is susceptible to suffer from the system and kit of the biological sample of cone rod cell muscular dystrophy

According to the third aspect of the invention we, the present invention, which proposes one kind and can effectively implement above-mentioned screening, is susceptible to suffer from cone retinal rod The system of the method for the biological sample of the bad disease of cytotrophy.

With reference to Fig. 1, embodiments in accordance with the present invention, the screening is susceptible to suffer from the biological sample of cone rod cell muscular dystrophy System 1000 include nucleic acid-extracting apparatus 100, nucleotide sequence determining device 200 and judgment means 300.

Embodiments in accordance with the present invention, nucleic acid-extracting apparatus 100 is used for from extraction from biological material sample of nucleic acid.According to this hair Bright embodiment, the type of biological sample is not particularly restricted, as long as it is biological that reflection can be extracted from the biological sample Sample of nucleic acid of the sample CRX with the presence or absence of mutation.Embodiments in accordance with the present invention, biological sample can be selected from human body blood Liquid, skin, the preferably at least one of hypodermis, peripheral blood.Thus, it is possible to be easily sampled and detect, so as to Further improve the efficiency that screening is susceptible to suffer from the biological sample of cone rod cell muscular dystrophy.Embodiments in accordance with the present invention, Term " sample of nucleic acid " used herein above should be interpreted broadly, and it can any can reflect in biological sample whether is CRX In the presence of the sample of mutation, for example, can be the complete genome DNA or the full-length genome directly extracted from biological sample In include the parts of CRX coded sequences, can be the total serum IgE that is extracted from biological sample or from biological sample The mRNA of extraction.According to one embodiment of present invention, the sample of nucleic acid is complete genome DNA.Thus, it is possible to expand biology Sample carrys out source range, and can the much information of biological sample be determined simultaneously, is susceptible to suffer from so as to improve screening The efficiency of the biological sample of cone rod cell muscular dystrophy.In addition, embodiments in accordance with the present invention, the type of sample of nucleic acid It is not particularly restricted, for using RNA, as sample of nucleic acid, then nucleic acid-extracting apparatus further comprises RNA extraction units 101 With reverse transcription unit 102, wherein, extraction unit 101 is used for from extraction from biological material RNA samples, reverse transcription unit 102 and RNA Extraction unit 101 is connected, for carrying out reverse transcription reaction to RNA samples, to obtain cDNA samples, resulting cDNA samples Constitute sample of nucleic acid.The nutrition of cone rod cell is susceptible to suffer from thus, it is possible to further improve by the use of RNA as sample of nucleic acid screening not The efficiency of the biological sample of good disease.

Embodiments in accordance with the present invention, nucleotide sequence determining device 200 is connected with nucleic acid-extracting apparatus 100, for core Acid sample is analyzed, to determine the nucleotide sequence of sample of nucleic acid.Embodiments in accordance with the present invention, it is determined that resulting nucleic acid sample The method and apparatus of this nucleotide sequence is not particularly restricted.According to a particular embodiment of the invention, can be using sequencing Method determines the nucleotide sequence of sample of nucleic acid.Thus, according to one embodiment of present invention, the nucleotide sequence determining device 200 may further include：Library construction unit 201 and sequencing unit 202.Library construction unit 201 is used to be directed to nucleic acid Sample, builds nucleic acid sequencing library；Sequencing unit 202 is connected with library construction unit 201, for being carried out to nucleic acid sequencing library Sequencing, to obtain the sequencing result being made up of multiple sequencing datas.

On for sample of nucleic acid, building the method and flow of sequencing library, those skilled in the art can be according to difference Microarray dataset suitably selected, on the details of flow, may refer to be sequenced such as Illumina companies of manufacturer of instrument The code provided, for example, see Illumina companies Multiplexing Sample Preparation Guide (Part# 1005361；Feb 2010) or Paired-End SamplePrep Guide (Part#1005063；Feb 2010), pass through ginseng According to be incorporated into herein.Embodiments in accordance with the present invention, from the method and apparatus of extraction from biological material sample of nucleic acid, also not by spy Do not limit, can be carried out using the nucleic acid extraction kit of commercialization.

It should be noted that used term " nucleotide sequence " should broadly understood herein, it can be to core Acid sample carries out that the complete nucleic acid sequence information obtained after obtained sequencing data is assembled is sequenced or directly adopted It is used as nucleotide sequence with the sequencing data (reads) obtained by by the way that sample of nucleic acid is sequenced, as long as these nucleotide sequences In containing correspondence CRX coded sequence.

In addition, embodiments in accordance with the present invention, can be screened to sample of nucleic acid, CRX extrons are enriched with, the screening is rich Collection can be before sequencing library be built, during structure sequencing library, or builds progress after sequencing library.Thus, it is literary Storehouse construction unit 201 may further include PCR amplification module (not shown)s, and CRX is provided with PCR amplification modules Extron specific primer, to utilize CRX extron specific primers, performing PCR amplification is entered to the sample of nucleic acid.Thus, It can be expanded by PCR, be enriched with CRX extrons, cone rod cell malnutrition is susceptible to suffer from so as to further improve screening The efficiency of the biological sample of disease.According to a particular embodiment of the invention, CRX extrons specific primer has such as SEQ ID NO： Nucleotide sequence shown in 3 and 4：

Forward primer：5’-CCTCTGGGCATCTCAGAT-3’(SEQ ID NO：3)；

Reverse primer：5’-AGGAAAGGGGTGGTCTCT-3’(SEQ ID NO：4).

It is surprisingly found by the inventors that, by using above-mentioned primer, can significantly it be effectively completed in PCR reaction systems pair The amplification of CRX extrons.It should be noted that these SEQ ID NO：3 and SEQ ID NO：Nucleotide sequence shown in 4 is this The inventor of invention surprisingly obtains after arduous labor has been paid.

Embodiments in accordance with the present invention, the equipment that can be used for being sequenced is not particularly restricted.According to the present invention's Embodiment, can use second generation microarray dataset, it would however also be possible to employ the third generation and forth generation or more advanced microarray dataset. According to the specific example of the present invention, sequencing unit 202 can be selected from HISEQ2000, SOLiD, 454 and single-molecule sequencing device At least one.Thus, with reference to newest sequencing technologies, higher sequencing depth, detection spirit can be reached for Single locus Sensitivity and accuracy are greatly improved, it is thus possible to the characteristics of utilizing high flux, the deep sequencing of these sequencing devices, are further carried The efficiency that height is tested and analyzed to sample of nucleic acid.So as to improve the accuracy and standard when subsequently analyzing sequencing data Exactness.

Embodiments in accordance with the present invention, judgment means 300 are connected with nucleotide sequence determining device 200, suitable for by nucleic acid sample This nucleotide sequence is compared, so as to the nucleotide sequence based on sample of nucleic acid and SEQ ID NO：1 difference judges biological sample Whether product are susceptible to suffer from cone rod cell muscular dystrophy.Thus, using the system, it can effectively screen that to be susceptible to suffer from cone retinal rod thin The biological sample of born of the same parents' muscular dystrophy.

Specifically, the nucleotide sequence based on sample of nucleic acid and SEQ ID NO：1 compares, if with c.766C>T is mutated, Judge whether biological sample is susceptible to suffer from cone rod cell muscular dystrophy.As it was previously stated, according to one embodiment of present invention, core The nucleotide sequence of acid sample and SEQ ID NO：1 compares, with c.766C>T is mutated, and is that biological sample is susceptible to suffer from cone rod cell The instruction of muscular dystrophy.Embodiments in accordance with the present invention, to nucleotide sequence and SEQ ID NO：1 equipment being compared is not It is particularly limited, can be operated using the software of any conventional, can be soft using SOAP according to the instantiation of the present invention Part is compared.

According to the fourth aspect of the invention, the present invention proposes one kind and is susceptible to suffer from cone rod cell malnutrition for screening The kit of the biological sample of disease.Embodiments in accordance with the present invention, this is used for screening and is susceptible to suffer from cone rod cell muscular dystrophy The kit of biological sample include：Be adapted to detect for the reagent of CRX gene mutation bodies, wherein with SEQ ID NO：1 compares, should C.766C CRX gene mutation bodies have>T is mutated.Using kit according to an embodiment of the invention, can effectively it screen easily Suffer from the biological sample of cone rod cell muscular dystrophy.Herein, used term " is adapted to detect for CRX gene mutations The reagent of body " should be interpreted broadly, you can be the reagent of detection CRX encoding genes or detect CRX mutant polypeptides Reagent, for example can using identification specific position antibody.According to one embodiment of present invention, the reagent is nucleic acid Probe or primer, it is preferable that the nucleic acid probe or primer have such as SEQ ID NO：Nucleotide sequence shown in 3-4.Thus, The biological sample for being susceptible to suffer from cone rod cell muscular dystrophy can efficiently be screened.

It should be noted that in the system that screening is susceptible to suffer from the biological sample of cone rod cell muscular dystrophy herein above Feature and advantage described in part, are equally applicable to screen the biological sample that is susceptible to suffer from cone rod cell muscular dystrophy Kit, will not be repeated here.

Construct and recombinant cell

According to the fifth aspect of the invention, the invention also provides a kind of construct.Embodiments in accordance with the present invention, the structure Build the nucleic acid of coding CRX mutant of the body comprising foregoing separation, i.e. CRX gene mutation bodies of the invention.Thus, utilize The recombinant cell that the construct transformed acceptor cell of the present invention is obtained, can be efficiently used for screening treatment cone rod cell battalion Support the medicine of bad disease.Wherein, the species of the recipient cell is not particularly limited, for example, can be Bacillus coli cells, the food in one's mouth Newborn zooblast, preferably this receptor cell derived is in mammal.

Used term " construct " refers to a kind of such genetic carrier in the present invention, and it includes specific nucleic acid sequence Row, and purpose nucleic acid sequence can be transferred in host cell, to obtain recombinant cell.Embodiments in accordance with the present invention, structure The form for building body is not particularly limited.Embodiments in accordance with the present invention, it can be plasmid, bacteriophage, artificial chromosome, clay (Cosmid), viral at least one, preferred plasmid.Plasmid, with simple to operate, can carry larger piece as genetic carrier The property of section, is easy to operate and handles.The form of plasmid is also not particularly limited, and both can be circular plasmids or line Property grain, you can be single-stranded or double-strand.Those skilled in the art can be selected as needed.At this Term " nucleic acid " used in invention can be any polymer comprising deoxyribonucleotide or ribonucleotide, bag Include but be not limited to by modification or unmodified DNA, RNA, its length is not any particular limitation.For for building The construct of recombinant cell, preferably described nucleic acid is DNA, because DNA is for RNA, its is more stable, and is easy to behaviour Make.

According to the sixth aspect of the invention, the invention also provides a kind of recombinant cell.Embodiments in accordance with the present invention, should Recombinant cell is obtained by foregoing construct transformed acceptor cell.So as to which recombinant cell of the invention can CRX gene mutation bodies entrained by expression construct.According to some embodiments of the present invention, using the recombinant cell of the present invention, The medicine for the treatment of cone rod cell muscular dystrophy can effectively be screened.Embodiments in accordance with the present invention, recipient cell Species is not particularly limited, for example, can be Bacillus coli cells, mammalian cell, preferably described recipient cell is from non- People mammal.

The method for building medicaments sifting model

According to the seventh aspect of the invention, present invention also offers a kind of method for building medicaments sifting model.According to this The embodiment of invention, this method includes：At least a portion cell of animal is set to express the nucleic acid of foregoing coding CRX mutant. Embodiments in accordance with the present invention, the animal is mouse, pig, dog, primate.According to some embodiments of the present invention, profit With the medicaments sifting model of the present invention, the medicine for the treatment of cone rod cell muscular dystrophy can be effectively screened.

It should be noted that the method that the present invention builds medicaments sifting model is not particularly limited, as long as making animal At least a portion cell expresses foregoing CRX gene mutation bodies.For example, the method using genetic transformation can be used, by before Construct of the invention described in face is transferred to receptor (inhuman), so that at least a portion cell expression up to animal is foregoing CRX gene mutation bodies；Marker-free transgenic technology, CRISPR/Cas9 gene editings technology, site-directed integration skill can also be used Art etc., makes the CRX genes of receptor occur c.766C>T is mutated, and the foregoing polypeptide of effective expression, so that this receptor animal Can occur cone rod cell muscular dystrophy, and then it is malnutritive to be efficiently used for screening treatment cone rod cell The medicine of disease, namely medicaments sifting model can be acted effectively as.

It should be noted that full sequencing of extron group technology of the present invention using a new generation, for an ADCORD patient Family carries out full-length genome sequencing of extron group analysis, so that the new Disease-causing gene CRX of an ADCORD are found that, and the base Because of upper pathogenic mutation.Thus, the present invention enriches the Disease-causing gene and pathogenic mutation collection of illustrative plates of CRX genes, further illustrates The Molecular pathogenesis of cone rod cell muscular dystrophy, for the early stage Disease-causing gene of cone rod cell muscular dystrophy Examination and therapeutic intervention provide scientific basis.

Below with reference to specific embodiment, the present invention will be described, it is necessary to which explanation, these embodiments are only explanation Property, and be not considered as limiting the invention.

Unless otherwise specified, the conventional hand that the technological means employed in embodiment is well known to those skilled in the art Section, is referred to《Molecular Cloning:A Laboratory guide》The third edition or Related product are carried out, and the reagent and product used is also Available commercial.The various processes and method not being described in detail are conventional methods as known in the art, agents useful for same Source, trade name and it is necessary to list its constituent person, indicate on the first appearance, identical reagent used thereafter is such as without spy Different explanation is identical with the content indicated first.

Embodiment 1 determines cone rod cell muscular dystrophy pathogenic mutation

1st, sample is collected

The ill familys of adCORD that inventor one occupy southern china, its pedigree chart is shown in Fig. 2.As shown in Fig. 2 wherein, Zero represents normal female, and represents normal male, and ■ represents male patient, ● female patient is represented,Represent that late male suffers from Person；Represent late normal male；Represent late normal female；Arrow meaning is propositus (III-12).

The family is existing 9 patients, and continuous 3 generation morbidity, men and women is involved, and meets Mendel's autosomal dominant Hereditary pattern.The propositus (III-12) of the family is male, 50 years old.Gradual visual impairment is diagnosed as during propositus 40 years old With slight color defect, eye-ground photography (fundus photography) finds that patient's macula lutea has 2 × 2PD goldleaf sample Image, diffusivity hypopigmentation；Peripherality retinal manifestations go out spicule shape pigmentation and arteria retina decay, see figure 3A- Fig. 3 B；Optical coherence tomography (optical coherence tomography, OCT), which is checked, to be found, its macular area is regarded Nethike embrane is thinning, sees Fig. 3 C- Fig. 3 D；Fundus autofluorescence (fundus autofluorescence) finds that macular area has strong glimmering Light, but choroidal atrophy around is had no, see Fig. 3 E- Fig. 3 G；Retinal current traces (electroretinography, ERG) hair The cone responses of existing propositus are slightly slow, and rod cell reaction is normal.Other patient's phenotypes of the family are similar.

Inventor acquires the peripheral blood of all members alive in the family.According to world medicine united organization《Hull Octyl group declaration》The requirement of (Declaration of Helsinki), subject and Hospital Ethical Committee, which endorsed, related to be known Feelings letter of consent.

2nd, target area capture sequencing

Inventor using Aglient SureSelect Human All Exon kit (Agilent Technologies, Santa Clara, CA, USA) Solexa high throughput sequencing technologies are combined, to above-mentioned cone rod cell muscular dystrophy patient The extron group sequence of 3 patients (III-4, III-12 and IV-7) and 1 normal person (III-10) in family is surveyed Sequence, wherein being used as control using normal person outside 100 familys.

It is specific as follows：

2.1 DNA are extracted

Under aseptic condition, extract family member III-4 (patient), III-10 (normal person), III-12 (propositus) and IV-7 (patient) periphery anticoagulation, carries out genome using peripheral blood DNA purification kit (German Qiagen companies) respectively DNA extraction and purifying, experimental procedure is carried out according to QIAGEN specifications.

The quality testing of DNA sample：With the Nanodrop ultramicrospectrophotometers of ultra-pure water school zero, the DNA of extracting is measured The ratio of sample light absorption value and sample light absorption value under 280nm ultraviolet lights under the concentration and 260nm ultraviolet lights of sample.Gained it is each The OD of sample genomic dna₂₆₀/OD₂₈₀It all should be located between 1.7-2.0, concentration is no less than 200ng/ μ l, total amount is no less than 30 μ g。

2.2 target areas are captured and sequencing

Each genomic DNA sample is broken at random using ultrasonoscope (CovarisS2, Massachusetts, USA) 150-200bp or so fragment, the operational manual then provided according to manufacturer connects top connection system respectively at fragment two ends Standby library (reference can be made to：http:The Illumina/Solexa standards that //www.illumina.com/ is provided build storehouse specification, lead to Cross reference to be incorporated by herein).Be available on the machine sequencing after library detection is qualified, to obtain raw sequencing data.Wherein, With reference to Illumina standards cluster and sequencing the step of be sequenced, microarray dataset be Illumina Hiseq 2000, read Length is 90bp, both-end sequencing.Wherein, 4 subjects (III-4, III-10, III-12 and IV-7) full extron group sequence Average sequencing depth is respectively 86.48 ×, 100.39 ×, 97.03 × and 94.38 ×.

3rd, variation detection, annotation and database compare

Using Illumina basecalling Software 1.7 to the raw sequencing data of above-mentioned acquisition at Reason, after filtering depollutes, using SOAPaligner/SOAP2 (reference can be made to：Li R,Li Y,Kristiansen K,et al,SOAP:short oligonucleotide alignment program.Bioinformatics 2008,24(5): 713-714；Li R,Yu C,Li Y,ea al,SOAP2:an improved ultrafast tool for short read alignment.Bioinformatics 2009,25(15):1966-1967, by referring to be incorporated by herein) compare To reference gene group UCSC NCBI37/hg19, compared to obtain to unique aligned sequences on genome.Then utilize SOAPsnp (reference can be made to：Li R,Li Y,Fang X,Yang H,et al,SNP detection for massively parallel whole-genome resequencing.Genome Res 2009,19(6):1124-1132, by referring to general It is incorporated by herein) determine the genotype of target region.

Using BWA (version 0.7.5) (referring to Li H, Durbin R.Fast and accurate long-read alignment with Burrows-Wheeler transform.Bioinformatics 2010,26(5):589-595, leads to Reference is crossed to be incorporated by herein) and GATK (Genome Analysis Toolkit) (version2.8-1) (referring to DePristo MA,Banks E,Poplin R,et al.A framework for variation discovery and genotyping using next-generation DNA sequencing data.Nat Genet 2011,43:491- 498, by referring to being incorporated by herein) determine Indel (insertion-deletion, insertion/deletion mark) class Type.

As a result, inventor has found 109560 SNPs (SNP) in III-4, is found in III-10 108907 SNPs (SNP), find 110292 SNPs (SNP), in IV-7 in III-12 108350 SNPs (SNP) of middle discovery.

Then pass through dbSNP databases (http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ Summary.cgi), thousand human genome database (http://www.1000genomes.org/) and HapMap databases (http://hapmap.ncbi.nlm.nih.gov/) public database filtering, remove all known and in database Gene frequency is more than 0.005 variation (variation MAF values are high, usually non-pathogenic common polymorphism).And then, to reduce The hunting zone of Disease-causing gene mutation, inventor will remove in mutation, intragenic mutation and same sense mutation between gene, to it is non-synonymous/ Splice site is mutated and small insertion and deletion carries out prioritizing selection.By comparing 3 patients (III-4, III-12, IV-7) and 1 The variation of example normal individual (III-10), it is found that 3 patients carry 26 identical mutators.Further, inventor's profit Harmfulness prediction has been carried out to this 26 mutation with forecasting software SIFT, finally thought：CRX genes are the cause of the adCORD familys Ospc gene.Specifically, for the adCORD familys, CRX genes there occurs nonsense mutation：c.766C>T (p.Q256X), causes many The termination of peptide chain translation.

Known CRX genes are located at No. 19 area 3 of chromosome long arm 1 of people with (19q13), containing 5 extrons, encode 299 Amino acid residue.CRX genes are expressed in the inner nuclear layer of retina, by cooperateing with work with other transcription factors (such as NRL and RX) For participating in the differentiation and maintenance of photosensory cell.CRX protein moleculars have a similar paired homology segment, a WPS knot Structure domain, and positioned at the OTX tails of c-terminus.OTX tails are the important features that CRX plays regulating and controlling effect as transcription factor. CRX plays activity by OTX tails and neural retina leucine zipper (NRL) formation dimer.Thus, OTX tails are to CRX Gene is extremely important.Inventor find, mutation (as c.504delA, c.587delCCCC, c.816delCACinsAA and C.C766T OTX tails) can be caused to disappear, the expression of photosensory cell specific gene is influenceed, because OTX tails can set up CRX and its The interaction of his transcription factor.Though the CTX albumen for lacking OTX tails is combined with target promoter, do not interact, The transcription activating of photosensory cell specific gene thus may be hindered, and then causes adCORD.

Further, Sanger checkings find that CRX genes are c.766C>T mutation exist only in adCORD patient's family In patient, and it is mutated in family in the outer normal control individuals of the family of normal individual and 100 affinity-less relations without this.

Thus, it has been recognised by the inventors that CRX genes are the Disease-causing gene of adCORD patient's family, CRX genes are c.766C>T (p.Q256X) it is mutated, is the pathogenic mutation of cone rod cell muscular dystrophy.

The Sanger method sequence verifications of embodiment 2

Respectively to all family members (including patient and the normal family in adCORD patient's family described in embodiment 1 Category), and the CRX genes of normal person are detected outside 100 familys：For CRX genes c.766C>T mutation design primers, Then expanded by PCR, the method for product purification and sequencing obtains the relevant sequence in mutational site, according to determining sequencing results Belong to saltant type or wild type, the correlation between checking CRX genes and the mutation and cone rod cell muscular dystrophy.

Specific method step is as follows：

1st, DNA is extracted

According to the method for the extraction DNA described in embodiment 1, the gene prepared in subject's peripheric venous blood is extracted respectively DNA is organized, it is standby.

2nd, design of primers and PCR reactions

First, with reference to human gene data unit sequence storehouse GRCh37/hg19, design is obtained with SEQ ID NO：3-4 institutes Show the CRX gene extron specific primers of nucleotide sequence, particular sequence is as follows：

Forward primer：5’-CCTCTGGGCATCTCAGAT-3’(SEQ ID NO：3)；

Reverse primer：5’-AGGAAAGGGGTGGTCTCT-3’(SEQ ID NO：4).

Then, the PCR reaction systems of each genomic DNA sample are prepared according to following proportioning respectively：

Reaction system (25 μ l)：

Then, each PCR reaction systems are entered into performing PCR reaction respectively according to following reaction condition：

95 DEG C 2 minutes；35 circulations：(94 DEG C of minutes, 62 DEG C 30 seconds, 72 DEG C 30 seconds)；72 DEG C, 7 minutes.

Thus, the pcr amplification product that each receptor gene organizes DNA sample is obtained.

3rd, it is sequenced

The pcr amplification product that each receptor gene obtained in step 2 is organized into DNA sample directly carries out DNA sequencing.Its In, sequencing is carried out using the sequenators of Genetic Analyzer 3500.

Based on sequencing result, CRX genes are somebody's turn to do in cone rod cell muscular dystrophy patient's family of the present invention Mutational site carries out mutation investigation, as a result finds that c.766C all existing cases carry in the family>T's (p.Q256X) is miscellaneous Mutation is closed, and its normal family members and the outer normal population of 100 familys do not carry the mutation.Wherein, Fig. 4 shows above-mentioned regard Bore in rod cell muscular dystrophy patient's family healthy member in propositus and family, and the outer normal person of family CRX bases Because c.766C>The representative Sanger sequence verifications peak figure in T mutational sites.

Thus, the Disease-causing gene that CRX genes are cone rod cell muscular dystrophy is further proved, CRX genes c.766C>T sports the sick pathogenic mutation.

The detection kit of embodiment 3

A detection kit is prepared, c.766C it is included can detect CRX genes>The primer of T mutation, for screening easily Suffer from the biological sample of cone rod cell muscular dystrophy, wherein these primers are CRX gene extron specific primers, its sequence Arrange SEQ ID NO as described in example 2 above：Shown in 3-4.

Screened using mentioned reagent box and be susceptible to suffer from the biological sample of cone rod cell muscular dystrophy and concretely comprise the following steps：Press Method described in 2.1 " the DNA extractions " according to the step 2 of embodiment 1 extracts person under test DNA, by template of the DNA that is extracted with it is upper The extron specific primer for stating CRX genes enters performing PCR reaction (PCR reaction systems and reaction condition are referring to embodiment 2), and presses PCR primer is purified according to this area conventional method, the product of purifying is sequenced, then by observing the sequence obtained by being sequenced C.766C whether row have>T is mutated, and can effectively detect whether the CRX gene mutation bodies of the present invention are deposited in person under test DNA , further, can be from person under test so as to effectively detect whether person under test is susceptible to suffer from cone rod cell muscular dystrophy In filter out the biological sample for being susceptible to suffer from cone rod cell muscular dystrophy.

In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show The description of example " or " some examples " etc. means to combine specific features, structure, material or the spy that the embodiment or example are described Point is contained at least one embodiment of the present invention or example.In this manual, to the schematic representation of above-mentioned term not Necessarily refer to identical embodiment or example.Moreover, specific features, structure, material or the feature of description can be any One or more embodiments or example in combine in an appropriate manner.

Although an embodiment of the present invention has been shown and described, it will be understood by those skilled in the art that：Not In the case of departing from the principle and objective of the present invention a variety of change, modification, replacement and modification can be carried out to these embodiments, this The scope of invention is limited by claim and its equivalent.

Claims (8)

1. the nucleic acid of the coding CRX mutant of a kind of separation, it is characterised in that the nucleic acid and SEQ ID NO：1 compares, and has c.766C>T is mutated.

2. the nucleic acid of the coding CRX mutant of separation according to claim 1, it is characterised in that the nucleic acid is DNA.

3. a kind of polypeptide of separation, it is characterised in that with SEQ ID NO：2 compare, and there is the polypeptide of the separation p.Q256X to dash forward Become.

4. a kind of kit for being used to screen the biological sample for being susceptible to suffer from cone rod cell muscular dystrophy, it is characterised in that contain Have：

Be adapted to detect for the reagent of CRX gene mutation bodies, wherein with SEQ ID NO：1 compares, and the CRX gene mutation bodies have c.766C>T is mutated.

5. kit according to claim 4, it is characterised in that the reagent is nucleic acid probe or primer.

6. kit according to claim 5, it is characterised in that the nucleotide sequence of the primer such as SEQ ID NO：3- Shown in 4.

7. a kind of construct, it is characterised in that the nucleic acid of the coding CRX mutant comprising the separation described in claim 1 or 2.

8. a kind of recombinant cell, it is characterised in that the recombinant cell is by the construct transformation receptor described in claim 7 Cell and obtain.