CN104745594B - CYP4V2 gene mutation body and its application - Google Patents

Abstract

The invention discloses CYP4V2 gene mutation body and its applications, more particularly to the nucleic acid of the coding CYP4V2 mutant of separation, isolated polypeptide, the method for screening the biological sample of susceptible primary crystalline degeneration of retina, the system for screening the biological sample of susceptible primary crystalline degeneration of retina, and the kit of the biological sample for screening susceptible primary crystalline degeneration of retina.Wherein, the nucleic acid of the coding CYP4V2 mutant of the separation is mutated compared with SEQ ID NO:1 with c.791del T.It whether there is by detecting the new mutant in the biological sample, the whether susceptible primary crystalline degeneration of retina of biological sample can be effectively detected.

Description

CYP4V2 gene mutation body and its application

Technical field

The present invention relates to CYP4V2 gene mutation body and its applications.In particular it relates to isolated coding CYP4V2 The nucleic acid of mutant, isolated polypeptide, the method for screening the biological sample of susceptible primary crystalline degeneration of retina, screening are easy The system for feeling the biological sample of primary crystalline degeneration of retina, for screening susceptible primary crystalline degeneration of retina The kit of biological sample, construct and recombinant cell.

Background technique

Bietti ' s crystalline degeneration of retina (Bietti ' s crystalline Corneoretinal Dystrophy, abbreviation BCD) it is rare heredity blinding disease, it is about 1/24000 in global disease incidence, high-incidence people Group is Chinese, and the disease incidence in China is about 1/4000.It falls ill within BCD patient usual 20-30 years old, 40-50 years old blindings, at present Have become and threaten the whole world young and the main eye disease of middle-aged population visual performance, visual quality, finally seriously affects trouble The quality of life of person brings huge burden to family and society.BCD is since (including the cone is thin for retinal photoreceptor cells Born of the same parents and rod cell) and retinal pigment epithelium denaturation, it is caused caused by the adjoint atrophy of progressive choroidal artery, hardening Blind property Genetic eye diseases.The mode of inheritance of BCD is mainly autosomal recessive inheritance (autosoma recessive BCD, letter Referred to as ARBCD).

Research at present in terms of the genetics of primary BCD is less, and the related mechanism that causes a disease is still indefinite.Thus, at present The research of primary BCD especially its Disease-causing gene is still needed to be goed deep into.

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 the method for the biological sample for proposing that one kind is capable of the susceptible primary crystalline degeneration of retina (BCD) of Effective selection.

The present invention is that the following work based on inventor is completed: inventor is sequenced by sequencing of extron group, Sanger Verify, the method for protein structural analysis has determined a new pathogenic mutation of primary crystalline degeneration of retina --- The c.791del T of CYP4V2 gene is mutated.

In turn, according to the first aspect of the invention, the invention proposes a kind of cores of the coding CYP4V2 mutant of separation Acid.According to an embodiment of the invention, the nucleic acid compared with SEQ ID NO:1, is mutated, i.e., relative to open country with c.791del T The 791st bit base of cDNA of raw type CYP4V2 gene, CYP4V2 gene mutation body of the invention lacks T.Reality according to the present invention Example is applied, inventor has determined the mutant of CYP4V2 gene, and the morbidity of the mutant and primary crystalline degeneration of retina is close Cut phase is closed, to whether there is in the biological sample by detecting the mutant, it is whether easy that biological sample can be effectively detected Feel primary crystalline degeneration of retina.

According to the second aspect of the invention, the invention proposes a kind of isolated polypeptides.According to an embodiment of the invention, institute The polypeptide for stating separation has the amino acid sequence as shown in SEQ ID NO:23, specifically, relative to wild type CYP4V2, this hair Bright isolated polypeptide (i.e. CYP4V2 mutant) the 264th amino acid sports leucine by phenylalanine, and cause 264 with Frameshift mutation occurs for amino acid afterwards, generates one section of abnormal peptide fragment containing 261 amino acid.By being in detection biological sample No expression polypeptide, can be effectively detected the whether susceptible primary crystalline degeneration of retina of biological sample.

According to the third aspect of the invention we, susceptible primary crystalline degeneration of retina is screened the invention proposes a kind of The method of biological sample.According to an embodiment of the invention, method includes the following steps: from the extraction from biological material nucleic acid sample This；Determine the nucleic acid sequence of the sample of nucleic acid；The nucleic acid sequence of the sample of nucleic acid has compared with SEQ ID NO:1 C.791delT mutation is the instruction of the susceptible primary crystalline degeneration of retina of the biological sample.By real according to the present invention The method for applying the biological sample of the susceptible primary crystalline degeneration of retina of screening of example, can effectively screen susceptible primary The biological sample of crystalline degeneration of retina.

According to the fourth aspect of the invention, susceptible primary crystalline degeneration of retina is screened the invention proposes a kind of The system of biological sample.According to an embodiment of the 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；Nucleic acid sequence determining device, the nucleic acid sequence determining device and the nucleic acid Extraction element is connected, for analyzing the sample of nucleic acid, to determine the nucleic acid sequence of the sample of nucleic acid；Judgement dress It sets, the judgment means are connected with the nucleic acid sequence determining device, so as to nucleic acid sequence and SEQ based on the sample of nucleic acid ID NO:1 is compared, if is mutated with c.791del T, is judged the whether susceptible primary crystalline retinal of the biological sample Denaturation.Using the system, it can effectively implement the biological sample of the aforementioned susceptible primary crystalline degeneration of retina of screening Method, so as to effectively screen the biological sample of susceptible primary crystalline degeneration of retina.

According to the fifth aspect of the invention, the invention proposes one kind becomes for screening susceptible primary crystalline retinal The kit of the biological sample of property.According to an embodiment of the invention, the kit contains: being adapted to detect for CYP4V2 gene mutation body Reagent, wherein compared with SEQ ID NO:1, which is mutated with c.791del T.Using according to this The kit of the embodiment of invention can effectively screen the biological sample of susceptible primary crystalline degeneration of retina.

According to the sixth aspect of the invention, the 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 CYP4V2 mutant of mentioned-above separation.As a result, using construct conversion of the invention by The recombinant cell that body cell obtains can be efficiently used for the drug of screening treatment primary crystalline degeneration of retina.

According to the seventh aspect of the 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.According to some embodiments of the present invention, sharp With recombinant cell of the invention, the drug for the treatment of primary crystalline degeneration of retina can be effectively screened.

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 shows the biological sample of the susceptible primary crystalline degeneration of retina of screening according to an embodiment of the present invention The schematic diagram of system and its component part, wherein

Fig. 1 I is to be according to the biological sample of the susceptible primary crystalline degeneration of retina of screening of the embodiment of the present invention The schematic diagram of system,

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

Fig. 1 III is the schematic diagram according to the nucleic acid sequence determining device of the embodiment of the present invention；

Fig. 2 shows the pedigree chart of BCD patient's family according to an embodiment of the invention；

Fig. 3 shows according to an embodiment of the present invention, the fundus image of propositus in BCD patient's family shown in Fig. 2；

Fig. 4 shows according to one embodiment of present invention, propositus in BCD patient's family shown in Fig. 2, normal in family The representative Sanger sequence verification peak figure in the CYP4V2 gene of the outer normal control of the people and family c.791del mutational site T, In,

Fig. 4 I is the representativeness according to the CYP4V2 gene of the propositus of the embodiment of the present invention c.791del mutational site T Sanger sequence verification peak figure,

Fig. 4 II is according to the CYP4V2 gene of normal person in the family of the embodiment of the present invention c.791del mutational site T Representative Sanger sequence verification peak figure,

Fig. 4 III is according to the CYP4V2 gene of normal control outside the family of the embodiment of the present invention c.791del mutational site T Representative Sanger sequence verification peak figure；

Fig. 5 shows according to one embodiment of present invention, the sequence comparison of multiple species CYP4V2 albumen.

Specific embodiment

Below with reference to specific embodiment, the present invention will be described, it should be noted that these embodiments are only illustrative , and be not considered as limiting the invention.

CYP4V2 gene mutation body

According to the first aspect of the invention, the invention proposes a kind of nucleic acid of the coding CYP4V2 mutant of separation.Root According to the embodiment of the present invention, the nucleic acid is mutated compared with SEQ ID NO:1 with c.791del T.It is used herein The expression way nucleic acid of CYP4V2 mutant " coding ", refer to nucleic acid object corresponding with the gene of CYP4V2 mutant is encoded The type of matter, i.e. nucleic acid is not particularly limited, and can be any comprising corresponding with the encoding gene of CYP4V2 mutant de- The polymer of oxygen ribonucleotide and/or ribonucleotide, including but not limited to DNA, RNA or cDNA.According to the present invention one The nucleic acid of a specific example, mentioned-above coding CYP4V2 mutant is DNA.According to an embodiment of the invention, inventor is true The mutant of CYP4V2 gene is determined, the morbidity of the mutant and primary crystalline degeneration of retina is closely related, thus logical It crosses and detects the mutant and whether there is in the biological sample, the whether susceptible primary crystalline of biological sample can be effectively detected Retinosis can also be whether there is in organism by detecting the mutant, effectively whether can predict organism Susceptible primary crystalline degeneration of retina.

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 coding CYP4V2 mutant is that present inventor is sequenced by sequencing of extron group, Sanger The Disease-causing gene CYP4V2's for the primary crystalline degeneration of retina that the method for verifying and protein structural analysis determines is new Pathogenic mutation.The pathogenic mutation site is not mentioned in the prior art.

Wherein, the cDNA of wild type CYP4V2 gene has nucleotide sequence as follows:

ATGGCGGGGCTCTGGCTGGGGCTCGTGTGGCAGAAGCTGCTGCTGTGGGGCGCGGCGAGTGCCCTTTC CCTGGCCGGCGCCAGTCTGGTCCTGAGCCTGCTGCAGAGGGTGGCGAGCTACGCGCGGAAATGGCAGCAGATGCGG CCCATCCCCACGGTGGCCCGCGCCTACCCACTGGTGGGCCACGCGCTGCTGATGAAGCCGGACGGGCGAGAATTTT TTCAGCAGATCATTGAGTACACAGAGGAATACCGCCACATGCCGCTGCTGAAGCTCTGGGTCGGGCCAGTGCCCAT GGTGGCCCTTTATAATGCAGAAAATGTGGAGGTAATTTTAACTAGTTCAAAGCAAATTGACAAATCCTCTATGTAC AAGTTTTTAGAACCATGGCTTGGCCTAGGACTTCTTACAAGTACTGGAAACAAATGGCGCTCCAGGAGAAAGATGT TAACACCCACTTTCCATTTTACCATTCTGGAAGATTTCTTAGATATCATGAATGAACAAGCAAATATATTGGTTAA GAAACTTGAAAAACACATTAACCAAGAAGCATTTAACTGCTTTTTTTACATCACTCTTTGTGCCTTAGATATCATC TGTGAAACAGCTATGGGGAAGAATATTGGTGCTCAAAGTAATGATGATTCCGAGTATGTCCGTGCAGTTTATAGAA TGAGTGAGATGATATTTCGAAGAATAAAGATGCCCTGGCTTTGGCTTGATCTCTGGTACCTTATGTTTAAAGAAGG ATGGGAACACAAAAAGAGCCTTCAGATCCTACATACTTTTACCAACAGTGTCATCGCTGAACGGGCCAATGAAATG AACGCCAATGAAGACTGTAGAGGTGATGGCAGGGGCTCTGCCCCCTCCAAAAATAAACGCAGGGCCTTTCTTGACT TGCTTTTAAGTGTGACTGATGACGAAGGGAACAGGCTAAGTCATGAAGATATTCGAGAAGAAGTTGACACCTTCAT GTTTGAGGGGCACGATACAACTGCAGCTGCAATAAACTGGTCCTTATACCTGTTGGGTTCTAACCCAGAAGTCCAG AAAAAAGTGGATCATGAATTGGATGACGTGTTTGGGAAGTCTGACCGTCCCGCTACAGTAGAAGACCTGAAGAAAC TTCGGTATCTGGAATGTGTTATTAAGGAGACCCTTCGCCTTTTTCCTTCTGTTCCTTTATTTGCCCGTAGTGTTAG TGAAGATTGTGAAGTGGCAGGTTACAGAGTTCTAAAAGGCACTGAAGCCGTCATCATTCCCTATGCATTGCACAGA GATCCGAGATACTTCCCCAACCCCGAGGAGTTCCAGCCTGAGCGGTTCTTCCCCGAGAATGCACAAGGGCGCCATC CATATGCCTACGTGCCCTTCTCTGCTGGCCCCAGGAACTGTATAGGTCAAAAGTTTGCTGTGATGGAAGAAAAGAC CATTCTTTCGTGCATCCTGAGGCACTTTTGGATAGAATCCAACCAGAAAAGAGAAGAGCTTGGTCTAGAAGGACAG TTGATTCTTCGTCCAAGTAATGGCATCTGGATCAAGTTGAAGAGGAGAAATGCAGA TGAACGCTAA (SEQ ID NO: 1),

Its protein encoded has amino acid sequence as follows:

MAGLWLGLVW QKLLLWGAAS ALSLAGASLV LSLLQRVASY ARKWQQMRPI PTVA RAYPLVGHALLMKPDG REFFQQIIEY TEEYRHMPLL KLWVGPVPMV ALYNAENVEV ILTSSKQIDK SSMYKFLEPW LGLGLLTSTG NKWRSRRKML TPTFHFTILE DFLDIMN EQA NILVKKLEKH INQEAFNCFF YITLCALDII CETAMGKNIG AQSNDDSEYV RAV YRMSEMI FRRIKMPWLW LDLWYLMFKE GWEHKKSLQI LHTFTNSVIA ERANEMNA NE DCRGDGRGSA PSKNKRRAFL DLLLSVTDDE GNRLSHEDIR EEVDTFMFEG HDT TAAAINW SLYLLGSNPE VQKKVDHELD DVFGKSDRPA TVEDLKKLRY LECVIKETL R LFPSVPLFAR SVSEDCEVAG YRVLKGTEAV IIPYALHRDP RYFPNPEEFQ PERFFPE NAQ GRHPYAYVPF SAGPRNCIGQ KFAVMEEKTI LSCILRHFWI ESNQKREELG LEG QLILRPS NGIWIKLKRR NADER(SEQ ID NO:2).

The CYP4V2 gene mutation body of inventor's discovery is mutated, i.e. phase compared with SEQ ID NO:1 with c.791del T For wild type CYP4V2 gene, the 791st bit base of cDNA of CYP4V2 gene mutation body of the invention lacks T.Its institute as a result, Compared with the CYP4V2 of wild type, frame internal shift occurs the product of coding for the 264th amino acids by phenylpropyl alcohol ammonia due to base deletion Acid mutation is leucine, and all amino acid sequences thereafter is made to change.

It is known that CYP4V2 coded by said gene overall length 19.28kp, includes 11 exons, coded product is 525 amino acid Protein, which belongs to CYP450 family, plays a significant role in fatty acid metabolism.Currently, people are for intracorporal The cognition of CYP4V2 gene is less, also there is not yet CYP4V2 gene and c.791del T thereon sport primary crystalline view The relevant report of the pathogenic mutation of nethike embrane denaturation.

According to the second aspect of the invention, the invention proposes a kind of isolated polypeptides.According to an embodiment of the invention, should Isolated polypeptide has the amino acid sequence as shown in SEQ ID NO:23:

MAGLWLGLVWQKLLLWGAASALSLAGASLVLSLLQRVASYARKWQQMRPIPTVARAYPLVGHALLMKP DGREFFQQIIEYTEEYRHMPLLKLWVGPVPMVALYNAENVEVILTSSKQIDKSSMYKFLEPWLGLGLLTSTGNKWR SRRKMLTPTFHFTILEDFLDIMNEQANILVKKLEKHINQEAFNCFFYITLCALDIICETAMGKNIGAQSNDDSEYV RAVYRMSEMIFRRIKMPWLWLDLWYLMFKEGWEHKKSLQILHTLPTVSSLNGPMKMTPMKTVEVMAGALPPPKINA GPFLTCFLVVLMTKGTGLVMKIFEKKLTPSCLRGTIQLQLQITGPYTCWVLTQKSRKKWIMNWMTCLGSLTVPLQA KTERNFGIWNVLLRRPFAFFLLFLYLPVVLVKIVKWQVTEFRKALKPSSFPMHCTEIRDTSPTPRSSSLSGSSPRM HKGAIHMPTCPSLLAPGTVPVKSLLQWKKRPFFRASLGTFGRNPTRKEKSLVRKDSGFFVQVMASGSSGRGEMQMNX (SEQ ID NO:23).

Specifically, relative to wild type CYP4V2, isolated the 264th ammonia of polypeptide (i.e. CYP4V2 mutant) of the invention Base acid sports leucine by phenylalanine, and causes 264 later amino acid that frameshift mutation occurs.According to the present invention one A little specific examples, the polypeptide are by the nucleic acid encode of the coding CYP4V2 mutant of aforementioned separation.By detecting biological sample In whether express the polypeptide, the whether susceptible primary crystalline degeneration of retina of biological sample can be effectively detected, can also be with It whether there is in organism by detecting these polypeptides, can effectively predict the whether susceptible primary crystalline view of organism Nethike embrane denaturation.

According to the third aspect of the invention we, susceptible primary crystalline degeneration of retina is screened the invention proposes a kind of The method of biological sample.According to an embodiment of the invention, method includes the following steps:

From the 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 can extract reflection biological sample CYP4V2 from the biological sample whether there is the sample of nucleic acid of mutation i.e. It can.According to an embodiment of the invention, biological sample can be at least one selected from blood of human body, skin, subcutaneous tissue, preferably Peripheral blood.Thus, it is possible to easily be sampled and detect, so as to further increase the susceptible primary crystalline view of screening The efficiency of the biological sample of nethike embrane denaturation.According to an embodiment of the invention, term " sample of nucleic acid " used herein above should do extensively Reason and good sense solution, can be it is any be able to reflect CYP4V2 in biological sample with the presence or absence of the sample of mutation, such as can be from life The complete genome DNA directly extracted in object sample is also possible to a part in the full-length genome comprising CYP4V2 coded sequence, It can be the total serum IgE extracted from biological sample, be also possible to the mRNA extracted from biological sample.One according to the present invention Embodiment, the sample of nucleic acid are complete genome DNA.Thus, it is possible to expand the source range that comes of biological sample, and can be simultaneously The much information of biological sample is determined, so as to improve the biology for screening susceptible primary crystalline degeneration of retina The efficiency of sample.In addition, according to an embodiment of the invention, for RNA is used as sample of nucleic acid, from extraction from biological material nucleic acid Sample may further include: from extraction from biological material RNA sample, preferably RNA sample is mRNA；And based on obtained RNA sample obtains cDNA sample by reverse transcription reaction, and obtained cDNA sample constitutes sample of nucleic acid.Thus, it is possible into One step improves the efficiency that the biological sample of susceptible primary crystalline degeneration of retina is screened using RNA as sample of nucleic acid.

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, enrichment CYP4V2 exon, screening enrichment can according to an embodiment of the invention, can screen to sample of nucleic acid To be carried out during constructing sequencing library, or after building sequencing library before constructing sequencing library.It is according to the present invention One embodiment, for sample of nucleic acid, constructing nucleic acid sequencing library further comprises: being drawn using CYP4V2 exon specificity Object carries out PCR amplification to sample of nucleic acid；And it is directed to obtained amplified production, construct nucleic acid sequencing library.Thus, it is possible to By PCR amplification, it is enriched with CYP4V2 exon, so as to further increase the susceptible primary crystalline degeneration of retina of screening Biological sample efficiency.According to an embodiment of the invention, the sequence of CYP4V2 exon specific primer is not particularly limited, Preferred embodiment in accordance with the present invention, the CYP4V2 exon specific primer have nucleosides shown in SEQ ID NO:13 and 14 Acid sequence:

Forward primer: 5 '-GCTTCATGGGATGCGTAATAGC-3 ' (SEQ ID NO:13)；

Reverse primer: 5 '-GAAATGAACGGTGGGGATGGT-3 ' (SEQ ID NO:14).

It is surprisingly found by the inventors that by using above-mentioned primer, can significantly be effectively completed in PCR reaction system pair The amplification of CYP4V2 exon.

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；Feb2010), 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 CYP4V2 in column.

Finally, after determining the nucleic acid sequence of sample of nucleic acid, by the nucleic acid sequence and SEQ of obtained sample of nucleic acid The sequence of ID NO:1 compared to pair.If be mutated in obtained nucleic acid sequence with c.791del T, biological sample is indicated Susceptible primary crystalline degeneration of retina.It is regarded as a result, by the susceptible primary crystalline of screening according to an embodiment of the present invention The method of the biological sample of nethike embrane denaturation, can effectively screen the biological sample of susceptible primary crystalline degeneration of retina. According to an embodiment of the invention, nucleic acid sequence is not particularly restricted with the SEQ ID NO:1 method and apparatus being compared, It can be operated, specific example according to the present invention, can be compared using SOAP software using the software of any conventional.

It should be noted that according to an embodiment of the present invention " screen the biology of susceptible primary crystalline degeneration of retina The purposes of the method for sample " is not particularly limited, such as may be used as the screening technique of non-diagnostic purpose.

Screen the system and kit of the biological sample of susceptible primary crystalline degeneration of retina

According to the fourth aspect of the invention, the invention proposes one kind can effectively implement the susceptible primary knot of above-mentioned screening The system of the method for the biological sample of brilliant sample retinosis.

With reference to Fig. 1, according to an embodiment of the invention, the biological sample of the susceptible primary crystalline degeneration of retina of the screening System 1000 include 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, CYP4V2 exon can be enriched with, further by PCR amplification Improve the efficiency for screening the biological sample of susceptible primary crystalline degeneration of retina.Library construction unit 201 can be into as a result, One step includes PCR amplification module (not shown), and CYP4V2 exon specificity is provided in the PCR amplification module and is drawn Object carries out PCR amplification to the sample of nucleic acid to utilize CYP4V2 exon specific primer, according to the present invention specific Embodiment, CYP4V2 exon specific primer have the nucleotide sequence as shown in SEQ ID NO:13 and 14.According to this hair Bright embodiment, sequencing unit 202 may include at least one selected from HISEQ2000, SOLiD, 454 and single-molecule sequencing device Kind.Newest sequencing technologies are combined as a result, can achieve higher sequencing depth, detection sensitivity and standard for single locus True property greatly improves, it is thus possible to the characteristics of using the high throughput of these sequencing devices, deep sequencing, further increase to nucleic acid The efficiency that sample is tested and analyzed.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 difference of nucleic acid sequence and SEQ ID NO:1 based on sample of nucleic acid judges biological sample The whether susceptible primary crystalline degeneration of retina of product.Specifically, based on the nucleic acid sequence of sample of nucleic acid and SEQ ID NO:1 phase Than, if it is mutated with c.791del T, judges the whether susceptible primary crystalline degeneration of retina of biological sample.Such as preceding institute It states, according to one embodiment of present invention, the nucleic acid sequence of sample of nucleic acid has c.791del T compared with SEQ ID NO:1 Mutation, is the instruction of the susceptible primary crystalline degeneration of retina of biological sample.As previously mentioned, according to an embodiment of the invention, Nucleic acid sequence is not particularly restricted with the SEQ ID NO:1 equipment being compared, can using any conventional software into Row operation, specific example according to the present invention can be compared using SOAP software.

The system is utilized as a result, can effectively implement the life of the aforementioned susceptible primary crystalline degeneration of retina of screening The method of object sample, so as to effectively screen the biological sample of susceptible primary crystalline degeneration of retina.

According to the fifth aspect of the invention, the invention proposes one kind becomes for screening susceptible primary crystalline retinal The kit of the biological sample of property.According to an embodiment of the invention, this is used to screen susceptible primary crystalline degeneration of retina The kit of biological sample include: the reagent for being adapted to detect for CYP4V2 gene mutation body, wherein compared with SEQ ID NO:1, The CYP4V2 gene mutation body is mutated with c.791del T.It, can be effective using the kit of embodiment according to the present invention Screen the biological sample of susceptible primary crystalline degeneration of retina in ground.Herein, used term " is adapted to detect for The reagent of CYP4V2 gene mutation body " shall be understood in a broad sense, it can is the reagent for detecting CYP4V2 encoding gene, is also possible to The reagent of CYP4V2 mutant polypeptide is detected, such as can be using the antibody of identification specific position.One according to the present invention Embodiment, the reagent are nucleic acid probe or primer, it is preferable that the nucleic acid probe or primer have such as SEQ ID NO:13- Nucleotide sequence shown in 14.Thus, it is possible to efficiently screen the biological sample of susceptible primary crystalline degeneration of retina.

It should be noted that in the method for the biological sample for screening susceptible primary crystalline degeneration of retina herein above Feature and advantage described in part are equally applicable to screen the biological sample of susceptible primary crystalline degeneration of retina System or kit, details are not described herein.

Construct and recombinant cell

According to the sixth aspect of the invention, the 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 CYP4V2 mutant of mentioned-above separation, i.e., CYP4V2 gene mutation body of the invention.By This, the recombinant cell obtained using construct transformed acceptor cell of the invention can be efficiently used for screening treatment primary The drug of crystalline degeneration of retina.Wherein, the type of the recipient cell is not particularly limited, such as can be Escherichia coli Cell, 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 the seventh aspect of the 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 CYP4V2 gene mutation body entrained by expression construct.According to some embodiments of the present invention, thin using recombination of the invention Born of the same parents can effectively screen the drug for the treatment of primary crystalline degeneration of retina.According to an embodiment of the invention, recipient cell Type be not particularly limited, such as can be Bacillus coli cells, mammalian cell, the preferably described recipient cell derives from Non-human mammal.

The embodiment of the present invention is described below in detail.The embodiments described below is exemplary, and is only used for explaining this hair It is bright, and be not considered as limiting the invention.Particular technique or condition are not specified in embodiment, according to text in the art It offers described technology or conditions or is carried out according to product description.Reagents or instruments used without specified manufacturer, For can be with conventional products that are commercially available.

Embodiment 1 determines primary crystalline degeneration of retina (BCD) pathogenic mutation

1, sample collection

2010, inventor was collected into 1 BCD patient in Chongqing, the family member of the patient totally 4 people, wherein patient 1 People, normal 3 people of member (wherein father is dead, and eyesight and the visual field are normal before death, no yctalopia).Participate in the present invention studies totally 3 People, wherein 1 people of patient, 2 people of normal member (mother and younger brother) are all to participate in the family members that the present invention studies and endorsed to know Feelings letter of consent.The pedigree chart of the patient is as shown in Figure 2, wherein indicates that normal male, ■ indicate male patient, and zero indicates just Normal women,Indicate that the normal male that died, M indicate mutation ,+it is expressed as wild type.

In the family, 49 annual expenditure of patient shows yctalopia, and funduscopy shows as typical crystalline degeneration of retina and changes.Figure 3 show the fundus image of the BCD patient.As seen from Figure 3, steel gray to lose normal retina red for the double eye grounds of the patient Polishing is damp, and more yellow-white crystalline sample particle deposition, retinal pigment epithelium atrophy, Posterior pole are seen in Posterior pole retina Choroidal sclerosis, a large amount of retina osteocyte sample pigment depositions of Posterior pole.The phenotype table of crystalline degeneration of retina in the family It is now recessive inheritance mode, the non-illness of mother of patient is that heterozygosis carrier (because father is dead, can not trace to the source and carry heterozygosis Mutation situation)；Children's not illness that parents are disease-free meets the complete recessive inheritance feature of autosome.

2, pathogenic mutation is determined

Inventor is using HISEQ2000, SOLiD, 454 or single-molecule sequencing device, using Sanger sequencing approach to elder generation The exon group sequence of card person (member representated by ■ in Fig. 2) is sequenced.It is specific as follows:

2.1DNA extracting

The peripheral blood for acquiring patient in patient's family shown in Fig. 2, is extracted from peripheral blood sample using DNA extraction kit Genomic DNA, and utilize the concentration and purity of spectrophotometer measurement DNA, the OD of each genomic DNA of gained₂₆₀/OD₂₈₀It should all Between 1.7-2.0, concentration be no less than 200 nanograms/microlitre, total amount is no less than 30 micrograms.

The capture of 2.2 target areas and sequencing

Each genomic DNA sample is broken into the segment of 100~500bp or so at random using ultrasonoscope, then according to Manufacturer provide operational manual, segment both ends be separately connected top connection preparation library (reference can be made to: http: // The Illumina/Solexa standard that www.illumina.com/ is provided builds library specification, by referring to being incorporated by this Text).By PCR amplification and capture reagent SureSelect Biotiny lated RNA Library after library is purified (BAITS) hybridization enrichment is carried out, using PCR amplification, be available on the machine sequencing after library detection is qualified, to obtain primitive sequencer Data.Wherein, microarray dataset HiSeq, reading length are 35~100bp, the average sequencing depth of each sample is at least 50 ×.

3, variation detection, annotation and database compare

It is handled using raw sequencing data of the Illumina basecalling Software1.7 to above-mentioned acquisition, After filtering depollutes, using SOAPaligner/SOAP2(reference can be made to: Li R, Li Y, Kristiansen K, et al, SOAP:short oligonucleotide alignment program.Bioinformatics2008,24(5):713- 714；Li R,Yu C,Li Y,ea al,SOAP2:an improved ultrafast tool for short read Alignment.Bioinformatics2009,25 (15): 1966-1967, by referring to be incorporated by herein) compare arrive The UCSC mankind refer to genome (hg19, build37.1, http://genome.ucsc.edu/), compare to obtain to gene Unique aligned sequences in group.Then using 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 Res2009,19 (6): 1124-1132, by referring to being incorporated by herein) genotype that determines target region, obtain the mononucleotide of patient Polymorphism (SNPs) and insertion/deletion sequencing result.

Then pass through dbSNP database (http://hgdownload.cse.ucsc.edu/goldenPath/hg19/ Database/snp135.txt.gz.), HapMap database (ftp: //ftp.ncbi.nlm.nih.gov/hapmap), thousand people Genome database (ftp: //ftp.1000genomes.ebi.ac.uk/vol1/ftp), Yan Di and Huang Di, two legendary rulers of remote antiquity's database (http: // Same sense mutation in the sequencing result of above-mentioned acquisition, non-is removed in the filtering of public databases such as yh.genomics.org.cn/) The variation of code area and all known and variation of the gene frequency greater than 0.005 in the database, and utilize SIFT Software carries out SNP function prediction, finally obtains 3 de novo SNP sites that may have pathogenic meaning.

Above-mentioned 3 de novo SNP site is swept in family member and in normal population control group genomic DNA It retouches, it is final to determine that c.791del T heterozygous mutant causes CYP4V2 albumen that deletion mutation, this gene mutation occurs to CYP4V2 gene It is isolated in the autosomal recessive inheritance crystalline degeneration of retina family with disease phenotype, and in normal control population The mutation is not detected.To sum up speculate, CYP4V2 gene c.791del T mutation be BCD pathogenic mutation.

Embodiment 2: Sanger method sequence verification is carried out to candidate CYP4V2 gene

For CYP4V2 gene (exons 1-11) primers, pass through PCR amplification, product purification, the side of sequencing Method obtains related sequence.Concrete operations are as follows:

1.DNA is extracted

Propositus (member representated by ■ in Fig. 2) peripheral blood in above-mentioned BCD family is acquired, conventional phenol-chloroform method is utilized Extract the genomic DNA in peripheral white blood cells, using the concentration and purity of spectrophotometer measurement DNA, resulting each mark The OD of this genomic DNA₂₆₀/OD₂₈₀Be respectively positioned between 1.7-2.0, concentration be no less than 200 nanograms/microlitre, it is micro- that total amount is no less than 30 Gram.

2. design of primers

PCR reacts design of primers and refers to human genomic sequence, is specifically shown in the following table 1:

Table 1

Then, the PCR reaction system of each genomic DNA sample is prepared according to following proportion respectively and carry out PCR reaction:

PCR reaction system:

System composition	Volume
		DNA profiling (20 nanograms/microlitre)	1 microlitre
2 × GC buffer I (joined Mg2+)	2.5 microlitre
		2mM dNTP	2 microlitres

LA Taq enzyme (5U/ microlitres)	0.25 microlitre
		Primer (100 nanograms/microlitre) it is positive/negative to	Each 1 microlitre
Deionized water	Add to 25 microlitres

Reaction condition:

The pcr amplification product that each receptor gene organizes DNA sample is obtained as a result,.

3. sequencing

It will be in step 2 using Sanger sequencing approach using HISEQ2000, SOLiD, 454 or single-molecule sequencing device The pcr amplification product that each receptor gene obtained organizes DNA sample directly carries out DNA sequencing.

Based on sequencing result, and further combined with CYP4V2 c.791 del T protein structural analysis, above-mentioned mutation is prompted On CYP4V2 gene extron 6, since encoding base lacks on exon, therefore lead to the password after 791 base of catastrophe point It is sub to be all displaced, make 264 and subsequent amino acid sequence all changes, generates one section and contain 261 amino acid Abnormal polypeptide.The anomaly sxtructure of exon 6 is not only generated, but also protein translation is abnormal to influence proteinase activity.As a result, Show the CYP4V2 gene mutation body Disease-causing gene that c.791 del T is BCD.

Embodiment 3: Sanger method sequence verification is carried out to pathogenic mutation

It is (including normal in patient and family to all family members in BCD patient's family described in embodiment 1 respectively People) and the CYP4V2 gene of the outer normal person of 100 familys detect: using in embodiment 2 for CYP4V2 gene extron The primer of 6 design of son obtains the related sequence in mutational site by the method for PCR amplification, product purification and sequencing, according to determining sequence Column measurement result belongs to saltant type or wild type, verifies the correlation between CYP4V2 gene and the mutation and BCD.

Steps are as follows for specific method:

1. DNA is extracted

According to the method for extracting DNA described in embodiment 2, the gene in preparation subject's peripheric venous blood is extracted respectively DNA is organized, it is spare.

2, PCR reacts

Using the primer and PCR condition that CYP4V2 gene extron 6 designs is directed in embodiment 2, PCR reaction is carried out.By This, obtains the pcr amplification product that each receptor gene organizes DNA sample.

3, it is sequenced

Using HISEQ2000, SOLiD, 454 or single-molecule sequencing device, each receptor gene's group that will be obtained in step 2 The pcr amplification product of DNA sample directly carries out Sanger method sequence verification.Propositus in family, normal person and family in family The representative Sanger sequence verification peak figure in the CYP4V2 gene of the outer normal control c.791 mutational site del T is as shown in Figure 4.

As shown in Figure 4, CYP4V2 gene wild type is that 791T/T is homozygous, and saltant type is 791T/C heterozygous, and mutation is led It causes 264 phenylalanines of CYP4V2 albumen to change into leucine, and causes subsequent base that frame internal shift occurs, lead to amino Sour unusual combination.In addition, c.791del T heterozygous mutant does not appear as disease phenotype in family by verifying discovery CYP4V2 It isolates, i.e., patient carries the heterozygous mutant (2 allele), and uninvolved family member (mother and younger brother) is respective Carry another pathogenic allelic mutation.Meanwhile inventor is in 100 and the normal control of BCD family's affinity-less relation Middle investigation does not find the mutational site.

Further prove that CYP4V2 gene is the Disease-causing gene of BCD as a result, the c.791del T of CYP4V2 gene is sported The pathogenic mutation of BCD.

Embodiment 4:CYP4V2c.791del T(, that is, F264) species conservative Analysis

Be compared using species homologies of the ncbi database to CYP4V2 gene (http: // www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=homologene&dopt= Ent&list_uids=5859 MultipleAlignm), as a result see Fig. 5.Fig. 5 shows the sequence of multiple species CYP4V2 albumen Column comparing result.As shown in Figure 5, CYP4V2c.791T is highly conserved in mammals, shows 264 paddy of CYP4V2 albumen Glutamine is conservative between species.It is prominent to further demonstrate that the c.791del T of CYP4V2 gene sports causing a disease for BCD as a result, Become.

Embodiment 5: detection kit

A detection kit is prepared, it includes the primers that the c.791del T for being able to detect CYP4V2 gene is mutated, and are used for The biological sample of susceptible primary crystalline degeneration of retina is screened, wherein these primers are the special of CYP4V2 gene extron Property primer, shown in sequence SEQ ID NO:13-14 as described in example 2 above.

The specific steps of the biological sample of susceptible primary crystalline degeneration of retina are screened using mentioned reagent box are as follows: press Method described in step 1 according to embodiment 2 extracts person under test DNA, using extracted DNA as template and above-mentioned CYP4V2 gene 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 c.791del T mutation by observation, this hair can be effectively detected Bright CYP4V2 gene mutation body whether there is in person under test DNA, so as to which the whether susceptible original of person under test is effectively detected Hair property crystalline degeneration of retina further can filter out susceptible primary crystalline degeneration of retina from person under test Biological sample.

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 Centainly refer to identical embodiment or example.Moreover, particular features, structures, materials, or characteristics described can be any One or more embodiment or examples in can be combined in any suitable 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 A variety of change, modification, replacement and modification can be carried out to these embodiments in the case where being detached from the principle of the present invention and objective, this The range of invention is defined by the claims and their equivalents.

Claims (9)

1. a kind of system for the biological sample for screening susceptible primary crystalline degeneration of retina characterized by comprising

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 is compared with SEQ ID NO:1, if is mutated with c.791del T, judges the whether susceptible primary of the biological sample Crystalline degeneration of retina.

2. system according to claim 1, 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.

3. system according to claim 1, which is characterized in that 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.

4. system according to claim 3, which is characterized in that the library construction unit further comprises:

PCR amplification module is provided with CYP4V2 gene extron specific primer in the PCR amplification module, to utilize institute Specific primer is stated, PCR amplification is carried out to the sample of nucleic acid.

5. system according to claim 4, which is characterized in that the specific primer is such as SEQ ID NO:13-14 institute The nucleotide sequence shown.

6. system according to claim 3, which is characterized in that the sequencing unit include selected from HISEQ2000, SOLiD, 454 and single-molecule sequencing device at least one.

7. a kind of for screening the kit of the biological sample of susceptible primary crystalline degeneration of retina, which is characterized in that contain Have:

It is adapted to detect for the reagent of CYP4V2 gene mutation body, wherein compared with SEQ ID NO:1, the CYP4V2 gene mutation body With being c.791delT mutated.

8. kit according to claim 7, which is characterized in that the reagent is nucleic acid probe or specific primer.

9. kit according to claim 8, which is characterized in that the specific primer is such as SEQ ID NO:13-14 Shown in nucleotide sequence.