CN103290031B - CARD9 gene mutant and application thereof - Google Patents
CARD9 gene mutant and application thereof Download PDFInfo
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- CN103290031B CN103290031B CN201210055659.8A CN201210055659A CN103290031B CN 103290031 B CN103290031 B CN 103290031B CN 201210055659 A CN201210055659 A CN 201210055659A CN 103290031 B CN103290031 B CN 103290031B
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Abstract
The invention relates to an isolated nucleic acid for coding a CARD9 mutant, an isolated polypeptide, a method for screening a biological sample susceptible to dematiaceous fungi, a system for screening a biological sample susceptible to dematiaceous fungi, a kit for screening a biological sample susceptible to dematiaceous fungi and a method for screening a drug for treating or preventing phaeohyphomycosis, wherein compared with SEQ ID NO: 1, the isolated nucleic acid for coding the CARD9 mutant has at least one mutant selected from c.G241A, c.G104A, c.G472T, c.191insTGCT and c.818insG. Whether the biological sample is susceptible to dematiaceous fungi can be effectively detected by detecting whether the mutants exist in the biological sample.
Description
Technical field
The present invention relates to CARD9 gene mutation body and application thereof.Particularly, the present invention relates to a kind of nucleic acid of coding CARD9 mutant of separation, a kind of isolated polypeptide, a kind of screen the biological sample of susceptible Dematiaceous fungi method, a kind ofly screen the system of the biological sample of susceptible Dematiaceous fungi, a kind of test kit of the biological sample for screening susceptible Dematiaceous fungi, a kind of method of screening the medicine for the treatment of or prevention phaeohyphomycosis.
Background technology
Phaeohyphomycosis (phaeohyphomycosis) be one group of Dematiaceous fungi cause with tissue in have dark-coloured mycelia to be feature skin, subcutis or systemic infection.Within 1974, Ajello proposes this name.Phaeohyphomycosis is dispersed in Case report all over the world, but is more common in the torrid zone.The ground such as China Shandong, northeast and Zhanjiang have Sporadic cases to report.Pathogenic pathway is exogenous infection and opportunistic sexuality dye mainly.Route of infection may be that pathogenic bacteria is implanted through skin injury place or sucks fungal spore.Clinical manifestation is fester, ecchymosis, brown-black spot or verrucous hyperplasia, consciously micro-ly to itch or slight distending pain, and what have can no conscious sympton.By Mycology examination and tissue pathology checking's diagnosis.Mostly insensitive to antifungal drug, curative effect is not good enough.Majority shows as falls ill since the childhood, repeatedly chance of occurrence pathogenic infection, and antifungal therapy effectively but recur very soon after drug withdrawal.
Therefore, at present the research of phaeohyphomycosis is still needed deeply and improved.
Summary of the invention
The present invention is intended at least to solve one of technical problem existed in prior art.For this reason, one object of the present invention be to propose a kind of can the method for biological sample of Effective selection susceptible Dematiaceous fungi.
The present invention completes based on the following work of contriver: contriver determines the new mutant on the Disease-causing gene of phaeohyphomycosis by the method that exon group checks order.
According to a first aspect of the invention, the present invention proposes a kind of nucleic acid of coding CARD9 mutant of separation.C.191insTGCT and c.818insG according to embodiments of the invention, compared with SEQ ID NO:1, this nucleic acid has and is selected from the sudden change of following at least one: c.G241A, c.G104A, c.C472T.According to embodiments of the invention, contriver determines the new mutant of multiple CARD9 gene, the morbidity of these new mutant and phaeohyphomycosis is closely related, thus whether exist in biological sample by detecting these new mutant, effectively can detect biological sample whether susceptible Dematiaceous fungi.
According to a second aspect of the invention, the present invention proposes a kind of isolated polypeptide.According to embodiments of the invention, compared with SEQID NO:2, this isolated polypeptide has and is selected from the sudden change of following at least one: p.E81K, p.R35Q, p.Q158X, p.L64fsX59 and p.D274fsX60.According to concrete examples more of the present invention, this polypeptide is by the nucleic acid encoding of the coding CARD9 mutant of aforementioned separation.By detecting in biological sample whether express this polypeptide, biological sample whether susceptible Dematiaceous fungi effectively can be detected.
According to a third aspect of the invention we, the present invention proposes a kind of method of screening the biological sample of susceptible Dematiaceous fungi.According to embodiments of the invention, the method for the biological sample of this screening susceptible Dematiaceous fungi comprises the following steps: from extraction from biological material sample of nucleic acid; The nucleotide sequence of definite kernel acid sample; The nucleotide sequence of this sample of nucleic acid compared with SEQ ID NO:1, have be selected from c.G241A, c.G104A, c.C472T, at least one sudden change c.191insTGCT and be c.818insG the instruction of biological sample susceptible Dematiaceous fungi.By the method for the biological sample of the screening susceptible Dematiaceous fungi according to the embodiment of the present invention, the biological sample of susceptible Dematiaceous fungi effectively can be screened.
According to a forth aspect of the invention, the present invention proposes a kind of system of screening the biological sample of susceptible Dematiaceous fungi.According to embodiments of the invention, the system of the biological sample of this screening susceptible Dematiaceous fungi comprises: nucleic acid-extracting apparatus, and this nucleic acid-extracting apparatus is used for from extraction from biological material sample of nucleic acid; Nucleotide sequence determining device, this nucleotide sequence determining device is connected with nucleic acid-extracting apparatus, for analyzing sample of nucleic acid, so that the nucleotide sequence of definite kernel acid sample; Judgment means, this judgment means is connected with nucleotide sequence determining device, so that based on the nucleotide sequence of sample of nucleic acid compared with SEQ ID NO:1, whether have and be selected from c.G241A, c.G104A, c.C472T, at least one sudden change c.191insTGCT and c.818insG, judge biological sample whether susceptible Dematiaceous fungi.Utilize this system, effectively can implement the method for the biological sample of aforementioned screening susceptible Dematiaceous fungi, thus effectively can screen the biological sample of susceptible Dematiaceous fungi.
According to a fifth aspect of the invention, the present invention proposes a kind of test kit of the biological sample for screening susceptible Dematiaceous fungi.According to embodiments of the invention, this test kit being used for the biological sample screening susceptible Dematiaceous fungi comprises: be suitable for the reagent detecting CARD9 gene mutation body, wherein compared with SEQ ID NO:1, CARD9 gene mutation body have be selected from c.G241A, c.G104A, c.C472T, c.191insTGCT and c.818insG at least one sudden change.Utilize test kit according to an embodiment of the invention, effectively can screen the biological sample of susceptible Dematiaceous fungi.
According to a sixth aspect of the invention, the present invention proposes a kind of method of screening the medicine for the treatment of or prevention phaeohyphomycosis.According to embodiments of the invention, the method of the medicine of this screening treatment or prevention phaeohyphomycosis comprises the following steps: cultivated when there is candidate agent by biological sample, and wherein biological sample is expressed and have the polypeptide being selected from following at least one and suddenling change compared with SEQ ID NO:2: p.E81K, p.R35Q, p.Q158X, p.L64fsX59 and p.D274fsX60; Biological sample is cultivated when there is not candidate agent; Determine that biological sample is when existing candidate agent and there is not candidate agent, to the susceptibility of Dematiaceous fungi; And compare to the susceptibility of Dematiaceous fungi, susceptibility when wherein there is candidate agent, lower than susceptibility when there is not candidate agent, is candidate agent as the instruction of the medicine for the treatment of or prevention phaeohyphomycosis.Utilize the method for the medicine of this screening treatment or prevention phaeohyphomycosis, effectively can screen the medicine for the treatment of or prevention phaeohyphomycosis.
Additional aspect of the present invention and advantage will part provide in the following description, and part will become obvious from the following description, or be recognized by practice of the present invention.
Accompanying drawing explanation
Above-mentioned and/or additional aspect of the present invention and advantage will become obvious and easy understand from accompanying drawing below combining to the description of embodiment, wherein:
Fig. 1: show the system of biological sample according to the screening susceptible Dematiaceous fungi of the embodiment of the present invention and the schematic diagram of integral part thereof, wherein,
Figure 1A is the schematic diagram of the system of the biological sample of screening susceptible Dematiaceous fungi according to the embodiment of the present invention,
Figure 1B is the schematic diagram of the nucleic acid-extracting apparatus according to the embodiment of the present invention,
Fig. 1 C is the schematic diagram of the nucleotide sequence determining device according to the embodiment of the present invention; And
Fig. 2: show and verify peak figure according to the Sanger to D6-3 patient CARD9 gene mutation site of the embodiment of the present invention.
Embodiment
Be described below in detail embodiments of the invention, the example of described embodiment is shown in the drawings, and wherein same or similar label represents same or similar element or has element that is identical or similar functions from start to finish.Being exemplary below by the embodiment be described with reference to the drawings, only for explaining the present invention, and can not limitation of the present invention being interpreted as.
CARD9 gene mutation body
According to a first aspect of the invention, the present invention proposes a kind of nucleic acid of coding CARD9 mutant of separation.C.191insTGCT and c.818insG according to embodiments of the invention, compared with SEQ ID NO:1, this nucleic acid has and is selected from the sudden change of following at least one: c.G241A, c.G104A, c.C472T.Phraseology " nucleic acid of coding CARD9 mutant " used in this article, refer to the nucleic acid substances corresponding with the gene of CARD9 mutant of encoding, namely the type of nucleic acid is not particularly limited, can be anyly comprise the deoxyribonucleotide corresponding with the encoding gene of CARD9 mutant and/or the polymkeric substance of ribonucleotide, include but not limited to DNA, RNA or cDNA.According to a concrete example of the present invention, the nucleic acid of foregoing coding CARD9 mutant is DNA.According to embodiments of the invention, the new mutant of contriver is true multiple CARD9 gene, the morbidity of these new mutant and phaeohyphomycosis is closely related, thus whether exist in biological sample by detecting these new mutant, effectively can detect biological sample whether susceptible Dematiaceous fungi, also whether can exist in organism by detecting these mutant, effectively can predict organism whether susceptible phaeohyphomycosis.It should be noted that, when the sporting c.818insG of DNA of the coding CARD9 mutant of separation of the present invention, represent and insert G after the 818th bit base of cDNA sequence, and rear two bit bases of the 818th bit base i.e. the 819th and the 820th bit base is also G, therefore c.820insG c.819insG this sudden change also can be represented as or, thus, in this article, c.818insG sudden change can represent and c.818insG, c.819insG or c.820insG suddenlys change.
The nucleic acid of these codings CARD9 mutant, is the method that present inventor is checked order by exon group, checks order for multiple clinical samples, the new mutant on the Disease-causing gene of the phaeohyphomycosis determined.
Caspase is raised territory albumen 9 (caspase recruitment domain protein 9, CARD9) and is belonged to a member in CARD albumen (CARD-containing proteins) family.The CARD9 assignment of genes gene mapping is as follows in karyomit(e) 9q34.3, CARD9 cDNA sequence:
ATGTCGGACTACGAGAACGATGACGAGTGCTGGAGCGTCCTGGAGGGCTTCCGG
GTGACGCTCACCTCGGTCATCGACCCCTCACGCATCACACCTTACCTGCGGCAGTGCA
AGGTCCTGAACCCCGATGATGAGGAGCAGGTGCTCAGCGACCCCAACCTGGTCATCC
GCAAACGGAAAGTGGGTGTGCTCCTGGACATCCTGCAGCGGACCGGCCACAAGGGCT
ACGTGGCCTTCCTCGAGAGCCTGGAGCTCTACTACCCGCAGCTGTACAAGAAGGTCAC
AGGCAAGGAGCCGGCCCGCGTCTTCTCCATGATCATCGACGCGTCCGGGGAGTCAGG
CCTGACTCAGCTGCTGATGACTGAGGTCATGAAGCTGCAGAAGAAGGTGCAGGACCT
GACCGCGCTGCTGAGCTCCAAAGATGACTTCATCAAGGAGCTGCGGGTGAAGGACAG
CCTGCTGCGCAAGCACCAGGAGCGTGTGCAGAGGCTCAAGGAGGAGTGCGAGGCCGG
CAGCCGCGAGCTCAAGCGCTGCAAGGAGGAGAACTACGACCTGGCCATGCGCCTGGC
GCACCAGAGTGAGGAGAAGGGCGCCGCGCTCATGCGGAACCGTGACCTGCAGCTGGA
GATTGACCAGCTCAAGCACAGCCTCATGAAGGCCGAGGACGACTGCAAGGTGGAGCG
CAAGCACACGCTGAAGCTCAGGCACGCCATGGAGCAGCGGCCCAGCCAGGAGCTGCT
GTGGGAGCTGCAGCAGGAGAAGGCCCTGCTCCAGGCCCGGGTGCAGGAGCTGGAGGC
CTCCGTCCAGGAGGGGAAGCTGGACAGGAGCAGCCCCTACATCCAGGTACTGGAGGA
GGACTGGCGGCAGGCGCTGCGGGACCACCAGGAGCAGGCCAACACCATCTTCTCCCT
GCGCAAGGACCTCCGCCAGGGCGAGGCCCGACGCCTCCGGTGCATGGAGGAGAAGG
AGATGTTCGAGCTGCAGTGCCTGGCACTACGTAAGGACTCCAAGATGTACAAGGACC
GCATCGAGGCCATCCTGCTGCAGATGGAGGAGGTCGCCATTGAGCGGGACCAGAGCA
CACAAATGGAGGGGCTGTGA(SEQ ID NO：1)，
The aminoacid sequence of its coding is as follows:
MSDYENDDECWSVLEGFRVTLTSVIDPSRITPYLRQCKVLNPDDEEQVLSDPNLVIRK
RKVGVLLDILQRTGHKGYVAFLESLELYYPQLYKKVTGKEPARVFSMIIDASGESGLTQLL
MTEVMKLQKKVQDLTALLSSKDDFIKELRVKDSLLRKHQERVQRLKEECEAGSRELKRCK
EENYDLAMRLAHQ SEEKGAALMRNRDLQLEIDQLKHSLMKAEDDCKVERKHTLKLRHA
MEQRPSQELLWELQQEKALLQARVQELEASVQEGKLDRSSPYIQVLEEDWRQALRDHQE
QANTIF SLRKDLRQGEARRLRCMEEKEMFELQCLALRKDSKMYKDRIEAILLQMEEVAIE
RDQSTQMEGL(SEQ ID NO：2)。
In rodent, also identify mouse rCARD9 to encode 536 amino-acid residues, in sequence, have 88% homology with hCARD9.HCARD9 structurally has two functional zone at least, and one is the N-terminal district of CARD9, has 7 ~ 98 amino-acid residues, has remarkable similarity with many apoptotic proteins (as BCL10 and RAIDD etc.) CARD die body; Another is the central zone of CARD9, includes 140 ~ 420 amino-acid residues, primarily of exercising protein oligomerization function, having the coiled coil domain of seven peptide repeated characteristics composition.Known by COILS2 programanalysis, the central zone of CARD9 has three amino-acid residue sections at least, is respectively the coiled coil domain of 140 ~ 230,243 ~ 277,332 ~ 419, spaced apart by low coiled-coiled structure between them.These structural domains and myosin, clathrin have great similarity.CARD9 is positioned in tenuigenin, is present in various human adult tissue, as spleen, liver, placenta, lung, peripheral blood lymphocyte, brain etc., has great expression simultaneously, in tire hepatic tissue, also have expression in HL60 cancerous cell line.CARD is one of protein molecule regulating antiapoptotic signals, and it can regulate apoptosis by many A signal pathways.Dectin-1 is the main pattern recognition receptors of Mammals, can identify the zymosan composition of fungi, and CARD9 is the sensor of Dectin-1 signal path, and this signal path has mediated the inherent immunity of opposing fungi infestation.Contriver predicts that CARD9 is very likely the Disease-causing gene of phaeohyphomycosis.
For this reason, contriver carries out the order-checking of exon group for multiple patient, thus determine multiple CARD9 gene mutation body, i.e. c.G241A, c.G104A, c.C472T, c.191insTGCT and c.818insG, the morbidity height correlation of these mutant and phaeohyphomycosis.Detailed description about these transgenations sees the following form 1.
Further, contriver finds, when there are at least two in said mutation type in CARD mutant simultaneously, then the susceptibility of phaeohyphomycosis is higher.Whether the CARD mutant having multiple mutational site by detecting these exists in biological sample, effectively can detect biological sample whether susceptible Dematiaceous fungi further, also whether can exist in organism by detecting these mutant, effectively can predict organism whether susceptible phaeohyphomycosis.
According to a second aspect of the invention, the present invention proposes a kind of isolated polypeptide.According to embodiments of the invention, compared with SEQID NO:2, this isolated polypeptide has and is selected from the sudden change of following at least one: p.E81K, p.R35Q, p.Q158X, p.L64fsX59 and p.D274fsX60.According to concrete examples more of the present invention, this polypeptide is by the nucleic acid encoding of the coding CARD9 mutant of aforementioned separation.Detailed description about these protein mutants sees the following form 1.By detecting in biological sample whether express this polypeptide, effectively can detecting biological sample whether susceptible Dematiaceous fungi, also whether can exist in organism by detecting these polypeptide, effectively can predict organism whether susceptible phaeohyphomycosis.
Table 1CARD9 gene mutation body
The method of the biological sample of screening susceptible Dematiaceous fungi
According to a third aspect of the invention we, the present invention proposes a kind of method of screening the biological sample of susceptible Dematiaceous fungi.According to embodiments of the invention, the method for the biological sample of this screening susceptible Dematiaceous fungi can comprise the following steps:
First, from extraction from biological material sample of nucleic acid.According to embodiments of the invention, the type of biological sample is also not particularly limited, as long as can extract the sample of nucleic acid whether reflection biological sample CARD9 exists sudden change from this biological sample.According to embodiments of the invention, biological sample can for being selected from blood of human body, skin and hypodermic at least one.Thus, can carry out easily sampling and detecting, thus the efficiency of the biological sample of screening susceptible Dematiaceous fungi can be improved further.According to embodiments of the invention, here used term " sample of nucleic acid " should be interpreted broadly, it can be anyly can reflect in biological sample, whether CARD9 exists the sample of sudden change, it can be such as the complete genome DNA of extracting directly from biological sample, also can be the part comprising CARD9 encoding sequence in this full-length genome, can be the total serum IgE extracted from biological sample, also can be the mRNA extracted from biological sample.According to one embodiment of present invention, described sample of nucleic acid is complete genome DNA.Thus, what can expand biological sample carrys out source range, and can determine the much information of biological sample simultaneously, thus can improve the efficiency of the biological sample of screening susceptible Dematiaceous fungi.In addition, according to embodiments of the invention, for employing RNA as sample of nucleic acid, may further include from extraction from biological material sample of nucleic acid: from extraction from biological material RNA sample, preferred RNA sample is mRNA; And based on obtained RNA sample, by reverse transcription reaction, obtain cDNA sample, the cDNA composition of sample sample of nucleic acid obtained.Thus, the efficiency of the biological sample utilizing RNA as sample of nucleic acid screening susceptible Dematiaceous fungi can be improved further.
Next, after obtaining sample of nucleic acid, can analyze sample of nucleic acid, thus the nucleotide sequence of obtained sample of nucleic acid can be determined.According to embodiments of the invention, determine the method and apparatus of the nucleotide sequence of obtained sample of nucleic acid and be not particularly limited.According to a particular embodiment of the invention, sequence measurement can be passed through, the nucleotide sequence of definite kernel acid sample.According to embodiments of the invention, may be used for the method and apparatus that carries out checking order and be not particularly limited.According to embodiments of the invention, s-generation sequencing technologies can be adopted, also can adopt the third generation and forth generation or more advanced sequencing technologies.According to concrete example of the present invention, can utilize be selected from Hiseq2000, SOLiD, 454 and at least one of single-molecule sequencing device nucleotide sequence is checked order.Thus, in conjunction with up-to-date sequencing technologies, can reach the higher order-checking degree of depth for Single locus, detection sensitivity and accuracy improve greatly, thus can utilize the feature that the high-throughput of these sequencing devices, the degree of depth check order, improve further and carry out detecting the efficiency analyzed to sample of nucleic acid.Thus, follow-up accuracy when sequencing data is analyzed and accuracy can be improved.Thus, according to embodiments of the invention, the nucleotide sequence of definite kernel acid sample may further include: first, for obtained sample of nucleic acid, builds nucleic acid sequencing library; And checked order in obtained nucleic acid sequencing library, to obtain the sequencing result be made up of multiple sequencing data.According to some embodiments of the present invention, can adopt be selected from Hiseq2000, SOLiD, 454 and at least one of single-molecule sequencing device checked order in obtained nucleic acid sequencing library.In addition, according to embodiments of the invention, can screen sample of nucleic acid, enrichment CARD9 exon, this screening enrichment before structure sequencing library, can build in sequencing library process, or carries out after building sequencing library.According to one embodiment of present invention, for sample of nucleic acid, build nucleic acid sequencing library and comprise further: utilize CARD9 exon Auele Specific Primer, pcr amplification is carried out to sample of nucleic acid; And for obtained amplified production, build nucleic acid sequencing library.Thus, can pcr amplification be passed through, enrichment CARD9 exon, thus the efficiency of the biological sample of screening susceptible Dematiaceous fungi can be improved further.According to embodiments of the invention, the sequence of CARD9 exon Auele Specific Primer is not particularly limited, according to a preferred embodiment of the invention, the sequence of these CARD9 exon Auele Specific Primers has nucleotide sequence as shown in table 2 below, i.e. nucleotide sequence shown in SEQ IDNO:3-26.Contriver is surprised to find, and by adopting these primers, can complete the amplification to CARD9 exon in a PCR reaction system by multiplex PCR, and the homogeneity of amplification is good.It should be noted that, these nucleotide sequences shown in SEQ ID NO:3-26 be the present inventor after having paid arduous labor, unexpected to obtain.
Table 2CARD9 exon Auele Specific Primer
About for sample of nucleic acid, build method and the flow process of sequencing library, those skilled in the art suitably can select according to different sequencing technologies, about the details of flow process, the code that can provide see the such as Illumina company of manufacturer of order-checking instrument, for example, see Illumina company Multiplexing Sample Preparation Guide (Part#1005361; Feb 2010) or Paired-End SamplePrep Guide (Part#1005063; Feb 2010), by referring to being incorporated to herein.According to embodiments of the invention, from the method and apparatus of extraction from biological material sample of nucleic acid, be also not particularly limited, commercial nucleic acid extraction kit can be adopted to carry out.
It should be noted that, term " nucleotide sequence " used here should make broad understanding, it can be after the sequencing data that obtains of checking order to sample of nucleic acid is assembled, the complete nucleic acid sequence information obtained, also can be directly adopt sequencing data (reads) by checking order obtained to sample of nucleic acid as nucleotide sequence, as long as the encoding sequence containing corresponding CARD9 in these nucleotide sequences.
Finally, after the nucleotide sequence of definite kernel acid sample, the sequence of the nucleotide sequence of obtained sample of nucleic acid and SEQ ID NO:1 is compared.If have in obtained nucleotide sequence be selected from c.G241A, c.G104A, c.C472T, c.191insTGCT and c.818insG at least one sudden change, then indicator organism sample susceptible Dematiaceous fungi.Thus, by the method for the biological sample of the screening susceptible Dematiaceous fungi according to the embodiment of the present invention, the biological sample of susceptible Dematiaceous fungi can effectively be screened.According to embodiments of the invention, the method and apparatus of compare to nucleotide sequence and SEQ ID NO:1 being also not particularly limited, and the software of any conventional can be adopted to operate, according to specific examples of the present invention, SOAP software can be adopted to compare.
Term " Dematiaceous fungi " used in this article should be interpreted broadly, it can be all pathogenic bacterium of phaeohyphomycosis, such as, include but not limited to: Phialophora verrucosa (Phialophora verrucosa), Exophiala jeanselmei (Exophialajeanselmei), Exophiala dermatitides (Exophiala dermatitidis).
It should be noted that, the purposes according to " method of the biological sample of screening susceptible Dematiaceous fungi " of the embodiment of the present invention is not particularly limited, such as, can be used as the screening method of non-diagnostic object.
The system of the biological sample of screening susceptible Dematiaceous fungi and test kit
According to a forth aspect of the invention, the present invention proposes a kind of system effectively can implementing the method for the biological sample of above-mentioned screening susceptible Dematiaceous fungi.
With reference to figure 1, according to embodiments of the invention, the system 1000 of the biological sample of this screening susceptible Dematiaceous fungi comprises nucleic acid-extracting apparatus 100, nucleotide sequence determining device 200 and judgment means 300.
According to embodiments of the invention, nucleic acid-extracting apparatus 100 is for from extraction from biological material sample of nucleic acid.As previously mentioned, according to embodiments of the invention, the type of sample of nucleic acid is also not particularly limited, and for employing RNA as sample of nucleic acid, then nucleic acid-extracting apparatus comprises RNA extraction unit 101 and reverse transcription unit 102 further, wherein, extraction unit 101 is for from extraction from biological material RNA sample, and reverse transcription unit 102 is connected with RNA extraction unit 101, for carrying out reverse transcription reaction to RNA sample, to obtain cDNA sample, the cDNA composition of sample sample of nucleic acid obtained.
According to embodiments of the invention, nucleotide sequence determining device 200 is connected with nucleic acid-extracting apparatus 100, for analyzing sample of nucleic acid, so that the nucleotide sequence of definite kernel acid sample.As previously shown, the nucleotide sequence of the method definite kernel acid sample of order-checking can be adopted.Thus, according to one embodiment of present invention, described nucleotide sequence determining device 200 may further include: library construction unit 201 and order-checking unit 202.Library construction unit 201, for for sample of nucleic acid, builds nucleic acid sequencing library; Order-checking unit 202 is connected with library construction unit 201, for checking order to nucleic acid sequencing library, to obtain the sequencing result be made up of multiple sequencing data.As previously mentioned, can pcr amplification be passed through, enrichment CARD9 exon, improve the efficiency of the biological sample of screening susceptible Dematiaceous fungi further.Thus, library construction unit 201 may further include pcr amplification module (not shown), CARD9 exon Auele Specific Primer is provided with in this pcr amplification module, to utilize CARD9 exon Auele Specific Primer, pcr amplification is carried out to described sample of nucleic acid, according to a particular embodiment of the invention, CARD9 exon Auele Specific Primer has the nucleotide sequence as shown in SEQ ID NO:3-26.According to embodiments of the invention, order-checking unit 202 can comprise and is selected from HISEQ2000, SOLiD, 454 and at least one of single-molecule sequencing device.Thus, in conjunction with up-to-date sequencing technologies, can reach the higher order-checking degree of depth for Single locus, detection sensitivity and accuracy improve greatly, thus can utilize the feature that the high-throughput of these sequencing devices, the degree of depth check order, improve further and carry out detecting the efficiency analyzed to sample of nucleic acid.Thus, improve follow-up accuracy when sequencing data is analyzed and accuracy.
According to embodiments of the invention, judgment means 300 is connected with nucleotide sequence determining device 200, be suitable for the nucleotide sequence of sample of nucleic acid to compare, to judge biological sample whether susceptible Dematiaceous fungi based on the nucleotide sequence of sample of nucleic acid and the difference of SEQ ID NO:1.Particularly, based on the nucleotide sequence of sample of nucleic acid compared with SEQ ID NO:1, whether have and be selected from c.G241A, c.G104A, c.C472T, at least one sudden change c.191insTGCT and c.818insG, judge biological sample whether susceptible Dematiaceous fungi.As previously mentioned, according to one embodiment of present invention, the nucleotide sequence of sample of nucleic acid compared with SEQ IDNO:1, have be selected from c.G241A, c.G104A, c.C472T, c.191insTGCT and c.818insG at least one sudden change, be the instruction of biological sample susceptible Dematiaceous fungi.As previously mentioned, according to embodiments of the invention, the equipment of compare to nucleotide sequence and SEQ ID NO:1 being also not particularly limited, and the software of any conventional can be adopted to operate, according to specific examples of the present invention, SOAP software can be adopted to compare.
Thus, utilize this system, effectively can implement the method for the biological sample of aforementioned screening susceptible Dematiaceous fungi, thus effectively can screen the biological sample of susceptible Dematiaceous fungi.
According to a fifth aspect of the invention, the present invention proposes a kind of test kit of the biological sample for screening susceptible Dematiaceous fungi.According to embodiments of the invention, this test kit being used for the biological sample screening susceptible Dematiaceous fungi comprises: be suitable for the reagent detecting CARD9 gene mutation body, wherein compared with SEQ ID NO:1, this CARD9 gene mutation body have be selected from c.G241A, c.G104A, c.C472T, c.191insTGCT and c.818insG at least one sudden change.Utilize test kit according to an embodiment of the invention, effectively can screen the biological sample of susceptible Dematiaceous fungi.In this article, the term used " is suitable for detecting the reagent of CARD9 gene mutation body " and should be interpreted broadly, namely can be the reagent detecting CARD9 encoding gene, also can be the reagent detecting CARD9 mutant polypeptide, such as, can adopt the antibody in identification specificity site.According to one embodiment of present invention, described reagent is nucleic acid probe, thus, can screen the biological sample of susceptible Dematiaceous fungi efficiently.
It should be noted that, before this paper, screen the feature and advantage described in method part of the biological sample of susceptible Dematiaceous fungi, be equally applicable to system or the test kit of the biological sample screening susceptible Dematiaceous fungi, do not repeat them here.
The method of the medicine of screening treatment or prevention phaeohyphomycosis
According to a sixth aspect of the invention, the present invention proposes a kind of method of screening the medicine for the treatment of or prevention phaeohyphomycosis.According to embodiments of the invention, the method for the medicine of this screening treatment or prevention phaeohyphomycosis can comprise the following steps:
First, cultivated when there is candidate agent by biological sample, wherein this biological sample is expressed and have the polypeptide being selected from following at least one and suddenling change compared with SEQ IDNO:2: p.E81K, p.R35Q, p.Q158X, p.L64fsX59 and p.D274fsX60.Here used term " cultivation " should be interpreted broadly, and refers to and biological sample is existed with activated state.According to embodiments of the invention, the type of biological sample is not particularly limited, as long as this biological sample can express such peptide species, this polypeptide has and is selected from following at least one and suddenlys change compared with SEQ ID NO:2: p.E81K, p.R35Q, p.Q158X, p.L64fsX59 and p.D274fsX60.According to concrete examples more of the present invention, biological sample can for being selected from blood of human body, skin, hypodermic at least one.
Secondly, above-mentioned biological sample is cultivated when there is not this candidate agent.
Then, determine that above-mentioned biological sample is when existing candidate agent and there is not candidate agent, to the susceptibility of Dematiaceous fungi, such as, can pass through Dematiaceous fungi and biological sample co-cultivation, test organisms sample infects the ratio change of Dematiaceous fungi.
Then, obtained compared the susceptibility of Dematiaceous fungi, susceptibility when wherein there is candidate agent, lower than susceptibility when there is not this candidate agent, is candidate agent as the instruction of the medicine for the treatment of or prevention phaeohyphomycosis.
Utilize the method for the medicine of screening treatment of the present invention or prevention phaeohyphomycosis, effectively can screen the medicine for the treatment of or prevention phaeohyphomycosis.
Below with reference to specific embodiment, the present invention will be described, it should be noted that, these embodiments are only illustrative, and can not be interpreted as limitation of the present invention.
If do not specialize, the conventional means that the technique means adopted in embodiment is well known to those skilled in the art, can carry out with reference to " Molecular Cloning: A Laboratory guide " third edition or related products, the reagent adopted and product be also can business obtain.The various process do not described in detail and method are the ordinary methods of public office in this area, source, the trade(brand)name of agents useful for same and be necessary to list its moiety person, all indicate when occurring first, identical reagent used if no special instructions thereafter, all identical with the content indicated first.
Mutational site is determined in the order-checking of embodiment 1 exon
((wherein, D6-1 and D6-3 is the medical patient from Peking University First Hospital for D6-1, D6-3, D6-4 to select three examples; D6-4 is the medical patient from Shandong, Shandong hospital)) patient that distributes that is diagnosed as phaeohyphomycosis carries out the order-checking of exon group and data analysis, and specific practice is as follows:
First, contriver checks order in conjunction with the exon group sequence of Solexa high throughput sequencing technologies to this three routine patient with NimbleGen 2.1M Human Exome Array, specific as follows:
1) genomic dna of Patient Sample A is broken at random the fragment of about 200-300bp, subsequently according to the process specifications that manufacturers provides, connects top connection respectively at fragment two ends and prepare Hybrid Library.Hybridization enrichment is carried out through the linear amplification of LinkerMediated PCR (LM-PCR) and Biotinylated DNA Library after library is purified, again through the linear amplification of LM-PCR, namely be available on the machine after library detection is qualified order-checking, to obtain raw sequencing data.Order-checking platform is Illumina Hiseq 2000, and reading length is 90bp, and the average order-checking degree of depth of each sample is minimum is 50.
2) Illumina Basecalling Software 1.7 is utilized to process the raw sequencing data of above-mentioned acquisition, filtration depollutes, then use SOAPaligner 2.20 (can be see: Li R, Li Y, Kristiansen K, et al, SOAP:shortoligonucleotide 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., is incorporated to herein in full by referring to by it) it is compared with reference to genome, thus, comparison is obtained to the unique comparison sequencing data (Unique mapped reads) on genome.Then, (can be see: Li R by SOAPsnp, Li Y, Fang X, Yang H, et al, SNP detection for massively parallel whole-genomeresequencing.Genome Res 2009,19 (6): 1124-1132., be incorporated to herein in full by referring to by it) determine target region, i.e. the genotype of full exon region.
After obtaining sequencing result, nonsynonymous mutation, acceptor splicing site/donor site sudden change, coding region are inserted and studied with the most possible sudden change relevant to pathology of this three class of deletion mutantion.As a result, discovery respectively in patient is distributed in three examples: D6-1 has the insertion/deletion at 95667 single nucleotide polymorphism (SNPs) and 7290 places; D6-3 has the insertion/deletion at 97878 single nucleotide polymorphism (SNPs) and 7193 places; D6-4 has the insertion/deletion at 93646 single nucleotide polymorphism (SNPs) and 7011 places.Subsequently by the above results by four public databases (dbSNP (v131):
http:// hgdownload.cse.ucsc.edu/goldenPath/hg19/database/snp131.txt.gz.; 1000 people:,
ftp: //ftp.1000genomes.ebi.ac.uk/vo11/ftp or
ftp: //ftp-trace.ncbi.nih.gov/1000genomes/ftp; Hapmap:
ftp: //ftp.ncbi.nlm.nih.gov/hapmap, YH database:
http:// yh.genomics.org.cn) filtration, remove all known and gene frequency variations of being greater than 0.005 in a database, thus, removal can not cause rare pathogenetic common variation.
After acquisition SNP result, utilize SIFT software prediction to screen the harmless sudden change of removing wherein, then by GO and KEGG, function prediction is carried out in remaining sudden change.Wherein, SIFT (Sorting Intolerant From Toleran) is one and replaces the software on protein function impact for predicted amino acid, it can judge that this amino-acid substitution is harmless (functionally neutral) or harmful (deleterious) on protein function, investigator can infer whether will do further research to this replacement by this result, and the details about this software individual can be see
http:// sift.jcvi.org/.SIFT is calculated as a standardized score value for predicting the outcome of an amino-acid substitution, and variation range, from 0 to 1, represents that when this value is greater than 0.05 time this sudden change can be tolerated, namely do not affect protein function or affect very little; Then illustrate when being less than or equal to 0.05 that this sudden change is harmful, namely have considerable influence to protein function.GO is described from cellular component (cellular component), molecular function (molecular function), biological procedures (biological process) three aspects gene product attribute.KEGG (the Kyoto Encyclopedia ofGenes and Genomes) utilizes computer to describe biosystem, it comprises three parts: at present for the information of the information of the understanding of Molecular interaction network, gene and albumen, chemical complex and reaction, detailed content is shown in link
http:// www.genome.jp/kegg/kegggg1a.html.
In conjunction with the candidate gene that mark, GO and KEGG of SIFT prediction obtain, contriver finds that CARD9 gene is closely related with this disease, and, in 3 patients, CARD9 gene has and isozygoty or the sudden change of compound heterozygosis, so contriver selects CARD9 gene (its exon sequence is as shown in SEQ ID NO:1) as the candidate gene of phaeohyphomycosis, further study.
Embodiment 2Sanger method sequence verification
Respectively to 3 patient (D6-1, D6-3, D6-4), normal people (the i.e. father and mother of this 3 routine patient in 6 familys, they all do not fall ill) and a gene distributing patient (from Wuhan City No.1 Hospital) detect, for all exon sequence design primers of CARD9 gene, then CARD9 relevant sequence is obtained by the method for pcr amplification, product purification and order-checking, according to determining that sequencing results belongs to saltant type or wild-type, the dependency between checking CARD9 and phaeohyphomycosis disease.Concrete grammar step is as follows:
2.1DNA extract
Gather the peripheral blood of normal people in above-mentioned 4 routine phaeohyphomycosis patients and 6 familys respectively, then the genomic dna in conventional phenol-chloroform method extracting peripheral blood leucocyte is utilized, and utilize spectrophotometer measurement extract concentration and the purity of DNA, the OD260/OD280 of each sample genomic dna of gained is all between 1.7-2.0, concentration is no less than 200ng/ microlitre, and total amount is no less than 30 μ g, thus, obtain 10 kinds of DNA sample, for subsequent use.
2.2 design of primers and PCR reaction
First, reference men and women's genoid data unit sequence storehouse hg19/build36.3, design obtains the CARD9 exon Auele Specific Primer (see above and state table 2) with the nucleotide sequence shown in SEQ ID NO:3-26.
Then, 10 of said extracted kinds of DNA sample are configured respective PCR reaction system according to the following ratio respectively:
Reaction system: 25 μ L
Then, each PCR reaction system is carried out PCR reaction respectively according to following reaction conditions:
Thus, obtain 3 routine phaeohyphomycosis patients and normal father and mother thereof, and a pcr amplification product distributing patient (from Wuhan City No.1 Hospital).
The order-checking of 2.3Sanger method and the result
Obtained 10 kinds of pcr amplification products are directly carried out DNA sequencing, to obtain sequencing result.
Then, based on sequencing result, to 3 routine phaeohyphomycosis patients and normal father and mother thereof, and one is distributed patient and carries out the comparison of CARD9 gene coded sequence.Contriver finds, CARD9 (cDNA sequence of CARD9 is as shown in SEQ ID NO:1) gene and proteins encoded thereof distribute in patient (D6-1, D6-3, D6-4) at 4 all exists significant sudden change, particularly, D6-1 patient sports c.G241A, c.G104A, p.E81K and p.R35Q, D6-3 sport c.C472T, c.191insTGCT, p.Q158X and p.L64fsX59, c.818insG D6-4 sports and p.D274fsX60, and distributes the mutational site of patient (from Wuhan City No.1 Hospital) and the just the same of D6-4.It can thus be appreciated that differ to establish a capital and concentrate on above an amino acid in the mutational site of a disease, same gene can exist different mutational site, and result causes same disease.
Phaeohyphomycosis nationwide number of the infected is little, and is and is dispersed in distribution.So most probable hereditary pattern is autosomal recessive homozygous mutation or autosomal recessive compound heterozygous mutations.Thus, next, based on sequencing result and Relational database and software, in patient family member, sudden change investigation is carried out to all encoding sequences of CARD9 gene and the albumen of flanking sequence and this genes encoding, the father and mother of result display patient are the heterozygosis carrier of corresponding sudden change, showing the CARD9 transgenation in aforementioned 3 routine patients, is also respectively from its father and mother.Particularly, the c.G241A (p.E81K) of D6-1 patient is from father, and c.G104A (p.R35Q), from mother, meets the mode of inheritance of recessive sick compound heterozygosis; The c.C472T (p.Q158X) of D6-3 is from mother, and c.191insTGCT (p.L64fsX59) is from father, meets the mode of inheritance of recessive sick compound heterozygosis; C.818insG (p.D274fsX60) pure and mild sudden change of D6-4, respectively from father and mother, meets the mode of inheritance of recessive sick homozygous mutation.Thus, contriver verifies, confirms the new mutant site of falling ill relevant to phaeohyphomycosis.
Wherein, Fig. 2 shows and verifies peak figure to the Sanger of D6-3 patient CARD9 gene mutation site.As shown in Figure 2, D6-3 patient has c.C472T (p.Q158X) and c.191insTGCT (p.L64fsX59) sudden change.It should be noted that, shown in Fig. 2 is the sequencing result of complementary strand, and what above illustrate is single base mutation, so only have a heterozygosis peak in the place of arrows.Under what illustrate is insertion mutation, so start in the place of arrows all to occur heterozygosis peak left.
In the description of this specification sheets, specific features, structure, material or feature that the description of reference term " embodiment ", " some embodiments ", " example ", " concrete example " or " some examples " etc. means to describe in conjunction with this embodiment or example are contained at least one embodiment of the present invention or example.In this manual, identical embodiment or example are not necessarily referred to the schematic representation of above-mentioned term.And the specific features of description, structure, material or feature can combine in an appropriate manner in any one or more embodiment or example.
Although illustrate and describe embodiments of the invention, those having ordinary skill in the art will appreciate that: can carry out multiple change, amendment, replacement and modification to these embodiments when not departing from principle of the present invention and aim, scope of the present invention is by claim and equivalents thereof.
Claims (11)
1. the nucleic acid of the coding CARD9 mutant be separated, it is characterized in that, c.191insTGCT and c.818insG compared with SEQ ID NO:1, described nucleic acid has and is selected from the sudden change of following at least one: c.G241A, c.G104A, c.C472T, described nucleic acid is DNA.
2. an isolated polypeptide, is characterized in that, compared with SEQ ID NO:2, described isolated polypeptide has and is selected from the sudden change of following at least one: p.E81K, p.R35Q, p.Q158X, p.L64fsX59 and p.D274fsX60.
3. isolated polypeptide according to claim 2, is characterized in that, described polypeptide is by nucleic acid encoding according to claim 1.
4. screen a system for the biological sample of susceptible Dematiaceous fungi, it is characterized in that, comprising:
Nucleic acid-extracting apparatus, described nucleic acid-extracting apparatus is used for from described extraction from biological material sample of nucleic acid;
Nucleotide sequence determining device, described nucleotide sequence determining device is connected with described nucleic acid-extracting apparatus, for analyzing described sample of nucleic acid, to determine the nucleotide sequence of described sample of nucleic acid;
Judgment means, described judgment means is connected with described nucleotide sequence determining device, so that based on the nucleotide sequence of described sample of nucleic acid compared with SEQ ID NO:1, whether have and be selected from c.G241A, c.G104A, c.C472T, at least one sudden change c.191insTGCT and c.818insG, judge described biological sample whether susceptible Dematiaceous fungi.
5. system according to claim 4, is characterized in that, described nucleic acid-extracting apparatus comprises further:
RNA extraction unit, described RNA extraction unit is used for from described extraction from biological material RNA sample; And
Reverse transcription unit, described reverse transcription unit is connected with described RNA extraction unit, for carrying out reverse transcription reaction to described RNA sample, to obtain cDNA sample, sample of nucleic acid described in described cDNA composition of sample.
6. system according to claim 4, is characterized in that, described nucleotide sequence determining device comprises further:
Library construction unit, described library construction unit is used for for described sample of nucleic acid, builds nucleic acid sequencing library; And
Order-checking unit, described order-checking unit is connected with described library construction unit, for checking order to described nucleic acid sequencing library, to obtain the sequencing result be made up of multiple sequencing data.
7. system according to claim 6, is characterized in that, described library construction unit comprises further:
Pcr amplification module, is provided with CARD9 exon Auele Specific Primer in described pcr amplification module, to utilize CARD9 exon Auele Specific Primer, carry out pcr amplification to described sample of nucleic acid.
8. system according to claim 7, is characterized in that, described CARD9 exon Auele Specific Primer has the nucleotide sequence as shown in SEQ ID NO:3-26.
9. system according to claim 6, is characterized in that, described order-checking unit comprises and is selected from HISEQ2000, SOLiD, 454 and at least one of single-molecule sequencing device.
10. for screening a test kit for the biological sample of susceptible Dematiaceous fungi, it is characterized in that, containing:
Be suitable for the reagent detecting CARD9 gene mutation body, wherein compared with SEQ ID NO:1, described CARD9 gene mutation body has and is selected from c.G241A, c.G104A, c.C472T, at least one sudden change c.191insTGCT and c.818insG.
11. test kits according to claim 10, is characterized in that, described reagent is nucleic acid probe.
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| Drummond RA,et al.The role of Sky/CARD9 coupled C-type lecins in antifungal immunity.《Eur J Immunol》.2011,第41卷(第2期), * |
| Erik-Oliver Glocker,et al.A Homozygous CARD9 Mutation in a Family with Susceptibility to Fungal Infections.《N Engl J Med》.2009,第361卷(第18期), * |
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