CN107058259A - Molecular marker MK2 genes and its application - Google Patents

Molecular marker MK2 genes and its application Download PDF

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CN107058259A
CN107058259A CN201710351805.4A CN201710351805A CN107058259A CN 107058259 A CN107058259 A CN 107058259A CN 201710351805 A CN201710351805 A CN 201710351805A CN 107058259 A CN107058259 A CN 107058259A
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genes
disease
huppert
protein
chip
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顾春艳
杨烨
芦婷
杨洪宝
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Nanjing University of Chinese Medicine
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Nanjing University of Chinese Medicine
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Abstract

Molecular marker MK2 genes and its application, belong to molecular diagnosis field, are related to application of the molecular marker MK2 genes in myeloma cell in the product for preparing diagnosis Huppert's disease.The experiment proves that compared with normal bone marrow tissue, MK2 genes expression quantity in multiple myeloma patients is significantly raised.Pass through the expression of MK2 in subject, it can be determined that whether subject is with Huppert's disease or in the presence of the risk with Huppert's disease, so as to instruct clinician to provide prevention scheme or therapeutic scheme to subject.MK2 genes are a kind of new biomarkers, compared to traditional detection means, and gene diagnosis is not only stablized, conveniently, and sensitive, special, can realize the early diagnosis of Huppert's disease, so as to control the process of Huppert's disease in time, reduce the death rate.

Description

Molecular marker MK2 genes and its application
Technical field
The present invention relates to the molecular diagnosis field of tumour, more particularly it relates to the molecule mark in myeloma cell Will thing MK2 genes and its application in the product for preparing diagnosis Huppert's disease.
Background technology
Huppert's disease (multiple myeloma, MM) is a kind of plasma-cell malignancy, is characterized in marrow Thick liquid cell clonal expansion, and secrete monoclonal immunoglobulin or light chain.Due to abnormal plasmocyte infiltration bone, marrow and complete The multiple organ-tissues of body, cause in pathologic bone destruction, the serum of patient and abnormal monoclonal immunoglobulin occur, from And the normal polyclonal immunoglobulin of body is inhibited, occur albumen in the urine of patient, patient finally shows to cause poor Blood, kidney function damage and pathologic fracture etc..MM is that the whole world incidence of disease is number two the neoplastic hematologic disorder of position, current China 50 years old MM patient occupies about 3% or so in tumour patient above, and with the aging of China human mortality, the MM incidences of disease are with annual 1% Speed rise.It is now recognized that MM is a process being progressively in progress, the list of interrogatory occurs in early stage for many patients Globulin disease is cloned, MM is then progressed to, some patientss finally develop into marrow dermoskeleton myeloma (plasma cell leukemia), whole process Middle Bone Marrow of Patients thick liquid cell with several genes mutation.
The diagnosis of current Huppert's disease relies primarily on the Clonal thick liquid cell ratio of marrow and (or) living tissue pathology Inspection is used as MM diagnostic bases.International Myeloma working group (IMWG) has revised the unknown monoclonal immunological ball of meaning in 2014 Proteinemia (MGUS), MM diagnostic criteria.MM diagnostic criteria includes Clonal thick liquid cell ratio >=10% and/or group living in marrow Knit pathological examination and turn out to be plasmacytoma；Simultaneously with one or more of situation：It is hypercalcinemia, renal insufficiency, poor Blood and osteolytic Bone destruction.Monoclonal immunoglobulin (M albumen) is clinical conventional MM diagnosis markers, but revised is examined Disconnected standard emphasizes that M albumen is not the necessary requirement for diagnosing MM, but can be used to distinguish secreting type or nonsecreting type MM.In addition, MRI, The modern imaging technologies such as low dosage whole body CT, FDG/PET and PET/CT are included into inspection householder method.
Method complexity, the inspection project of current Diagnosis of Multiple Myeloma are more, lack a kind of reliable, convenient clinical diagnosis Method.In recent years, this seminar is based on gene chip expression spectral, finds in multiple myeloma patients, the expression of MK2 genes Significantly increase, the expression of MK2 genes with it is closely related with multiple myeloma patients pathogenesis and drug resistance.Show MK2 is a kind of high-risk marrow tumor gene, and reliable judgement can be provided for the clinical diagnosis of Huppert's disease.
MK2 (mitogen-activated protein kinase-activated protein kinase 2, MAPKAPK2) found and first MAPK activated protein kinase being purified in 1992, it is mainly direct by p38 protein kinases Phosphorylation activation, is the important substrate of p38 protein kinases, in cell propagation, differentiation caused by mediation extracellular signal, aging, is withered Die and the reaction such as inflammation in play an important role.MK2 is oncogene, can pass through phosphorylation pathways direct activation kinds of tumors phase Close the factor such as cell division cycle protein 25B/C and Heat shock protein 27 etc.；It can also be raised many by post-transcriptional control mechanism Individual oncogene and the expression of proinflammatory factor, and promote tumour to develop including lung cancer；MK2, which is overexpressed, can activate lim kinase And the angiogenesis of VEGF inductions are activated, so as to promote the invasion and attack and transfer of tumour.
The content of the invention
The technical problem of solution：Examined it is an object of the invention to provide a kind of molecular marker MK2 genes and its in preparation Application in the product of disconnected Huppert's disease.
Technical scheme：Molecular marker MK2 genes, sequence is as shown in SEQ ID NO.1.
Molecular marker MK2 albumen, sequence is as shown in SEQ ID NO.2.
Application of the molecular marker MK2 genes in the product for preparing diagnosis Huppert's disease.
The said goods include：Measured by RT-PCR, real-time quantitative PCR, SABC, in situ hybridization, chip or high pass Sequence detection of platform MK2 gene expression doses are to diagnose the product of Huppert's disease.
The above-mentioned product for diagnosing Huppert's disease with real-time quantitative PCR at least includes a pair of specific amplified MK2 genes Primer；The product of the use immunodiagnosis Huppert's disease includes：The antibody combined with MK2 protein-specifics；It is described The product for diagnosing Huppert's disease with situ hybridization includes：With the probe of the nucleic acid array hybridizing of MK2 genes；It is described to use chip The product of diagnosis Huppert's disease includes：Protein chip and genetic chip, wherein protein chip include and MK2 protein-specifics With reference to antibody, genetic chip includes probe with the nucleic acid array hybridizing of MK2 genes.
The above-mentioned product for diagnosing Huppert's disease with real-time quantitative PCR at least includes a pair of specific amplified MK2 genes Primer, sequence is such as：Shown in SEQ ID NO.3 and SEQ ID NO.4.
A kind of instrument for being used to diagnose Huppert's disease, the instrument can detect the expression of MK2 genes in sample.
Above-mentioned instrument includes chip, kit, test paper or high-flux sequence platform.
Said chip includes genetic chip or protein-chip, and the genetic chip includes solid phase carrier and is fixed on The oligonucleotide probe of phase carrier, the oligonucleotide probe include be used for detect MK2 gene transcription levels be directed to MK2 genes Oligonucleotide probe；The protein-chip includes solid phase carrier and is fixed on the specificity of the MK2 albumen of solid phase carrier Antibody；The kit includes gene detecting kit and protein immunization group kit；The gene detecting kit includes Reagent for detecting MK2 gene transcription levels；The protein immunization group kit includes the specific antibody of MK2 albumen； The test paper includes the test paper for being used to detect MK2 gene transcription levels；The high-flux sequence platform includes being used to detect MK2 bases Because of the reagent of transcriptional level.
The reagent of above-mentioned detection MK2 gene transcription levels includes the primer and/or probe for MK2 genes.
Beneficial effect：Present invention firstly discovers that MK2 gene expressions are related to Huppert's disease.Present invention finds one The molecular marker MK2 of diagnosis Huppert's disease is planted, the morning that can occur using the molecular marker in Huppert's disease Phase can be used as judge, or judge subject with the presence or absence of suffer from Huppert's disease risk there is provided the existence of patient Rate.Present invention finds a kind of new biomarker MK2 genes, compared to traditional detection means, gene diagnosis not only stablize, It is convenient and sensitive, special, so as to control the process of Huppert's disease in time, reduce the death rate.
Brief description of the drawings
Fig. 1 displays detect MK2 genes multiple using genetic chip (Gene expression profiling, GEP) Differential expression figure in patients with malignant myeloma and normal person.Analyze the unknown monoclonal third of 22 normal bone marrows (NP), 44 meanings Bulb albumen disease is (before monoclonal gammopathy of undetermined significance, MGUS, myeloma Body disease) and 351 first visit MM clinical samples, MK2 expressions significantly raise in MM clinical samples.
Fig. 2 displays detect differential expression figure of the MK2 genes in multiple myeloma patients and normal person using QPCR.
Fig. 3 displays analyze MK2 tables in normal bone marrow and MM Bone Marrow of Patients samples using SABC (IHC) colouring method Up to situation map, as a result show that MK2 expresses notable rise in MM clinical samples.
Embodiment
The present invention is further detailed explanation with reference to the accompanying drawings and examples.Following examples are merely to illustrate this Invention rather than limitation the scope of the present invention.The test method of unreceipted actual conditions in embodiment, generally according to conventional strip Part, such as Sambrook et al., molecular cloning：Laboratory manual (New York：Cold Spring Harbor Laboratory Press, 1989) described in condition, or according to the condition proposed by manufacturer.
The invention provides the product of detection MK2 gene expressions answering in the instrument for preparing diagnosis Huppert's disease With.
Further, the product of detection MK2 gene expressions mentioned above includes：Pass through real-time quantitative PCR, immune inspection Survey, in situ hybridization, chip or high-flux sequence platform etc. detect MK2 gene expression doses to diagnose the production of Huppert's disease Product.
Further, the product for diagnosing Huppert's disease with real-time quantitative PCR at least includes a pair of specific amplified MK2 The primer of gene；The product for diagnosing Huppert's disease with immune detection includes：What is combined with MK2 protein-specifics is anti- Body；The product for diagnosing Huppert's disease with situ hybridization includes：With the probe of the nucleic acid array hybridizing of MK2 genes；Institute Stating the product for diagnosing Huppert's disease with chip includes：Protein chip and genetic chip；Wherein, protein chip includes and MK2 The antibody that protein-specific is combined, genetic chip includes the probe with the nucleic acid array hybridizing of MK2 genes.
It is described to diagnose the product of Huppert's disease at least with real-time quantitative PCR in specific embodiments of the present invention The sequence of primer including a pair of specific amplified MK2 genes is such as：Shown in SEQ ID NO.3 and SEQ ID NO.4.
Preferably, the diagnostic tool includes chip, kit, test paper or high-flux sequence platform.Wherein, high pass is measured Sequence platform is a kind of special diagnostic tool, and the product of detection MK2 gene expressions can apply to the platform and realize to MK2 genes Expression detection.With the development of high throughput sequencing technologies, ten will be turned into the structure of the gene expression profile of a people Divide and easily work.By contrasting the gene expression profile of Disease and normal population, the exception of which gene is easily analyzed It is related to disease.Therefore, know that the exception of MK2 genes is related to Huppert's disease in high-flux sequence and fall within MK2 bases The purposes of cause, equally within protection scope of the present invention.
Present invention also offers a kind of instrument of Huppert's disease, the product include chip, kit, test paper or High-flux sequence platform.
Wherein, the chip includes genetic chip, protein-chip；The genetic chip includes solid phase carrier and fixation In the oligonucleotide probe of solid phase carrier, what the oligonucleotide probe included being used for detecting MK2 gene transcription levels is directed to MK2 The oligonucleotide probe of gene；The protein-chip includes solid phase carrier and is fixed on the spy of the MK2 albumen of solid phase carrier Heterogenetic antibody；The genetic chip can be used for multiple genes of the detection including MK2 genes (for example, and Huppert's disease Related multiple genes) expression.The protein-chip can be used for multiple protein of the detection including MK2 albumen The expression of (such as multiple protein related to Huppert's disease).By will be multiple related to Huppert's disease Mark is detected simultaneously, is greatly improved the accuracy rate of Diagnosis of Multiple Myeloma.
Wherein, the kit includes gene detecting kit and protein immunization detection kit；The genetic test examination Agent box includes the reagent for being used to detect MK2 gene transcription levels；The protein immunization detection kit includes the special of MK2 albumen Property antibody.Further, the reagent detects MK2 including the use of real-time quantitative PCR, immune detection, in situ hybridization or chip method Reagent needed for during gene expression dose.Preferably, the reagent includes the primer and/or probe for MK2 genes.Root The primer and probe that can be used for detecting MK2 gene expression doses is easily designed according to the nucleotide sequence information of gene.
The high-flux sequence platform includes the reagent of detection MK2 gene expression doses.
The test paper includes test paper carrier and the oligonucleotides being fixed on test paper carrier, and the oligonucleotides can be detected The transcriptional level of MK2 genes.
Probe with the nucleic acid array hybridizing of MK2 genes can be DNA, RNA, DNA-RNA chimera, PNA or other spread out It is biological.The length of the probe is not limited, as long as completing specific hybrid, being specifically bound with purpose nucleotide sequence, is appointed What length can.The length of the probe can be as short as 25,20,15,13 or 10 bases longs.Equally, the length of the probe Degree can be grown to 60,80,100,150,300 base-pairs or longer, or even whole gene.Because different probe lengths is to hybridization Efficiency, signal specificity have different influences, and the length of the probe is typically at least 14 base-pairs, most long to be usually no more than 30 base-pairs, complementary length is optimal with 15-25 base-pair with purpose nucleotide sequence.The probe self-complementary sequences Most preferably less than 4 base-pairs, in order to avoid influence hybridization efficiency.
Further, the specific antibody of the MK2 albumen includes monoclonal antibody, polyclonal antibody.The MK2 albumen Specific antibody includes complete antibody molecule, any fragment of antibody or modified (for example：Chimeric antibody, scFv, Fab, F (ab ') 2, Fv etc.).As long as the fragment can retain the binding ability with MK2 albumen.Antibody for protein level Preparation when well known to a person skilled in the art and the present invention can use any method to prepare the antibody.
In specific embodiments of the present invention, the primer sequence for MK2 genes is as follows：Forward primer sequence is such as Shown in SEQ ID NO.3, reverse primer sequences are as shown in SEQ ID NO.4.
The source of MK2 genes and its expression product for diagnosing Huppert's disease include but is not limited to marrow, blood, Tissue fluid, urine, saliva etc. can obtain the body fluid of genomic DNA.It is many for diagnosing in specific embodiments of the present invention Hair property the MK2 genes of myeloma and its source of expression product be marrow.
In the context of the present invention, " MK2 genes " includes many of any functional equivalent of MK2 genes and MK2 genes Nucleotides.MK2 genes include and MK2 genes (NC_000001.11) DNA sequences in current international public nucleotide sequence GeneBank Row have more than 70% homology, and coding identical function protein DNA sequence；
Preferably, the coded sequence of MK2 genes include it is following any one：
(1) DNA sequence dna in sequence table shown in SEQ ID NO.1；
(2) the DNA sequence dna hybridization limited under strict conditions with (1) and the DNA sequence dna of coding same protein；
(3) DNA sequence dna limited with (1) or (2) has 70%, preferably, more than 90% homology, and encodes identical work( Can protein DNA molecule.
In specific embodiments of the present invention, the coded sequence of the MK2 genes is the DNA shown in SEQ ID NO.1 Sequence.
In the context of the present invention, MK2 gene expression products include the partial peptide of MK2 albumen and MK2 albumen.It is described The partial peptide of MK2 albumen contains the functional domain related to Huppert's disease.
" MK2 albumen " includes any functional equivalent of MK2 albumen and MK2 albumen.The functional equivalent includes MK2 Albumen conservative variation protein or its active fragment, or its reactive derivative, allelic variant, natural mutation, induction are prominent Variant, can be with the protein coded by the DNA of MK2 DNA hybridization under high or low high stringency conditions.
Preferably, MK2 albumen is the protein with following amino acid sequences：
(1) protein being made up of the amino acid sequence in sequence table shown in SEQ ID NO.2；
(2) by the amino acid sequence shown in SEQ ID NO.2 is by the substitution of one or several amino acid residues and/or lacks Lose and/or addition and with the amino acid sequence shown in SEQ ID NO.2 have identical function as the ammonia shown in SEQ ID NO.2 The protein of base acid sequence derivative.The number of the amino acid of substitution, missing or addition is usually 1-50, preferably 1-20 It is individual, most preferably 1-10.
(3) at least there is 80% homology (also known as sequence identity) with the amino acid sequence shown in SEQ ID NO.2, It is highly preferred that the homology with the amino acid sequence at least about 90% to 95% shown in SEQ ID NO.2, be often 96%, 97%, 98%th, the polypeptide that the amino acid sequence of 99% homology is constituted.
In specific embodiments of the present invention, the MK2 albumen is with the amino acid sequence shown in SEQ ID NO.2 Protein.
It is known that, conventionally, the modification of one or more amino acid does not interfere with the function of protein in a protein. Those skilled in the art can approve the amino acid that changes single amino acids or small percentage or indivedual additions to amino acid sequence, Missing, insertion, replacement are conservative modifications, and the change of wherein protein produces the protein with identity function.Function phase is provided As the Conservative substitution tables of amino acid be well known in the art.
By adding the fusion egg that the example for the protein that an amino acid or more amino acid are modified is MK2 albumen In vain.Do not limited for the peptide or protein with MK2 protein fusions, as long as the fusion protein of gained retains the life of MK2 albumen Thing activity.
The MK2 albumen of the present invention also includes the non-conservative modification to the amino acid sequence shown in SEQ ID NO.2, as long as through The protein for crossing modification remains able to retain the biological activity of MK2 albumen.The ammonia being mutated in such modifying protein Base acid number is typically 10 or less, such as 6 or less, such as 3 or less.
In the context of the present invention, " Huppert's disease " both includes judging whether subject suffers from multiple bone Myeloma, also include subject whether there is with Huppert's disease risk.
Embodiment 1 screens the gene of differential expression in multiple myeloma patients and normal person
1st, research object：
Collect multiple myeloma patients 8, normal person 8
The inclusive criteria of multiple myeloma patients：The diagnosis of all Huppert's diseases diagnoses multiple bone according to WHO Myeloma standard, all multiple myeloma patients BMNC samples of just controlling obtain informed consent.
The exclusion standard of multiple myeloma patients：Metastatic carcinoma, reactive plasmocytosis and not qualitative monoclonal Immunoglobulino-pathy.
2nd, in marrow total serum IgE extraction
(1) separation of BMNC：The erythrocyte splitting of 3 times of volumes is added into the marrow specimen being collected into Liquid, mixing is blown and beaten with dropper after placing 10~15min on ice, the red blood cell in marrow specimen is fully cracked repeatedly； 1200rpm/min centrifuges 5min, abandons supernatant, PBS washings cell 1 time.Supernatant is abandoned, adds after 1mL PBS piping and druming is mixed and is transferred to In 1.5mL EP pipes, 1200rpm/min centrifugation 5min abandon supernatant.Each sample adds 1mL Trizol, and piping and druming is mixed.
(2) the μ L of chloroform 200 are added per 1mL Trizol, it is abundant to mix acutely concussion 15 seconds, it is stored at room temperature 10min and waits and divides Layer.
(3) in 4 DEG C of 12000rpm/min centrifugations 15min.Mixture liquid level is divided into 3 layers, the superiors in visible pipe after centrifugation For colourless aqueous phase, contain RNA；Intermediate layer is white flock, contains albumin layer and DNA；Orlop is organic for red phenol-chloroform Phase.
Take upper water to make an appointment 400 μ L into another EP pipes, note avoiding the albumin layer in the middle of suction, often pipe adds isometric Isopropanol, mixing of gently turning upside down, is stored at room temperature 10min, in 4 DEG C of 12000rpm/min centrifugations 10min, it is seen that ttom of pipe white Precipitation.
Supernatant is abandoned, the 75%DEPC alcohol 1mL of -20 DEG C of precoolings are added, precipitation, 4 DEG C of 7500rpm/min centrifugations has been hanged 5min。
Abandon supernatant, room temperature places 10-20min and air-dries precipitation, according to precipitation capacity number take the circumstances into consideration to add the dissolving of DEPC water it is heavy Form sediment.- 80 DEG C of refrigerators are preserved.
3rd, the quality analysis of RNA sample
UV spectrophotometer measuring RNA sample, OD260/OD280 is 1.8-2.0.
4th, gene chip hybridization and scanning
Rna level is detected using Affymetrix U133Plus 2.0microarrays.Multiple myeloma patients group Data can NIH gene expressions integrate (GEO) under GSE2658 searching numbers check in, normal group and not qualitative monoclonal immunological The microarray data of gammopathy is under GSE5900.
5th, chip data processing and analysis
The data analysis of genetic chip is based on GCO1.1 softwares (Affymetrix), includes a pair life related to disease The univariate log-rank detection deposited.For two groups of ratio natural logrithm absolute value be more than 2.0 or gene less than 0.5 make For difference expression gene.
6th, statistical procedures
Data analysis is carried out using the statistical softwares of SPSS 13.0, group difference, which compares, uses one-way analysis of variance method, P The significances of ＜ 0.05.
7th, result
As a result display is (as shown in figure 1,22 normal bone marrows of analysis, the unknown monoclonal gamma globulin disease of 44 meanings Sick (monoclonal gammopathy of undetermined significance, MGUS, MM precursors disease), 351 MM first visit patient's thick liquid cell microarray datas storehouse, it is found that the expression of MK2 genes will be apparently higher than normal in MM patient And MGUS samples), compared with normal person, the MRNA levels of multiple myeloma patients MK2 genes are significantly raised, and difference has Statistical significance (P ＜ 0.05).
The gene of differential expression in embodiment 2QPCR experimental verifications multiple myeloma patients and normal person
1st, research object
Screening criteria be the same as Example 1, multiple myeloma patients 46, normal person 66.
2nd, in marrow total serum IgE extraction
Step be the same as Example 1.
3rd, reverse transcription
RNA reverse transcriptions are carried out according to TAKARA Reverse Transcriptase kits.Using 10 μ L reaction systems, it is separately added into 5 × PrimeScript RT Master Mix 2 μ L, the μ g of total serum IgE 0.5, plus a certain amount of RNase Free Water to 10 μ L.37℃ React 15min, 85 DEG C of reaction 5s.Product cDNA puts -20 DEG C of refrigerator preservations.
4、QPCR
Using 10 μ L reaction systems, each sample sets 3 multiple holes, is loaded process lucifuge.Prepare following reaction system：2 The μ L of × SYBGREEN Master Mix 5, the μ L of forward primer 0.5, the μ L of reverse primer (5pm) 0.5, the μ of reverse primer (5pm) 0.5 L, template cDNA 1 μ L, ddH2O 3μL.The forward primer sequence for expanding MK2 genes is 5 '-TGGGGCGTTAACTGCATCTGG- 3 ' (SEQ ID NO.3), reverse primer sequences are 5 '-TGGGCCTGCTGTTCACAGTT-3 ' (SEQ ID NO.4)；Amplification The forward primer sequence of GAPDH genes is 5 '-GCACCGTCAAGGCTGAGAAC-3 ' (SEQ ID NO.5), reverse primer sequences For 5 '-TGGTGAAGACGCCAGTGGA-3 ' (SEQ ID NO.6), operations are carried out on ice.Amplification program is：95℃ 1min, 95 DEG C of 15s, 60 DEG C of 15s, 72 DEG C of 30s；Totally 40 circulations.Using SYBR Green as fluorescent marker, in Light Reacted on Cycler fluorescence real-time quantitative PCR instrument, purpose band, △ △ CT methods are determined by melt curve analysis analysis and electrophoresis Carry out relative quantification.
5th, statistical method
Result data is represented in the way of mean+SD, is united using SPSS13.0 statistical softwares Meter analysis, difference is using t inspections between the two, it is believed that have statistical significance as P ＜ 0.05.
6th, result
As a result as shown in Fig. 2 compared with normal person, the mRNA level in-site of the MK2 genes of multiple myeloma patients significantly increases Height, difference has statistical significance (P ＜ 0.05), as a result homogenic array experiment.
Above-described embodiment is preferably embodiment, but embodiments of the present invention are not by above-described embodiment of the invention Limitation, other any Spirit Essences without departing from the present invention and the change made under principle, modification, replacement, combine, simplification, Equivalent substitute mode is should be, is included within protection scope of the present invention.
SEQUENCE LISTING
<110>Nanjing University of Traditional Chinese Medicine
<120>Molecular marker MK2 genes and its application
<130>
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 49266
<212> DNA
<213>People source
<400> 1
gggcgccggg ccggtgggag ccagcggcgc gcggtgggac ccacggagcc ccgcgacccg 60
ccgagcctgg agccgggccg ggtcggggaa gccggctcca gcccggagcg aacttcgcag 120
cccgtcgggg ggcggcgggg agggggcccg gagccggagg agggggcggc cgcgggcacc 180
cccgcctgtg ccccggcgtc cccgggcacc atgctgtcca actcccaggg ccagagcccg 240
ccggtgccgt tccccgcccc ggccccgccg ccgcagcccc ccacccctgc cctgccgcac 300
cccccggcgc agccgccgcc gccgcccccg cagcagttcc cgcagttcca cgtcaagtcc 360
ggcctgcaga tcaagaagaa cgccatcatc gatgactaca aggtcaccag ccaggtcctg 420
gggctgggca tcaacggcaa agttttgcag atcttcaaca agaggaccca ggagaaattc 480
gccctcaaag taggtctggg gcccggggag gggaggcggg gccggtcccg ggccctggag 540
ctccacggcg tcgggtgccc gtcccggcct gggtcgcggc gcggggcgga ggggtggccg 600
cggcggggcg gggcggggcg gccgggccgg cggtgccgag tgcggcgcgg gcggcgggtc 660
ccagcgccct cgcctccctg accgcttccg gccgctcccg ggcgggagcc ctgggacccg 720
ctcctgggga ctcaggggac cctgccattt tggggtttga gcgggatcca gccgagtcag 780
aacggttggc ttcgggactt ctgctaattt cactccatct caaggaagtg attttggggt 840
tggtggttcc ccgtcctctc ctccctcctt tttctctctc tgcgggcaca aaaggcccat 900
tactcattga gccggttggt cggttggttt ggagaagtcg gatcgcagag gtttatttag 960
cctcgctcct tatttgggag ccggaatgtg tgatttcact tccccgagcc tcgcgagggc 1020
agccgggccg ccttggggcg cgccgggcag gaggtgtgcg gcgggagggt cctgggtgtc 1080
ccccggctgc gggggcggga ttatttggat cccggagctg gccccctcag ctggccccct 1140
cccacgcccg cgcggggcgg ctccggggag aggccggcgg tgtccgcggc ccggctgatc 1200
attgtgaaca gagtctgtaa cccgcggccg ggcccgtgct gattccgcct cctagagagg 1260
gccacttgct gtcctggact cgcgccgtga tgggcagtct gaggaaacat tatccttgag 1320
ccgggggagc ccatcccttt tctttcttta aggggtgtgg tggtggggag aataaggatt 1380
caagtgagaa tatcgacttc agaagttaaa cacaatggac tgggaggtct catgagatgg 1440
gtgggtccgc agtgaagtgt gagccggtct tgcagggtca ccaacaaggt tttcctctca 1500
caggggtttc aggtcaaacc aatctctaaa ggtgacgcct caggtctgtt tcccctgggg 1560
tgtctcaatg attggaggag gagaaaaata cttcctgtgt tatatttttc tcttcccctt 1620
cagcccccac agcgagctgt atttcagttg tcaagtacaa aactcagaaa tgtggtatgg 1680
gtggaactat tggaatatac gatcagatgt tttaaaagaa acaccagtac ttcggggttg 1740
tacggggcaa gcatgttcca tttctagatt ttaaggcctc tagaattgtt ttcctgctgc 1800
tctcatactc aaccctgtta cagtgctttg ctttctactg taagttattg ctctgggatc 1860
tcggttgttg tttagtgttg actgttggag aggggataag tatcttcaac acttcaacat 1920
ttgtgtctta gacttctctt tccctactca taactgaagg gaaaggagga ggaggagttg 1980
tttggattga gcatggctct gaccttgggg actggagttg ggtgaataca gtttgaccac 2040
agttaacctg tatcattgag gaagtgacag acggtcttta caggagtgtt tgctgtcccc 2100
tctgagacac catctgcctt cacccattag tagtttgggg acactggctg ttcattctct 2160
tggatatttg gaatattatg taaagacttc agaacaaaag ctcccaactt ctgctctgta 2220
ttctgtttaa tagacacacg cacaaaatca tcctataaaa tgcttttgag tggcaattca 2280
gatttctctt gtaactaacg agagagaggt ttacatattt ttctactttg aatagttttg 2340
gaaggaaaaa tccagcagcg tgttaaataa cactcatagt ttatccctct caggttagaa 2400
gtgggcgaag agtgaagtca gtctgttttg acaggcagcc tgctcccaga tcttttaagt 2460
tgactcaact gttaaattgt ggatttccca cacacgtctt tgtgttaaaa cttaataatg 2520
gaggaagaaa ggagagacaa ggcatttgga gactgacagc agtgttaaca gcacttccat 2580
atccaaatat tggctgagac ataattacag ctgtgggggg aatctggttc aaagaacgct 2640
gttccagaga cacacaccaa agagatttct agatcttgaa ttttgattaa cctcaggtgc 2700
tgagtagtga aatggttttc aaaaccagaa gtctagtgat tataaagcag gagaggaaga 2760
tggagtgggg caagagggat tacccccttg ggaaggtgcc tgagaaaagg ggaggagacc 2820
tctattctac agtgtgccga ggccaatgct gggggctagg gcagagctgg agggcaggag 2880
cccagcctct gctggggttc agaagtctgc tatgttgcag ccctgagagt tgatgggagc 2940
tggcagctgc ttagatgctc tcctctctgc ctatagtcgg tgacctctta gccctgttaa 3000
gtgaaatcag catttgaaaa agagtcccag ctcaccctgc tgcttctctc cctccacctc 3060
agagagctcc catttaaatg cttttggtgg gtgctttgac ctcacctcac catctctcct 3120
ggctgctttt gctaagttct gagtcatgct tccagtcaga gttgcaaggg acttgaaagg 3180
tcattttgtc cagccccctt attttgcaga agagaaaacc aaggcccaga gtgagggagt 3240
gacttgttca aggtaccata gccattcact tggcagaggc aggattggaa cccaggttgc 3300
ctgactccca gtctcaggct tcttcccact tacttcctaa ctgagccacg ggctcaggca 3360
catggccacc tgctgctcca gggtgcttcc agcctgagga gctgttctgt cacctcaggc 3420
cctgactgac aagaggggag gtcagcgaag ggggctggat gggtatttgc tgctcctgag 3480
aacagtcccc aaagttggag gggaacttca acagtagctt catgcatttt tttaaccact 3540
ccctccccct tttaagagag tatttgatag gcaccttact tactattttt tgtacaagaa 3600
catgggaatt ctattatttt tatttttatt ttttttgaga cagagtgttg ctctgtcgcc 3660
caggttagag tgcagtagca cgatcttggc tcactgcaag ctctgcttcc caggtccacg 3720
ccattctgcc tcagcctcct gagtagctgg gactacaggc gcccgtcacc atgcccggct 3780
aattttttgt atttttagta gagatggggt ttcactgtgt tattcaggat ggtcttgatc 3840
tcctgacctc gtgatctgcc tgcctcagcc tgcctaagtg ctgggattac aggtgtgagc 3900
taccgcgccc ggcctgtatt acttctttga taaggacacc tagtctcaac ccacttgtgt 3960
attaattagt tgtattcccc gggagggttc ctcagctgct tgccgcttca ctgtgtcatg 4020
gtgaccctta ggtgttagct caacaaattt aattaaagac tgtatcgttg ttctctgctg 4080
tgcaactctg attaggtgaa gcatgttact gctttataaa atatagaatg ttgtcgtaac 4140
ttatctctgt gtatctttcc attgcgtgga ttagagaaaa caaggtcctg tagtttcttt 4200
tcctttcctt tctccagcat tcctctctcc ctaggtcatg gaagtgccac tgcgatgtgg 4260
cttcctttgt tctggtgaag gggagatgat tggagcctct catgattatt ctggatgctg 4320
ttggagggaa atgccagagc tcccacatcc ccacagtaca ggcaccatct cctttccaac 4380
acacctcata ctgtgtacct gtggagggca agacagttag tgtggctagg tgagaggcag 4440
tgtcccaaac tggagtttgc tggagttgtc acaaggttag gaaaatttca ttgaccaagc 4500
cagcctcttt tatatgcaat atggttacag gtaggtaaca gtttgctgaa tatctatcat 4560
aagcttagtt gcatggagga tacacatgta aaaaatggcc tgtaaaattt tgagtcaaga 4620
aacttattta gagaaggtga aattaacaca tgtaggctag ggctgcactg tccaatctgg 4680
tagccactac cctcacatag ctaattaaat taaaattaat taacatgaat ggattaaact 4740
aaattaaatt tgaaaaatca gttgctagcc acacttcaag tgttcagtaa acatgtggct 4800
aatggctatc gaattgaaca atgcagaaga acattttcat ccttgcagaa cgttctccta 4860
gactttgttg gtctgggagc taaatcgtgt ggctgggaat agaggagctt tgaaagaggg 4920
gaatctgtgg tgactccagg agctaaggga ggctccagct ggggtcagtg ggacatggct 4980
gtgcctctga ggagtgagca agtctgtgag aggtgtggtg acattggcct ggctagaact 5040
gaggggagga acaggaggtc agagtggtca ggggttggtg gctccaaatt gaagctaact 5100
catcttgtgt caaagcaggc tttaaagtgt ttgaggggca tgaaggtgtc aaaaaaggtt 5160
aggaatcatt gaagatgggc ttacagaatt atgagggaag ggtcaggttt gtggtttcag 5220
agatcctgtc cctcctggcc gtagccctga ccccagccct gacccctggc tcctgcagca 5280
gggcccttgg catccactga ggcaagattg aagctgggtc agctcactgt ccttttttac 5340
ttgtgaattc tgggctttga ctgagcttgg agacaatctc cagctaagat acgggatcag 5400
tggctgacag gaggggagag acaagcagtc cctgggcctt agggccaaat attggtgggt 5460
gtctaggaaa caaaagaacc cctttgtggg aggaggaagg tattgggacc tgtgatgttt 5520
ctgctacttg ggacctgaga ggttgggcca tccataattg atgatttttc ttatgaagca 5580
ggtacagggg attgggagga gggcctggaa gagcccagcc tcagaactgc tggaggcaaa 5640
cttaatcctt gggctagggg aggggccaaa gctcctgtta gccagagccc aggagtgtgc 5700
tgagtgggtg ccaggaggaa tgtcagccaa gatggggaag agcagggcac agtcgtctgc 5760
atctgagcca ttgcccagtt cccaagcagc cttgccatgc tctgagctgg ggttcttcct 5820
ggtggcgtga tgtctgtggt gcaggattca ctctattctc ctacctttcc tctacttcag 5880
gccctgccaa gtgtcatgtg aggtctcttt tccccctctt ccctgcactg tatctttcca 5940
gatggttctg tgtcctggag ggaggtggat gggcagagaa gagttgttct ttgcagggac 6000
tgcagctttt cttggtaatg gtttgaggcc cagtggcaaa gcaacccaca cttcatagta 6060
acttagaacc cacactccct ggtcatcttt cttgactcct tgcacatcac tgaggaagaa 6120
gctggcctag tgcagaaatg ttcagtggca tgtccaaggt cttacatggt gtcagcatcc 6180
gagccacatc ttgagacctg gttccctggg acctgctctt ggtctgggcc cctttggttt 6240
ttggttgcta gaagactgta gacctggtgg agcaaaactg agctccacta acgcatcagc 6300
taggatcttt ccacatgggt taccttgtaa atgggggcat tggtgaagag aaggggccat 6360
gtcactggac agtgtaataa cgtatggcag ttatagaacc caaatcagat tgctgtgtac 6420
cttttctagt ttttaggaaa atactggtat tttgtatttt aagatatata tatatatata 6480
tatatacaca catacacaga tatagatatg tagagatgag cttgttacca tgggttatgt 6540
gctagataag aaagcaaaaa taagtgcata cataaataga aagcttccag tgtttgtatg 6600
ctgctaacaa gttacatgac cttgaatagg tcagctaaac tttccaggcc tcattgcaca 6660
gagggtcggc tcgagcagct cctgagcacc ggcagggtga ggcagtagaa ggcggtgctg 6720
caggccagag gtggagggca ggcaggatag cttgctgact tgctccgggt ctgaccatca 6780
gcagtatctg ggtcagcaca gatgcccttt cgtaagctta gtgcctggct ccagggcagc 6840
ctgtgtccag ttaatgaata atcatcactg accagctgct ccttctcttg tgtgaagact 6900
tagaaagtgt tctatgctgg tgactcggca gagaggatag actacgtaat ttagcagggt 6960
gtgcatcttc tctttcctta aaatcagaag aatcatagtg tagtggtggt gcaaagtaga 7020
gatgaggaac tctattccct gtttgagatt cctttctcaa gttatggggc atttgagggc 7080
cactgtgatt tctggtgcag gctgttccct gtgcacaggg atctggttga ggagctaagc 7140
gaaggctgaa gtccagccac tgtctccttg cttggctttg tctttgtgtg tggctgggtt 7200
ctttcaaagg agtgccttct aattcaccaa aggtatggtt atggattagg ggttgttctt 7260
aggttagagt tggggactga ttcacagagc tgatgaggaa attaggattt acattcaggt 7320
gaccctcagc ccctcagacc aggctgagta agaagagata tcaggaatcc aaggaagcag 7380
gaggaaaagg ggttgcctgg tgatgtctgt tctctttcct cctcttcctg cccatgcaag 7440
gctagagcag ctgtttcagc cccggaggtt tagactgtgg ggtcctgtgt gctgggccat 7500
caggtttggt gggaggagga agaaagccac agtagaagga gacataaagt tgggctgggc 7560
ctctggcagt gaaaggttct tggggtcttt attgtctgat cagtaaacac ctctgtgtgc 7620
cagaatgagc tgggacaagg ccccaaggtc agcccatatt tgtatctggt gttctcctcc 7680
aagaccagcc aagctgtgcg ctcattctgc tgggaacctg agccctccaa aagggtcata 7740
tgaaagggca tcttattctt gagggacgtt tccaagcatc tctgtggcca gtgtccatcc 7800
aggactcatc ttagaggtga agagtggtgg ccagaggcgg aagctagtgg ccacccgatg 7860
ggagcctttt ctgtttcaga gcatatgagg gagctgaggg agctgtactg ttgatgcctt 7920
ctctctggct catctccatg aagattcttt tgaaccagaa gcaaaggtga agtctctctg 7980
aggggagggg ctgaacagga gatctactga ctgtccccct gccgactcct ccactttcgt 8040
cctccggcgg ggctccttat cctcctgctt gtctacaggg agaagatcga gttgttcgtt 8100
tgctgccatt ttaatgatcc acattgttag gaggctctca gtacacaccg gaccttagca 8160
gcctggcctc ctgcccaggg cccagaactg ttactcagcc cctctgccga acagctaaaa 8220
ataaacctgc cactgctgca gtctcccctc acagccaggc cctgttagct gtcccagtgc 8280
ttcctgccac accgctgacc tgtttctctc actttctgct gatattctcc ctgcttgccc 8340
ctttctcttc cagcctgagc ttggtaggtg atttggggct aactcactct tctgagtgct 8400
gggagacgtg ctatggtttt tatgtctcca ggtgtctaga tttgtgtgta tgaaagctcc 8460
gtgaagctgt cctgcttatt tcccagtagg gaaagggtta gacaaaaagg agagtgacca 8520
agctcatcag agtggatcag accttctact gtgctcgcct ccagccctgg cttcctccag 8580
ttttacctcc aagcaggcct ctttgccaga gttacctggt tatggtaaca gagttaccat 8640
agttatatgc taggacagcc ccacatgggc agtctgcccc aggccttcaa cccgactgcc 8700
ctgtcaccga gcatgctacc taccgtgtac tcctgccctg ttgctacgtt atattctcca 8760
cctccgcagt gtgaggcttt gggctaccac attcaaaagt gtgacctcat cttgtcagcg 8820
tccaagaata actgggccct ggctctcagg atctccttcc tgagtcaggc catggcacct 8880
ctggcccttc cagctcagcc acatgcagaa tgtcaccgag tgcatgggtt tcttctacct 8940
cttgactgca gtattgatgt gtgtccatgg aggtgtggcc tcttcctcta ctccccaggg 9000
atcctggaga cttgtggagc aagatgagga aaggggctca ggtagctaga atatttgctt 9060
agctgataga tactgggcag agtctgacat tggcattctg ttcttcagcg tggcgttagt 9120
attctttctg attttttcta atccctagac aggtgttgat actaatcgtg gcctaggagg 9180
gctctatgtc tgctttcagg aattatggtt tatttggggg aaggggtgta tggtggagag 9240
tgatggcaat gacaaccaag acataggact tgaggaaccc actgccttca gctagagtta 9300
catactctga ccatgtgtcg atggcctcct tctcccacct ctcccctggg gccacatttc 9360
ctctgcaagg aaagatttat ctgccaaggc ccattggcct gtgcagagga agagttgggc 9420
atagttgcta agtgtttatt tttaactctg acagaaggag cccctgtcct cagaatagcc 9480
ataccctctg tctttcccta tcccgactgc cttccttggc ttctgtgatt cctagaagaa 9540
ggttagtagg attctaaagg ccaggcttca cgggagctgg agaaggctag cttctctaag 9600
tgaggaatct gcaggttgct cgtcatgctt ggcttctgca gagctcaaag ggtcctgcgg 9660
ggagagccct ggacactaaa ggccgtgtgt ctggaacttg gagaattcta ggaatgcagg 9720
tgacctgatg aggaggctgc atgtgccctg cagctcagag cagcctggct gcacagctga 9780
gttggacagc ccagcaaggg gtcatcccat accttctaca tccttacagg cacccagatc 9840
tctggcagat ggggaggcca cagggagaaa atgagcatat gtagtaacct gccctggaaa 9900
cacttgcgct tgcagacacg ccaagtgctg tcattccctc tgactgccag gctcacgagc 9960
atcatgtcct ttccagcaga gcccacctgc gactcttagt gacttcatac cctttgtgtt 10020
ctcttcccct gatgtgcttt agtgacctgt gctcacagag aggaggagga agggtggcag 10080
tgccccctct cctgggtctc agactttcct gactttatgc atttgatttg gctacctggg 10140
ctcggatact ttaaaagaaa ataaaattag gtttgaggtg taaccagaga gagaaggaat 10200
agagagtagt agggggtggg atgggtggtt gaaacatgtg gctaactctg agaaccctca 10260
gctatggaag gaaggtcttg cccccatgat tcttcggcgt gcagtggggg attagtttgc 10320
ccagagccca gctctgatca tgtagcttcc cagcttaagc ccttcagtgg gtctctctgc 10380
ctgcagaata catgagatat ccacatcttt cccagatctg gccctgatct gttttttttt 10440
ttttttttgg aacacatgct ttttttttct ccccagctta agtggcttat atatgttgtt 10500
tctttaacag aactgtgctt tccaccccca caccttagca tctatccatc tgcatcttct 10560
ctgtaccatg cctgtccccc gtcctgtcta tatctacctt ttccaccctg cacagatgct 10620
gcttttccag agcctttcgc accaaaagtg ggtcttccca tccttcccca tcaaacttgc 10680
tgatacactc ttacctttta tttctctgtt taagtgcatt tctccatgtg caaggcatct 10740
ctctctctct ctcttttttt ttttaacagg aaggtaattt attaacggaa aatatttgag 10800
gagtcctgtg cacagtgacc atggcaaccc cccttcctgc gggtgctgtc agaggggttg 10860
ggagtgacgt cctcaatctg tccgatcttc atacctgagc gggcaagggc cctgagggcc 10920
acctgggccc caggtccagg ggtcttggtt ctacttcctc ctatggctcg gagttcgatg 10980
tgtagggcag tgatacccag ctctgggcca catcccgggc agccaacata gcagcatgtg 11040
gtgaggattc atctcacctg ccttcacctt catcctacca gtcacacggc agatgatttc 11100
tttgccagaa agatcagtga catggacaaa agtgtcattg aaggatgcaa agatatggca 11160
gacaccaaat acattctctc cttcagccac ctgaggtctg aggctgatga cctgttcttc 11220
cttcttttcc ttcccctttc aaggtgccat ttctgcatgt tgtttccaga ctccgcacag 11280
gaacgcaagg cacctcttta gatatcggat tggactgtaa atcccctgaa aacctacatc 11340
ttcattattt ttgtgtcctc ttattttcat gtagtgaccg tatgtcctca ttaaaaaact 11400
caggccaggc atggtggctc acgcctgtaa tctcagtgct ttggaaggac aagtgggagg 11460
atcttttgaa ctcaggagtt tgatatcagc ctgggcaaca taacaagacc ccatctctac 11520
aaaaaattaa aaaggttagc cagctgtggt ggcacatacc catagtttca gctactcagg 11580
aggctgaggt gggaggatca cttgagccca ggaagtcacg gttacagtga gctatgatca 11640
tgtcagtgca ctccagcctg ggtgacagag cgagaccccg tctctgaaaa aagaatccta 11700
gagtattttg gcactgtgca ttatatgcat tatacttagc tgctgaataa aggtgtgata 11760
actgagtaat agtggatgcc accgatatta aatgtgatgt actagaggta atcctcagag 11820
tcagggactg tggcagccct gggagtggga tagtactggg ctgtgctctc tgagactgag 11880
ctggggtaga gtgatgatct ctcacattgt ctgttctcat tcttctgctg ctgaaaagct 11940
gctcttagtc ttctgagcat ttcttccctc tacccacttg gatctgctga ctcaagcagg 12000
caccttctat ttctgcttac agagttctca ttcttacaag gaccctgacc acagtttctc 12060
attgatgcct ctcctccttc tccctgttcc aaggcccctc ctattcctgt tcagtcatgg 12120
agatgaccct tttgcccttt ggagaacttt aactgaggga gggacagatg tggagcttgc 12180
caactttcct acagctcctg gcattttcta gttagctaca gacacggcac agacacacag 12240
atgcagtcca ttgttttcta tctgctggtc ctgctcctgt gtgttgcaag agggacgagt 12300
gttaaacact gtattagtcc attttgtgtt gcactaaagg agtatctgag actgggtaat 12360
ttatagagaa aagaggttta ttgctcacag ttctgcaggc tgtacaagga tcatggcacc 12420
agcatctgct tctggtgagg acctcaggaa gcttttgctc atgggggaag ttgaaggggg 12480
agcaggcatg tcacatgtcg agaagaggga gcatgggggt gggggaggcc ctcgactctt 12540
tttaacaacc agatcttcta tgaactcaga gcgagaactc actgccacaa ggagggcacc 12600
aagccattca tgagggatct gcccccaaga cccaaacatc tcccaccggg ccccacctcc 12660
aacactgggg atcatacttc aacatgagat ttggaggggc caaatatcca aactatatca 12720
agtacccagt tgctgggact gaggtcaaag gtttgaacct catggggctg cttaactcca 12780
tgccacaaca gatttacaca agaaagagaa cctgacacta gactggaatc tgggagacct 12840
ggctactaac tctacctgtg tggccccaag caagtcctag gacctgtctg ggcctcactc 12900
agtttcctca ttgtaaatca acaggactag acttgacctt gaaggactcc tgcaccgaaa 12960
gcctttcatc actgttgttg catccgcact gctgggaaaa taacctgatg ctgggaaaca 13020
agtggtcggc ctgggtccca gggagcctcg gggactaaga gccatcatct ctgtcctcag 13080
tttctgcagg acagtgttac ttcacctttt tcctggctgc agtgttttta ttcattcagc 13140
caatgtttat aaatgtttat tgttctccat gcctgcctcc tgtcttccct gctgactggc 13200
catcaggagg cccagatggc aggattagct tggaatggca gaacagtcaa gggtcatcct 13260
cttgggttta gaccccttta atacctctgc tatatgagat gagaagaatt tagaacattg 13320
atccttccag cagcttagac tagacccctg cctttctaag caggatttgc ctgtgggtac 13380
cttctatacc cttgagatcc aaagcaactt actactcaag aaaattaatt agcaatgcca 13440
agagtgactt gtacagctcc cctaaggata gctcaaaaca gcagatagac tttgatacaa 13500
tataacaaat ttctattatt cagatcactg taggaaactt agcaactctg tgaaggtttc 13560
ccagattaat aacaagtctt tattgagcag atactatgta tcaggcattg tgttaggtgt 13620
tttacttaca ttcttgttaa tccttaaaat aaccctttta ctgcaaagtg aaagtcccca 13680
ttttacagaa gaaacatgtt catgtacaga ataattcctt agcaattatt tattgagcac 13740
ctactaagtg ctaagcatta tcttgggctc tggatataca tcagtatata aaacagacaa 13800
aaaagctatt tggggggtgg gaagggggac caggtagagg aatagcaagt gcaaaggcaa 13860
aagcctgagg cttattaata agtgtgtcta actctaaaac ctaggctccc tccagctcac 13920
agtattgcct ctctaatggt cctttgtgac tccattataa ttgggtactt agatgaccta 13980
gcaattttaa gtttatccaa tatttatggg atacttattt tgtgcaaatt gctacgatga 14040
gatgaataag ttgtaggccc tgcctttgga aacatacagt ttagaggaac gaacacccgt 14100
gtgtataaaa atgaatgaaa gaggacttcc tctcaaaatc cagatcttac catttcttgc 14160
atttttgtca tttgaccctt gtaggccaga tggtgtggtt cctcgttttg tatctgaagg 14220
aagagtttca gggagtgggt gggggtaagt gtctgggcac cagggtggcc aataaaggat 14280
gcagagacac atgatgggct cttcaaggtg ggaggaagta gagggtggga tcactgcctg 14340
gcatgggcag aggatacttc atggacatat acctgtgaca cagccagggg agggtgcaga 14400
tgaaaatacg tccagtggga tggtttctaa ggagaagtgt gagggactcc actggggctc 14460
cgtattttgt ctggctgagg agggagagat gctgcctctc caggaatctg gtaataggca 14520
atgcttgaga ttggtgtgaa ccaacgcatc acatagagcc aggagaagtc acgaaggagg 14580
caagaaaaag aaatggctgg gctaaagctg tggcagagcc ctttttatcc tcctttgggc 14640
cctcatatgg cttctctttt ggtctgaaga atctcatcct tcttgagttt agccttctag 14700
gtgaaggagt gggcagactg catgcaggga gacagggagg ccgtgtttgt gtccttcagc 14760
tcaggagctg agagaacaga ggtcagggct tgggatggag ctgaccgtta gtgcagatgg 14820
gaaagtgaag caatgggtcg ggtctgtttg ttcctgagca tgtcctgtga caaaggaagt 14880
tgcaacatgt ctgtctgtct ttctctctct ttttcttttc tcctttcttt tcgtttcttt 14940
ctctctctct ctcttctttt tttctctttc tttcttcttc ttactttttt tttttttttt 15000
tttaaataaa gaaagaagca ggtaaaacca tcttccagaa aatgccagaa agaatatctg 15060
gtctgtgatg ggtggttctt gcccctcccg ctgcaatgct tttcctgtgt agtttttctt 15120
ttgtccttcc tttcccatcc acactttcca ctctcctttc ctctgagagg ttgtgtctct 15180
gcagcaggcc agctatgtct ccaaagggaa gtagtcagtg cttggactga gggaaccttg 15240
agagagaatc ctctcctttg tcctccaggt gagagagctg ttgcatccac tcacacctgg 15300
aaggttgctg gtctcctctc gctggacact gagagctgtt taaatttgac aggtggttcc 15360
tgattcccac ctggcatttt ttgctgctgc acaggtctgt gttgtgtctt agaaggctga 15420
gggaatactt acttctcaac tcagataacc accttgttgg taggttcagt gtgtgtctgt 15480
gtcaaggcag atgctgggaa aaaagggccc agaatgtgag tgggaggtag aaatcaaggc 15540
tagaagacca cacatgttaa tctggagtac ttggtcaggt ttgattttgg cctgtgtgct 15600
atagctgggc aagaagccag ttgtgtttgg aaatgtacag atgatctatt ttcctgatga 15660
aaccttttga ggtctggaat aaacatagct tgctttctct ctttccctct tttgagattc 15720
aatttctctc ccttggactt ctatacccta gtagagccaa aagttccagt acaaaccagt 15780
tttgagactc cagcattagt caactaaggg atgttgtagg tcatccctga gaacgagggc 15840
tgcttgctct ggggaagctg ggaaatggca ccaggtgtca ggccctactg cccaggatgg 15900
gatgacagct gctggtgtta attacgtttc tgggcagaac agggtaaagt ccttgggggt 15960
gagagttttt gtttaggcac tgtgttccct gtacctgttg cctctggtac agctgctgca 16020
gagcccctcc ttgcctgcag tcttctttga atttaatctc cctgtgatcc agtgcatctc 16080
ttcagactgg aggtgagccc agaggagtgg tggagggagt ggggccagaa tttacttgtt 16140
tgcctggatc ccactgagaa tacagaagct ctctttcacg agaagctgtg ctggcctctg 16200
tggtacccac tggccatatg tgactattaa aattgaaatt aagtgttaat aacatttaaa 16260
attcagttcc tcagtagtgc cagccacatt ttaagtgctc agtggttagc tagtggccac 16320
ggttttatgt ggcacagata gagaacattt tatcatcaca gaatagtctc ttagacaatg 16380
caagattctg agataatttt tctaatcctg gtagacactt agctggaagg ggatttgagg 16440
aagggtacag ggagaggttt tgaagcccag gtgagagggc aagtagaagg tattcattta 16500
gaaaaggatc ttaaaaagta tttagtttgt agggccaggt gcagtggctc atacctataa 16560
tcccagcagt tcatgaggct gaggcaggca aattgcttga gcccaggagt tcgagaccag 16620
cctgagcaac atggggaaac actgtctcta caaaaaatac aaaaattagc ctggcgtggt 16680
ggcgtacaca catggttcga tctactcagg aggctgaggt gggaggatca cttgagcctg 16740
ggaggccaag gctgcagaga gccgttattg cactgcactc caacctgggc aacagaatga 16800
gaccctgtct ctaaaaaaaa aaaaaaaaaa aaaaaaagag gagttcacct tgttgaggac 16860
tacatcctaa gagatggtca cctggcccag tggctcccaa accaggatga ttgttagact 16920
cacttgtggg tagcatttca agatacagat tcctgggcac cactctttgt gtctagttga 16980
atccaagact cttcagagga ggtaacctgg aatgtgcatt tttaatgtgc tccccaagtg 17040
ataattatct gggtttggca accaccaacc tatcatacct gtgcttaaat tcttccaatg 17100
gtaaggagct tatccctttt caagaaaacc ttaccattaa ggaaaaattc gctgttgtta 17160
aagttctatg tttttcgaat cttgagattt ggttctttga ggtcatgaaa tcctttggtg 17220
ctcattgcat ctttagagcc tcgcagaacc gtcatcttca tccttgcact cagtcttcag 17280
cagatatttg agggcagctg tggtgtggtc cctgccactc caagtcatgg catgcgtcag 17340
agtgcttgat ctgccaaggg ctctagggag atgcagagat gcaacatcat tccaggatcc 17400
ttccaattgt caggccttgc tgagagccag aggaaaggtc tgaatctcag tggagcccta 17460
cagggacagc ccaagggcca tccagccggt gagggccatg tgtgtgtctg cctctctgca 17520
tcctgctccg tagggaagag tccctgcttc tttgcatgtt agcacttgtt aacaccgacc 17580
tggttttctt cagagacatt gagagattca gccatccatt cccggtaatg acagatgtca 17640
tttctgttta gagaccttca gtagcctccc ttcacttcca gtgtaaaatt caaactcctg 17700
aacgtggcat tcaagcaagc tggtttgtcc tgccttccac actttgcctt ccaggagagc 17760
actccttccc gctctgcctt gcatttatct gcttccacat cttctcttgg atcttacatt 17820
tgcttatctt cagttttttc tggtagaact gtatttatcc ttcaaggctc ataatagatt 17880
ttaccccctt actcccctgc ccaattccag gcagaactgc ctccctcctc tgccctagtg 17940
agggttcatt ctgcctgcat gactgctatg cactcaaatt acagccccct gccaggccct 18000
ccctgctaga ccaagctcct tgcggacaag aactatgtct tacttggctt gatatcccca 18060
agtgcaagca agcaccagtg ctggagcctc cttttcctgc attggcattc ttaccctcag 18120
tggcccagag ctcatcagct ccaatgagaa aagtgctact tagcagctga taattaacag 18180
acaggacgtg aggtttggct acttcagagg caaaactggt atttaaacaa taaggttttt 18240
aaaaatcaag cattaaagtg tcagcttcag aagcagctac ttccaaacat aatcctgccc 18300
agttagagaa gacaaatgga ataatgagtg gaaccaaaga tttctctcta gccttgagaa 18360
gagttcaggg gaaagaggcc tgggtggcgt attggcagga gattggaaaa ggggccttca 18420
tatttctttt ctctggaatg gggctggggc tgcttggaga gcaggtggga ggtgcttcct 18480
tagaaagggc tgtctgtgtt ctcttccatt cccaatttct atagattgga ctagttttgc 18540
caagtgagca agcaagaaaa attgggaaac agagggtagg catcaaccct ggcatttatc 18600
ctaaggtgat gcagcacaca cttgtattca gcgatagaga cttttccgta atgcctggta 18660
atatctcatt gtattttctt ttgatgctcc tgggggcagg aggggagagc tgacatcaga 18720
gcctcctccc ttctagatca caaccagagc atgaggaatt ccccagcccc ttgaggtacc 18780
cagacactaa ccacaatctc catctctgct gaaggcgttg taagaaacat gcaggccagg 18840
aacagtcagt ggcttcagag caggggcagc ctgctaaaga ggcagaggct tcttgtgagg 18900
gcggggagct tgagagtgcc ctgcacccca tcctggggga ggtgagcatc tgcttctggg 18960
ttcagctgcc tgcggaagtc agtgggagac accctttcag ggtctgcttc attatgaatg 19020
aagcaaaaag cctatcccag gaaatcacag aggctgatgg gtatacccca gggtacacag 19080
gctatatttt tccaatccaa agtgagggtc tatctgatag tttttcctgg agttggggag 19140
gggtcctact ttcagatcca aaagaaaatc tgagatatgt catttgaata tgaacgcact 19200
agactttctg ggaaatgttg aagatttacg cctgtctgag tgtctctcac tcgttgttac 19260
ctgtccattt gttacctggc ccacggatgg aggaggtcgg atggggtctc ccagggtccc 19320
cttgctgggc acgcttggca ggcatcctgc ttttcctttc aggttgctgg aattagtggt 19380
ggtggtggct cagctcaccc agaccatgtg tgatggttat ggtggtgact tctcactgac 19440
tgctgatgtg tacagtacat tgaaagcagt cacccaactt cctttatagc cggatacaaa 19500
acagtgcctg aggtccaggg tcggggagga cttgatgagg gaataaaacc agccggctca 19560
tctaggggct gatatctgtc cgtggtgaca taagcacaca tgatctgcag tcaaggttga 19620
cttgagggtg tgcatgggtt cagtcccttt tatgaagtct aatggtcctt ccagaagctt 19680
catcatcatt tgaactgaat aagtgaagat gagtgcacca caagtgtgtg tgtgcacttc 19740
ttaatctgtc cattcatctg ttcactgcag gcttattgag ggagaaacat gtaagcaagg 19800
tatttcttgt gtactccaag tggtaggaga gctatgttca gggaactggg tgaacttgga 19860
agaaggagcc gcccgtttct ccaagtgcag cgccagtccc gagacactct gtgatcatct 19920
aaactgggga acagtctgca atgcacatta ctgaattaaa agctctgaga agttctacag 19980
tgaaagaatc tgtttaactt tgtttaacct ggtgcttctg ccatctatat gacccctttt 20040
tacttagcac ttattaacat cctacggaat actatttgag aaatggcaac agcccccctt 20100
ctctgggaga actgttcagt gattcagagc aaaagtcacc tgactctggg tcagcacaag 20160
ttggagaggt ttgaaaaagg tgtatgtatg tttgcatgtg tatgtacggt gggttggggg 20220
gaggggtgtg gcagaggagg ggccccggca ggtggcttct attgaaatca gctctgtctg 20280
cctccctgtt ggctttgtgc ttctggagaa gcttttgtcc tggcatgaaa acactgaata 20340
gcagctgcat ctgtcatgcc tgggaagcag caatgtgcag catggggttg catgagtaga 20400
cactcctgct ggagggccag gctgccgaga gtgcctgggt attggacaag ggagaggctg 20460
gtgattctga ggcagctgct tgctaatcct ggaagcagag gggaacaaga tttagctccc 20520
ttatctctct gccttctcct ttccgatggg tgcaattgtg ttgactcttg agggacagcc 20580
aggaagaagc aggtgggctg gatgctttct cctttctcca ccaacccaag ggcatctgga 20640
gggtggacca gggcagaaag gcgattagct ctccgttacc cagggagctg agacaaaggc 20700
attttgggtt aaattcaagg aggaattcag aattgtgaac tggatttatg gaagtttgtg 20760
gagtttgaaa attcgagagt cctggctgga cttgtgcttg gaacgttggt cggaggtgga 20820
tatgaatgat gggtcctgca gatggccgac tcctgagaaa tgactgggag gctttactgg 20880
atttttcgct tggaacaaac ccaggcttag ggaattcccg gagcctgttg caggcctcaa 20940
cacccttctg gtgcagcttc gctgtgaggc agtggttatt tctcaggaaa ctggcaactg 21000
ggagtaaatg ggagtgtggg gtgggaatca gctactcttc ctgaggatta tacacttcca 21060
gtgtaattaa ggcggggtca ggatttccta tctcttattt atttatttat ttatttattt 21120
atttatttat ttatttattt gcttaatctt aaaaggctgc ttttcccctc ctggtcttcc 21180
cacagggaca ggagttagtc tgttcccaga aggagctagg ggaggtgtcc ttcatctccc 21240
attccttctt gccctctccc tgcctttccc tggccacagc cacagagacc cagagtcact 21300
cagatggggg aggagcaaag ccagtccctc tgatcattgc ctctttgtta gtggagaaag 21360
ggcagatgct gctctgcagt tatcccgacc accctgcagg acgtctgggt gggccgggct 21420
ttggtttggg aatcgggagt attagctgca gctgccaact gggcttgtac ttgcagactc 21480
ccaggctcca ggctctctgg aggggaccag cagctctgac ccccgtgggc ttgctgtggt 21540
ccccagggag caagcccttg ccaagaacct ctgagctcac cgaggcgggt atggggtaag 21600
gatggagaag gcctggggag cagaagtgtg cagatgactc cctccttccc ctcagctggg 21660
tatgcatcac cgctgctggg aagacgttca ggccacttct agacaaatcg gtcatgatgg 21720
aaattttttc ttgcccgtgg gtggggcttt acttgttcag aagtggcttc agatgtgctc 21780
tttctaggtc ctggcttgag tgctgagcat ttacctgcag cccctcgggg ctagaaggca 21840
gggcagtgtc tgagcccctt ccctacccct tggggctaga aggcagggca gtgtctgagc 21900
cccttcccta cccctcattc attctctccc ctcacctgtc attccatccg tcagccccga 21960
acagcttggc agccttgcgg ctccagctgg agcttagtag gcatgtagtt ggaggaggga 22020
acggcactgc agccgcgctc ttctctctcc cacaggtctt cttcttgttg ggcctccact 22080
tcctgtcagg gccctgcttg cagctgtggc acatctggac ctagacgggg tatcctgttg 22140
tcctgcatga tgaggatccc agatttcccc taggtgatga ccacagtcat tccagggtgg 22200
gagcaggact tcccccaccc ccaggcactc cagagacttc ctccaggccc ccactaagta 22260
gtctgttccc aactccatct ctcccccatg gagaatggtg ggaaggaagt tctgccagag 22320
gttgtttttg gtttgtgagg taatagactt tggtttgaga aacatacatc tctccctctt 22380
ctttaagatg tgaagccaga agcaccagac tctggtttcc ttggaaacca gagcagcaaa 22440
ggtagagaaa aaaggggctg gttgtaggca ctgaaattgg ggaggattct ctggatccca 22500
gcacttttct ggggaatcca catggtctgc cctgggccct gggttgtagc cctcactgtg 22560
ttaatggact gtgtaacctt ggtaaaatgc ctgcaactct ctgcacctta gtttccgcca 22620
agatgcctaa ggtggggaac atgttggctc cactcagatt ctgtgcccca gagacctgct 22680
gggtgtgtgt gggggtttgg gggtagggca tggggctcct gctcactgat ttagcatttg 22740
ctgtggctaa actacaaagt ggacagtcca catggagaca gttgagagct acttgcaccc 22800
aggtcctttc tttgagaata cagtgatgta acaggagact tcttgcattt tgcctatgtg 22860
aaaccagctg gattttgctg aagctgtaat tgtgtctcta cattctttga ttcctgtcta 22920
aaaaaaatgg tttccacaaa tgctgtgaag aatagactga cagagctgga aagcgttaag 22980
tagggatcct ttaaacagat ccctacattg tacattgatc ccttcaacca cagccagaga 23040
ggggcaggga cttacttgct ctgatcacac agtcagaaca agaactaaca acagtaaagg 23100
tctgggtagc agcctcacat atgctaagat tccctgttgg ctaagtttgt ctgttcccag 23160
gtcaccctgg ccttttgcaa ttctgccctc cgcaggcccc tgtgttggct gaaaccagcc 23220
ctcttcttcc ccttgcgctt tcctcctgtg acctcccagc cacctcttgt ttcccctgtg 23280
gtgttcccca cctgttgtta cttcctattc ctcctcacaa ctttttatta tgaaacattt 23340
cagatgtgca gcaaactgga aagaattgta cagtgaacat gattatacca aaccacctag 23400
attcaaccat tagcctttta ctatgtttac tttatcatgt ctctacgtgt ttcaatctct 23460
ctatccattg accagtccat cttattttgg ggatgcattt tgtagtaaat tgcagacatc 23520
aatgtacttt acccctaaac atctcagcat acataggcat taatttgagt tcaatatttg 23580
ttcatgcatt tttttgatga taatatttat attaacagtg aaaaatgcac aactcttaag 23640
gtatcacctg ataagctttg ataaatgcct ctatctgtgc aacccaaacc tctgtcaact 23700
atagaacaaa aagttttatt acctaattaa ttccctcgtg ttgtttccca atcatcccaa 23760
cacatacccc ctcgcaagtg atcacagttc tgatgttttc tcactgtaga ttagttttgc 23820
tttttctgga acctcatata aatgggataa tcagtatgaa ctctcccttt tgtgtgaatt 23880
tcatctgcat ggtgacatgt atcagtagtt tgttatgctg agcagtattc cattgtatag 23940
atatgataaa atttgcttct ccattcccct gttgatggac atttgggctg tgtccagttt 24000
tgggctatga tgaataaagc tcctgttaac attcttgcac aaatcttgtt gtggatgtaa 24060
tgttttcatt tctcctgggt aaacaacgag gagtgtgatt gcagagttac aggaggctgt 24120
tacttattgt tcctatctga ctctctgccc tgccctctca ttgtactgtg cacccaatta 24180
ttttaggact gttatcacat ccagtccctc agttgctcct tcagggtttc tcttcttccg 24240
gcaattcacc ctagtttatt gaactgttgc tcttatgtta tgctcctcag atcttttata 24300
tcctggttgt cctcctctgg aagtataata ctcttttaca gatgtgttgc ccagaactga 24360
gttctcgctg ctcgatgtga tctacatgtg gattctaggt atgggagaaa tatagagtgt 24420
gggtgcattt ggatccctgt cttgagctag gtacttgatc tgtgagctga aggaaatatt 24480
aactttttgg cagccacgtc atggcatcag atcagatgag ttaatgtttg taaaaatacc 24540
acaaatctaa ggtcatatta agatatccat tcagcttgaa gtcagtccga gcctctgata 24600
ctaatggcat ttattatgaa ctttctactt gccattgtgt catttagttt ttgcctacca 24660
cttggtgaga aaggtactat tcaaattagc ttaataaaaa ctccttgatt taaaactagg 24720
gtacttagct ttggcactgt tgaccttttg gaccagataa gcctttgttt gttgtggggg 24780
attgtcctgt gctttgtagg agttagcagc atccctggcc tctacccact tgatgccagt 24840
tgcaaccatc tatctaccca tgcccccaag ttatgacaac agaaaatgcc ttcagacatt 24900
gccaaacgtc ccctgggggg caaattaaca ccaggttgag aactattccc aaggtgtaga 24960
gcttagtcac cgagaaaaaa tttaaacaat atagctcttc ctgaaaggta atcactgcca 25020
gctctttagc atatatactt acagattatg ctctttctat ggacagtcct catttttcat 25080
ggttctaatc tgtgtgaatt tcagttacta tggtttggtt aaataacatc agtcctcgaa 25140
gaatttggct caaatttgag ttgccaggat atatgtatta caagtaactg catgcagtac 25200
aaactttgct gttagctctt cagtccacag atcactatgt acataacaga tgctcttcat 25260
ggtcactgac taatcatgtt tcttcttgaa gagtcttttg gtgattggtc actgcctatc 25320
tgttcacttc acacgcagac aggaaagtgt gtagttgtgt tgcctccctg tctcctagtg 25380
agaaactcgt gacattttac aaaaatggac aattgatgag ggaattggcc aagaaagatg 25440
aaatttcaaa tgaaatgtgg taatgctggt agtgaatttc aagccaaacg taatggaatt 25500
atatagacta gatagctgac tgtgggaatg tcaacactgt cactgttcaa gactacatat 25560
acagcctggg aaactcagtg aaggtggcat gtcagcataa atgaggaaag tggtttgatg 25620
aaaaggatga agaggtccca gaggaggtga cactgcagaa ggaaacttca cattaaaggc 25680
accttgaaga tatttcacaa catcgaaagt gcaaaggata aaacgttaga agcagatcca 25740
aacttagaaa ggagtatgac aatttgcaaa ggcatatgaa agatactcat tccatattgg 25800
atgttaaaca acaagaaggc aagtgctgtt caaattactc tggataaggt ttttacaaag 25860
caataaaaca ctcattctca gtgactctaa tgttttagag tatacacaat aaatagtttt 25920
actgttttct catttcctta tttataattt gacagcaaga gagtttttaa tgttttgacc 25980
gaaattttta aatgtcgtgg aaaaatcata attttcctca ttggtgatta gggttgcttt 26040
gcatggtcat ttttatggtt ccgcattact gtgcaaagca agagctgcct gtatttacaa 26100
aaagtgtggg tgaatacaca tgcacacatg cacacaccat gctttttctc tttttaaaac 26160
attttacaaa atgggatcag actatatgta tcaaattaca acttgctttt ttcatggaag 26220
ttttttttgt ttgtttgttt gtttgttttt gagacaccat ctctctgtcg cccaggctgg 26280
agcgcagtgg cgctatctca gcccactgca acctccacct ctcggagttc aagtgattct 26340
catgcctcag cctcccaagt agctggtacc acaggtgcgt accaccacac ccagctaatt 26400
tttgtgtttt taatagagat ggggtttcgc catgttggct aggctggtct cgaactcctg 26460
acctcaagtg acccacccac ctcggcctcc cagagtgctg ggattacagg tgtgagccac 26520
tgtgcccagc cagaagaatg tatttttgaa gaacttttga tttcagtatg tcttaatcta 26580
cctcattctt tttttttttt ttttttttta tgtttttgag acaggtctca ctctgtcccc 26640
catgctggag ttcagtggtg tgatctctgc tcagtgaaac ccctgcctcc caggctcaag 26700
tgatcctcat gtctcagctt cccaagtagc taggactact ggcctgtgcc accacaccca 26760
gctagttttt tctgtatttt attcgtagag acagggtttt gccatgttgc ccaggttggt 26820
ctcaaactcc tgggctcaag tgatccatct gcctcggcct ctcaaagtgc tgggattaca 26880
ggtgtgcgtc actgtgcctg gccccgttgt ctattaacag ctgtttagtt tatttccttc 26940
tgtcccctaa tttgaaataa tgctaaagtg aatatttttg atggatacta tcataaactc 27000
catttaatcg atgagttcaa tggagattta gaatgtgaag taactggcta aggtcaggca 27060
gctactaagc tttagagctg gtgtttgaac ttggggcagt gtaatttcag agcccttgca 27120
atcacaatgc tttacacctt ctcttccttg ttcttagaag cagcttttaa caacgacaac 27180
aaataataaa aaaaagatct tactgagtca ggtacaattc taagtgcaat tatattgtta 27240
ttattcacta aataacaacc tgcgagttag ataatgttgt tatccctgta ttacagtgaa 27300
ggaaaccgga ggtataagaa ggttaggaaa ggtgctcagg gcccacagca gtttaatgga 27360
ggagctggga ctgcccccat gcagcctggc ttcagaacct tggctctcat tcatgttata 27420
aagatgtgac tagagaaaag tctctttcat cctgtgctgt tgaagatggg tttttttgtt 27480
gttgttgttg ttagttttgg cgtatatttt ctcctactgt aatcttttaa aaatctacta 27540
cctagcacag actgcctctc ccattgtttt cttagggggt gtcacttgga agtttgataa 27600
gcttactgcc tactatgtct ttagtcagct tgttggtaaa atattaatac taggcaaagc 27660
cagtctggtg tggtggctca tgcctgctgt aatcccagta ttttgggagg ccgaggtggg 27720
tgaatgactt gaggtcagga gtttgagacc agcctggcca atgtggtgaa accccatctg 27780
tactaaaaat ataaaaattt gctgagtgtg gtggtgggcg cctgtagtcc cagctactca 27840
ggaggctgag gcacgagaat ctcttggacc cgggaggtgg agatttcagt gagctgagat 27900
cgtgccactg cattccagcc tgggcgacag aatgagactc catcacacac acacacacac 27960
acacacacac acacacacac acacactaat aggtttgcca ctgaagttgt tctttgggtt 28020
tagtcaacaa gtagatactt attgaacacc tcctaaatgc catgtaccgg gtctaggatt 28080
tgaaatcaaa agatgaacaa gacattgtcc cagcttccaa gaggtacatg gtccacagtg 28140
aaaagagaaa ccgttttata aatcccactg gtgagtgctg aagtaggagt ttagcaagag 28200
tctgtgggca cagagaggca gaggggctga ctctgcctgg aagtggtcag agaggcttca 28260
cagaggaggt gacattgcca tggttcttac agacgtgaag tcatgacaag gcctggcttg 28320
cctgaggtca ggaagtgcat tgtagcttca atatagggga tgtgttggga ggttagggta 28380
gcgatgatgt ggataggagc tagggaagac taagttaggg aatttggatt ttatcatgtc 28440
agccagtgga gatgtttaag gaggaaaatg tcctgctcag ctttgccttt cagcatgtga 28500
cccaagatgt actgggatgg agtacgtctc ccttctagaa accagttaga aaacacagaa 28560
gagtaggcca ggcatggtgg ctcatgcctg taatcctagt actttcggag gccgaggtgg 28620
acagatcact tgaggtcagg agattgagac cagcctggcc aacatgtgaa accccgtttc 28680
taccaaaaaa taaaaagtta gccaggcgtg gtggtgtatg tctgtagtcc cagctagtca 28740
ggtgcttgag gtgggagaat tgcttgaacc caagaggcgg agtttgcagt gagctgaaat 28800
catgccactg cactccagcc tgggggacag aatgagaccc tgtctcaaaa agagaaagca 28860
cacaagagaa aaggggctgc aacccagtaa gcgatagcag gactggaaaa ggaaagatgg 28920
atttgggaga tcttcccaga gacaaaattg gattgcttct gtgggtgacg gagggagaga 28980
gtctgaggat gagtactcaa gatttatatc tgggagcctg gctagatagt gatgtcattg 29040
ccgtttactg agatccggga gcaaattcaa aggggccgtg agttcagttt ggtgcctgtt 29100
cagcagcctg tcctagatcc accaaacagc actttattac tggccattta taacaactgc 29160
aaatattatc taaatgcttt gtcatctcac tgcctcctcc tttgtcatcc ctaaggtctg 29220
ggcaggagac tgtgttcata tacttcatag aggtccagac cccttgcgtc tggcattcct 29280
ttggtctata attcagtaaa ctctgctaaa aaggaaacga gactagcttg ctgtggcctc 29340
ttaagcgacc cagggtagct tgtgatggtt cagattatga tttgttctag agcttttcca 29400
gaggcagatg ttgaggagtt tatcctattt gtccccttcc ctttaaacaa acaaaagtgc 29460
cggctggacg cagtggctca tgctggtaat cccagcattt tgagaggctg aggcaggcgg 29520
atcacctgag gtcaggagtt cgaaactaag cctggccagc atggtgaaac cctgtctcta 29580
cttaaaaaaa aaaaaattag ttgggtgtgg tggtgggagc ctataatccc agccactcgg 29640
gaggctgagg tgggagaatt gcttgaatct gggaggtgga ggttgcagtg agctgagatc 29700
acaccattgc actccagcct gggtagcaga gtgaaactcg gtcacaaaaa aaaaaaaaaa 29760
aagaagtccc aaccccccag ttttttatat cctcttagtc atcatagttt attaacagtt 29820
ttgtttacat attgcttctt tttccctctt ggctagactg tgaaccttga agccctgtga 29880
gcttggtcta gtgcctggag cattgtgggt accaggtttc gagagaatgt gtgctgccag 29940
aaagcagatc tttgccacct cctctcccct cagctcctga accactgcca gcagtggttc 30000
tggaatcaac ctgtactttc cttctgcatc ctgggatgta attctgggtg attgtaagtt 30060
ggtttatggt ctagcttaga attgaggctt ctcttgagag agagcatgtg tgtaaatgtt 30120
tagcttgttc tagtctgtgg agcattctcc ttcatagaaa agatgggaaa tactggagat 30180
gaaaattctc cattctctgt cattttgtgt gttagagtct ctgtccccat cccagggcca 30240
cttcctccct ggttcttctt atggctttgg atgtagcttg aagacatgct cctagcattt 30300
tcacaagctc tgcttgttgt gtttagtgag cgcagactac tcccttcggg cctgtcctgg 30360
ctcatgggca gttgctcgtg tctttgcaca catcctttac tgtctcagct cttcacttct 30420
ccctgacatt tgtggctgcc tcctcctggt ttcttcttta agcgattatt gtgttttaga 30480
tgatcagaat tcacttttta gagacttgga ccttgtcttt caaaatgcat tcccttttgg 30540
gatctctgcc catcatactc cttggtctca acatctaaat attggttttt tttttctttt 30600
tctttctttc tttttttttt tttttgagat agggtctcac tctgttgccc aggctgaagt 30660
gcagtggtac gatcattggc tcactgcagc cttgacctcc tgggctcagt gatcttcctg 30720
cctcagcctc ctgagtagct ggggccacag gctagtgcca ccatgccctg ctaatttttt 30780
aaattattca tagagacggg gtctccctgt gttgcccagg ctggtcttga actcctcctt 30840
ctcctttttg tatcctctta gtcatcaagc catcctcctg cctcagcctc ccaaagtgct 30900
gggatttcag tgtgatccac tgcgcctgac ctgaatattg gtttttttga agtctaggct 30960
aaacaactgt ttttgtctca gttttttttt ttttaatttc cctcctttga actctgaatt 31020
gacaagctta cttcctccca atgtggtgtt cttgttatta ttgtttttgc tagctgaagt 31080
attttttttt tttttaaaca taatttacta ggtttttctt cctggccaga agtaagttca 31140
gggtaatagt tcccttcctt gtatcttcag ctgtgtgaga ggtaatgtag agtgagaatt 31200
tatttatcag gtgccctgct cgtagcagaa ggcatgacct cgacttctct ctcacagtga 31260
ccttgtgttt gtgttggtgg cttgaactca ctcttgtgtc tgggccattt ctctcactgt 31320
gtaggaaaga gtggttcctg tcttctccct ggccatgtag tctgctgtgt gtcaccataa 31380
caatcatcta tcttccggtt ctttttcctt ctctcctcac ccagttgtct ccagtcttag 31440
gcgtgtggaa ttgtgtactg ggtgggggat gtgtgtgtgt gggccgccct cttgatttgt 31500
aggatccttc tcttcctcca cattctctgt caactctcct ccaccccctg tgagatctgt 31560
ggactcaggt atacgttgct gtgagtctca ttctgctcag ctggggtgat gaaggtgaga 31620
ttgcttttgt cgcttcaaag taaaatttta gatttgcttt ggcagccgca cagggtcgta 31680
tgcatggggg ctgcttaggc cagaggcgca gtgttcctga cctccttccg tcttactttc 31740
tcttgctagt ttgtccatct ctgtggattt tttaaagtag atactagata cctagatact 31800
actaagaaca tgatctttct cttcttgcca cttggatttt agcttaaagc caacttggaa 31860
catttagggt gtttccttcc taccactgtc tgcctggtct cctagggaaa gatgccttgt 31920
atcttgttct ggtgtacagt ctgcccattt aattggtaag tgtttattgt gactcttctc 31980
tactgggctc tggccccagg cctgatgcga atagggcagg cctgtgaaca gagcaaagac 32040
gagtcactta ggaggggacg agagaggcca gcggtaaact aatattacca gctcagggtt 32100
tatttgtcaa agcctccttt gctgtgaata aaaggttaga tctgaattgg gtctgagtcc 32160
tggctttatt acctactagc catgtggcct tgggcaaatt attaatactt ccttgggcct 32220
tggttgccgc acctgtacga tggagtcagc aatggatcct gttctgtagg gctgctagga 32280
agcttaaata tagcaatatt tgctatatat aaagtgcacc tggtacataa ttgatgcggt 32340
tgaaatgaaa attcctcttt ctctttcagt gccatttgct ggaagaaaca gaagaaatat 32400
ctctgtcccc atctttcagt ttcctaccca agactcctgt gtggttgaca tcactgctgt 32460
gtgcagtgtt catgtccata taagtcagaa gtggcttgca gcagtggttt ggggctttgg 32520
ggcctctgct cgctccttat caattcttag ggttacatcc tctgggaaga tgccaggcct 32580
ggtcaagtgt ggcctctgac ctagagttat gcaaggcctc tcttcctcct gtggccagtg 32640
tgaaatttct catctgtgct ctcctgcctc tagcaggaag gttggcctac atttggggca 32700
catctctgtc actgtctcag gccaggggtt caaatataag acctgaatca tactgcacct 32760
cagaacccaa acccttttac ttaatttttt ttaaaaaatt tcattcagat ggtaaaggtt 32820
caaacaatac atagagatat aaaacagaaa gaaaaaaaaa tacacgcctc tctctagtac 32880
ctattcccca gtagtcaaca ctgctacctt ttttttgcag gggaggtaaa ttcctccaga 32940
aaaaaactta aacatctaca atttcatata tcctctcccc tcttccctaa aaaacaaaac 33000
aataacaaca aaactataga aatgggatta tgctatatat tctgttcagt acttttttgt 33060
ttttgaaaat ttaaaaaata tgcaaaagaa gaaaatgtca tccatattac taccacttat 33120
aattaaccac taattaactt ttaggtgtgt ttgcttcttg tctttatgaa tatactttaa 33180
aagctactta agtaaggtgg aagcagttct gtttaaaaaa ttaggatgtt gcccgggcgc 33240
tgtggcttac gcctgtaatc ccagcacttt gggaggccga ggcgggcaga tcacgaggtc 33300
aggagatcga gcccatcctg aataacatgg tgaaaccctg tctctactaa aaaatacaaa 33360
aaattagcca ggcatggtgg tgggcgcctg tagtcccagc tacttgggag gctgaggcag 33420
gagaatgacg tgaacccggg aggcagagct tgcagtgagc caagatcatg ccactgcagc 33480
tccagcctgg gcgacagagc aagactccat ctcaaaaaaa aaaaaaaaaa aaaaatagga 33540
tgttaaagat aaagccaaag tattatatca tcttggtcct tatctgcttt cccatttaat 33600
agcatcctgc tctccccgtc gccgtggatg gggagctgcc tctttcttct acgacaccgc 33660
acgttttcca ttgtatggat ttgctgtcat ttcatttatc tgaccaccct cttattggta 33720
ggcatttagg ttattaccag tttttttgcc attaaaatgc tttatgaata taatgtgtag 33780
gatggtttct tgctgggtaa ttactgggtc caaggtttga acatttacaa ttttgataga 33840
catggccaaa tacctcccca aaaatactag tttatttctc taccaactga atgtgtaaga 33900
gagtgccttt tctccccaga tctttgcata aacctgggag ttttattttg gtcaaacatt 33960
aaaatttttt ttttgccagt ctggtgaaat tatcatcttt tttataaaca tttctctttg 34020
agctacactg agtgtcttcc attcagccat ctgtgtttct cttgctttga actgcctgtt 34080
cttatcgttt ggccattttt atattaggtt gttgatcttc aacaattgat ttgcctgagt 34140
taagttagcc ttttatccat catgtgttgg aaatatttta aagatttgtc atctacagtg 34200
cgatttttag gcctcttgac tttttgttga gccctaggct tttgtgactg cctgtgacag 34260
ctctggagag ggggccgaca ggcacagtgg cccgcagtcg agccaggccc catcttctgg 34320
gcagtgacgc gtggttgttt taaggctttt ggtactggga ttccccgttg taagttttag 34380
ctttgttttg ggtagggact tttccccatc atattaatga acattaataa caaatagaga 34440
cttgcttttg cctcccaacc aaatgtttgg gaatgtgtgc acccacgtga agggtctgtg 34500
tgatatcttt atctcatcaa ccttctaagg cttttaaaac ctgttgttag atttatgttt 34560
agatcatggt tctaggggag aaccatcgtc actcctcatt gagctttgca gaggctctga 34620
aaggaggaca tactctcttc cccaatcaat gtagaggaac gtcagtagtt cagccaagat 34680
tgctctatct gccacctctt atgcttccaa gctgggctaa ttgagaagtg tgacatcttg 34740
ctccagccta taagcagctg tctgtaattt tgagagatga gaaggattct cccttaaacc 34800
attgctgctt ttgtggcagc atcttatttc tttcctttgc agcacaaatc acagtgtata 34860
attaaaacta ttttctttga gcttgttttt ggtgtacctc ctgcactaaa atgttaagtt 34920
ctgtggcggt cacggggctt atttgtcatg ttctctatta tacccttggc atcctctaaa 34980
ccagtgtgtg gcacatgcta ggcactccgg ggtttcactg agtgatccat tctgttagga 35040
tgggatcaag tgctcatgtg tctcctcctg tgtccctctc tggctccaga tcttccacct 35100
ggcagagttt ccggaagccc cagagatatg ctccatggcc atagggcctg agacgtggat 35160
ggctgccctc tgactaaaat ttagagagct aagcttgtct ctttaaaaag taacatatac 35220
taattgtaaa tcacctagaa aatacagaca taataaagga aattaaatct gtgattctgt 35280
ctttcagaga tcattaccat taacaactca ttgagtctta atgcctttat tgtttttgtt 35340
ttgttttgtt ttgttttgtt tttgagacag agtctcactc tgttgcccag gctggagtgc 35400
aggctcacta caacctccgc ttcctgggtt caagtgattt tcccacctca gcctcccaag 35460
tagctgggat tgcttacagg tgcgtgccac catgcctggc taattttttt gtattttttg 35520
atagagacag gctttcagca tgttggccag gctggtcatg aacttctgac ttcaagtaat 35580
ctgcccccct tggcctccca aagtgctggg gttatagatg tgagccacta tggccagccc 35640
ctatgccttt atttttttta tatacagatc caataaatat ccatagagca ctgagtttat 35700
gctgtacata agggtttgta actttcagtt accatcagac atgttttgat aatgaaattt 35760
aaagaaagta cacattggga tttgggatgc agaagaaaaa ataggatagt attctagaca 35820
atgacagtga tgatacctga gaatctagta gacactcagg aatccacatt tccccattta 35880
atattcatct gaaacaccat ttcagtggct gtagattgtg tagctaaact cacttttaca 35940
taaaacaacc tctgttactt atctctgggc cttaagtttg tgatatagtt tggatgtttg 36000
tttctcccaa atctcatgtt gaagtgtaat ccccagtgtg ggaggtgggg cctggtggga 36060
ggtgtttggg ccatgggggt aggtccctcg tgaatggctt ggtgctgttc ttaagatagt 36120
gagttcttag gagatctggt tgtttaaaag tgggtggcac cccacccctg ttgctcctgc 36180
tcttgccatg tgacatccct gctccccctt caccttccac catgattgta agcttcctga 36240
ggcctcacca aaagcagata ctagtgccaa gcttgtacca gctgcagaac tataagccag 36300
ttatatcttt tctttacaga ttacccagtc tcaggtattt cttaatagca atgcaaagta 36360
gactaataag tttgttagct atttctagta aaggtaaata actcttgaga tgtatatttt 36420
cttaaagata cgtctatgat taatacctga ggatgaaatt ctaaaagtag aattttaaca 36480
tcaaagggtt tgcactttaa ggcattttca gcctccattt ctggctgttg ctgttgtgga 36540
cagtcttggg cagattttga actctttgcc ttactgtggg gtggtggaaa ggaggcttta 36600
ggagcagcca gagcagggtt caaactccag ccctgccagg gacaactggc caaaaggtcg 36660
tgaactaatg actcagtgag ttttctcatt tataaaatag gatgtttcct cccagggtgg 36720
gggtgaagtg ggtgagatga cccatttgaa gcacagtgga cccttgaaca atgcaaaggt 36780
tgggcagtca attcacataa aacttttgat ctgagttggg cagatcacct gagattagga 36840
gtttgagacc agcgtgacca acatggagaa accctgtctc tactaaaaat acaaaattag 36900
ccgggcatgg tggcacacgc ctgtaatccc agctactcgg gaggcagagg caggagaatc 36960
acttgaaccc gggaggtgga ggttgcggtg agccgagatc gcaccattgc actccagcct 37020
gggtaatgag agtgaaactc tatttcaaaa aaaaaaaaag gccaggcacg gtggctcatg 37080
cctgtaatcc cagcactttg ggaggccgag gcgggcggat cacgaggtca ggagatcgag 37140
accatcctgg ctaacacggt gaaaccccgt ccctactaaa aatagaaaaa attagccagg 37200
cgtggtggtg ggcacctatg gtcccagcta ctcgggaagc tgaggcagga gaatggcgtg 37260
aacccgggaa gcggagcttg cagtgagcgg agatcgtgcc actgcactcc agcctgggca 37320
acagggcgag actctgtctc aaaaacaaaa acaaaaacaa aaaaaaacaa agaaacaaaa 37380
gaaaactttt gattccccta gaactactat taaataatag cctactgttg acccgaagcc 37440
ttactgatta catagttgat taacacatat tttgtctgtt gcgtgtatta tatctttatt 37500
cttgaataaa ataagctaaa gaaaataagc atcgttaaga aaatcacttt gagtaggctg 37560
aggaagagga ggaggaagag gaagggttgg tcttgctgct gcagggctgg cagaagtgga 37620
agaaaatccc tgtataaatg gatccacgca gttcaaacct gggttgttca agggccaaca 37680
gtacttagca gccatttagc acagagcact gccagccccc ctcggcctgc agcctgctcc 37740
agcaccttgc taagaggttg ctgaggctca cctgcagcag ctgccctccc gagtcctctt 37800
tcctttacac ccctcagagc acccgggcca gccatcctcc atgtggtgca gcagaggcgc 37860
cggtggtccg ggaaccagag tgggtggccc tggcctggag gcacggtggg ttagtgtgtg 37920
acacacgtgg ctttcccccg acccccagct gcctcctggg agtgaagagg aggtcttatt 37980
tgccaacaca taccgcacca cacacccaca acacacttgg tgccagaatc agccttttgc 38040
atcgctcttg gcgctgaatc agagtctgtc ttggggaggg atgtcttcct tctctcatta 38100
catgcgccca gcttaggtgg tgtagtgagg gagtgtacag ggctgggatg ccacagcctg 38160
ctttcctccc cgtgtcctgg ggtcccccgt ctgtccagtg cccagagcta ttaataacac 38220
cttcattgaa atgggtgtaa tggaatccca gttgcccccc ccagtgggac tccaacacat 38280
ggggagtggg gagcagaata ataatagcaa acacggatgc agtacttatt acgtgccaag 38340
cgttattctt agttatttac atcatgcaca tctattaact catcttgtat agcaagccca 38400
tgaggtgggg actattactg tcctattaat gggtaaggga accaaggcac aaaggggttg 38460
gaaacttttc ccagttcaca tagctagtga gcagtggcgc ttgtgaaggg ggtcctcctg 38520
agggcctggg gcctgctcct tcccagtctt gtcttcatgt ccaccttgct gacgagtggc 38580
acatcaggcc ctgggtgccg gctgctttcc tgtcctctgg ctcagccagg cttctaatgc 38640
tgaccactcc tgagtctttc cacgtggctc cacaatggca gacttgcagg aggagatgag 38700
gggaaccatg cataccttga atagggggag gggtcctgtc cttcttcccc tcaggtgctg 38760
gaagagacag agaggcatgg gactgtattt aggcagctcc cgcagctccc tcactgccgc 38820
tgtcctagag cttccttccc aggcaatgcg ctctgtggct tgggcccctg cccctgccac 38880
agttggcagg gggccttgcc tgtggcgttg cagctgagca cacgcaatca ttaacctttt 38940
ctacgtgtaa gagaatttca ggcacaaact gtggggctga ggtgagtcac aatgttggac 39000
cggggcttag tttgggctta aaatatttca caattcctcc aggcattctt tgactaatga 39060
tctatggctt tttagtggga agacccttcc ttcttctagt ctccagtcct cagagcatgg 39120
ttcagggcct gttggccttc actgcccatg gccatgccca tgcccaagga gcaggggcct 39180
tggcttctgg gtggacctgc cgcctcgctc tgctggtggc ctctggcagt gccctaggag 39240
tcctgctttc cttccctgtc tcttctttgg cttggagtgc aggccacctc ccctgctgtt 39300
ctttacttag ccctgtactc cagattagca cattcttggt ttcctctctc caggccttct 39360
gcgctccaca cttgtccaca gagccacttt ggcacttcag ggctgtgccc ccaggacgtc 39420
agccagcctg cctcgcccat ctgaagggct ttcattgctt gggttgattt tcttatccag 39480
tcagggattt ctgataaaca tgtgcctgga tggatgtcaa tgaccagtcc tttttttttt 39540
tttttttgtt aaatttaatt taagttctgg aatacaaatg caggatgtgc aatgaccagt 39600
cttcatggga aattcttgct aagtttctct aagaaattta aaatactacc ttttccaatt 39660
tatttcagga tggaaaggac agctcaccct cagtcactgg gggagagagt ttagtaggga 39720
gaagaagcaa cagaatgagg gggtgttagc tcagaagttc ctaaccacta ggtgagggaa 39780
ggaagagggg agggagtaaa cagaaatcaa attagatctt gcccattaga aactcagtta 39840
ggttgggcag ataaggcata aatatgcaaa atggtaaatt ttttcaaagt ggaaactacc 39900
ctatatagag tttctgaagc accttcaagg atattatttc acgaagttga atctaatttt 39960
acagatgagg aaaccctttc cagaaagata ggtgggcttg catgacacaa gctaggaagt 40020
gcagaactgg gattcaaaat gcatgcgatt gcaaagccta ggttcactgt gctacatccg 40080
tctcctctaa tgggtagaga gtttgtgcaa gtctggaagt gtgatgtgaa cctagccctt 40140
cttgcacaaa ctctcctttg ccttgtgcca cgcactccta acgtgttcag ggcagaaagt 40200
gcggaagttg cagtgagcgc ttggaattca gggggtgcta aggagcatgc atttagattt 40260
ggatcttggt gcatcctcac ttagcacagc tctggttggc agatgtcctg gtggtcgtca 40320
ctgggctgaa gacagaaacc cagcctacaa tcttggtgat aacttgtgct ggttacatcc 40380
acatgctcca agctcaactc agtggtgacg gaggaacccc cctgtactgt ggcattaatg 40440
gtactggttg acagtgatga atgcctttct gataataaag cacttttaga ggtattattt 40500
cattagatgc tcataatgtt tctatgaggg ggaggctagg caggtatcat cgctcctaaa 40560
tgcatgtggc acaggctctg aaaggtagac ctctctgtga ggctgtgcaa gaagaaattg 40620
acaaatttca gactgggcca agtcttccct gattgtaact ctacaaccag gcattgcttc 40680
tgagtaaatc tgacaacttt gattcttctc ctctgggctc cagttcctgt gtgcctgtag 40740
ggggcgatcg aggttctttc tgcttggctg ctctggaaac cttgttcctc ctgaaccctt 40800
tgaatttagc actcttacag catgtaatct cactggattt taaggatttt ctgtgtctcc 40860
cctcatatct tcttctgccc cccactagcc taggcttttc cagatcagga ctgctctatt 40920
tggttttgac attcagtgca atgcatggcc catagtagga gctcagtaaa tgcttgctgg 40980
attgaattat atacaaagga aattggccag tgccttctct tgctcccctt agagcatggg 41040
aaagtaaact tttttcctgg tacatccaaa tgaatgaagt tgaaggctgg gcatggtggc 41100
tcacacctgt aatcccagca cattgggagg ccaaggtggg gtatcacttg agaccaggag 41160
ttcgagacca gcctggccaa catggcgaaa cccggtctct actaaaaata ccaaaaaatt 41220
agccagacgt ggtggctcac acctgtaatc ccagctactc aggaggctga agtgggagaa 41280
ttgtttgaac ccgggaggcg gaggttacag tgagccaaga ttgtgccact gcactccagc 41340
ctgggcaaca gagcgagacc ctgtctcaga aacaaacaaa ccctaatgaa gttgaaagtg 41400
gcagaactca agacccatga ggtgtcctca tatggccaca aggtggcagt gtgacctcct 41460
gggaaccttg cctcctgcaa ctattggctc cggccacctg ttactccatg ggagagaccg 41520
agttatctag tcattgattc cttctagtca cctgtttggg gaattgcctc tgcaaagtca 41580
gtctgagaaa tcttggcttc cagcctcctc taaaagctgg gtggtgtggt ggaaagactt 41640
ggtctttgga gtctaccagg cctggattcc aaccttggct ctgccttttc tcagaaactt 41700
agccttgggc atgctacttc tctgagcctc agcttcccca tcctgtaaaa tggcaaatgt 41760
acagttagga aaaacccaac atatcatggg ccatagcata aatgtatgcc agcattcttg 41820
ttggaaatgc tcatttgtcc ttcccttccc tctgcaagct ggctccatca tggccatctg 41880
atcttatttg caagcttcag acactttcca gcaggttctt aggcctccct gctgtatcac 41940
cccagctcca ctccatctcg gtcagtcagc acctgcttcc cacgtttccc tctgccttca 42000
tgccttccct ctgcctgtcc atttccttcc cagcatgtct cttggacacc ccagccctct 42060
gaatgtccaa agcagttgga gtgtggactg tgtagggctc atcttgactt cccatgaaga 42120
tgctaaatct gtgagtgtcc ttgtcttaat tattttggca cctgcgcttg cacttagaca 42180
attggtgata aaaatgacag ctattagtga gtgcatgtca gatactctgt gaggtgcttt 42240
gtggaaatat ttcatttaat cctcacaaca aatctgtgaa acagaagcca ttatccatcc 42300
ccactttaca aaggaagggt agatcaggat gcttttggct ccaagcaata gacaatttca 42360
tgaaaagcag cttaaacagt aagaagagtt tattatcttg tcaaaaagaa gtactgaggg 42420
atccctggtg atggcagcga ggatccaggt gattgccatt tctcctggtt gccttcctca 42480
gtgcattggt ttatcctctt aggcccccct tactggtaat aatgtgactg tcatggtccc 42540
aggtgccata tgcaggagag atgatgtaca tttcttgtgc tccctttaag aagcctctta 42600
gcagatttct tttttttttg agatggagtc tcgctctgtt gcccaggctg gagtgcagtg 42660
gcgtgatctc ggctcgctgc aagctccacc tctcagggtt cacgccattc tcctgcctca 42720
gcctcctgag tagctggggc tacagtcgcc tgccaccatg cctggctaat tttttttgta 42780
ttgttagtag ggacggggtt tcaccgtgtt agccaggatg gtcttgatct cctgacctcg 42840
tgatctgcct gcctcggcct cccaaagtgc tgggattaca ggcacgagcc accgtgcccg 42900
gcagcagatt tcttacaagg ctcttggcca gttgagttgt aggcttgttc ccaagccagt 42960
cactagcaaa gggactggaa ttaatgagac tggcttagac aggtcaagat tgaacccctg 43020
aggctggtga gaagccagcc tcctttaata cccctagcca attggtccag ggagaagaag 43080
gggcatcagt gaggttcgac gagttaagta acttgctcaa gcttggaaat ctagtaataa 43140
ctggattcat actggcatcc ggctgacttc caaggccatc tgttgccagg ttgaagtcaa 43200
ctacagaggt agagagaata ttgctctgga tttgttgtgc tgggtctgag taattgctga 43260
ggggcctttt cctatggact ctggagtctc tcttctctct agcccccttt ccaatactgt 43320
attggttttt ctcagtctgc ctttctcccc gaagcccctt caccccctgc ttcccagtac 43380
ctgagtgctg cctgccttcc acagtagact ttatggccta agctggagac aggtctaagc 43440
tggaccgaat ggcctgaggg ctgatctcag tgtagctgtc aaggccccaa gagagtcatc 43500
atgagatttc atatagagca gataaggaac tgcagaggcc tcaggaaatg tgccaagggg 43560
gctggtgggg ggggccagca ggagtggggg gtttgtggta tgtcggtggc gatgtcaggg 43620
catgtgggcc aggggcttag agcaggccca tgtgacagcg cagttactca gaaagctgtt 43680
tggcctgcag atgcttcagg actgccccaa ggcccgcagg gaggtggagc tgcactggcg 43740
ggcctcccag tgcccgcaca tcgtacggat cgtggatgtg tacgagaatc tgtacgcagg 43800
gaggaagtgc ctgctgattg tcatggaatg gtaagcagcc actgttctag tcccggccca 43860
gcctgttccc actcctcact caggcacctt accctctgag gacagcccag ggatgggcct 43920
gaagcctgga atgtaaacac ttgatttttt ggggggcagt ggagagtggc ggcgggctgt 43980
tgtgttctct ggatctcact gtggcttcat ttcagtttgg acggtggaga actctttagc 44040
cgaatccagg atcgaggaga ccaggcattc acagaaagag gtagaaaggg tctgttgtgg 44100
gagcacgtgc aggcaggcag ggcagtgggg gtctgaaggg ggcctttgca gtgcctgctc 44160
ttggggaagg gtgctgaggc tgctgccccc tccagcatgc tgcaaggggt gcctcagctg 44220
accagggaag ctcctggtgg ctttgtttct ggggtgggtg gtggctgtgg ggagcctcag 44280
gctgcacaga ggtgggaccc ggtgcactgg gttttcaagc ctaatgatgt gtctttgtga 44340
ctttctttcc cactcactct tccctcccct ctacagaagc atccgaaatc atgaagagca 44400
tcggtgaggc catccagtat ctgcattcaa tcaacattgc ccatcgggat gtcaaggtgc 44460
cagctgttca taaccctgag cccgagtgct gtggggggca acaaaggggc tgtggccacc 44520
ccagagatgg ttgccaggcc accctgcgtc cttgctgcct ggttcctgag catgccattc 44580
tctgccttgt agggccttgc cctctctgcc catgggcaaa cccctaaacc cagtagtgaa 44640
gcattctctg gccccactca gccctgtggt tctcctcagg cagaacccct cagtcacact 44700
cttggtttct ctgttcctgt ggtgtttact gcgggttctg tgtgtcagtc tgttatctcc 44760
aggcactctg gaaaccctgc agaggcaggc acctgcccat agagagctgc cactcctcgt 44820
ggtgggcagt gcccaggata ggcaaatggt tgctggatga gtagaggaag ggaggaacca 44880
ggatcctttt cgtttctgat agcccctccc aggtccagca gggttgctat ttttctgtct 44940
ctctttctgt agcctgagaa tctcttatac acctccaaaa ggcccaacgc catcctgaaa 45000
ctcactgact ttggctttgc caaggaaacc accagccaca actctttgac cactccttgt 45060
tatacaccgt actatgtggg taagtccaca gggggcccag ggacctaggc ttttcccaga 45120
acttttctca gcccctcctc ttggctatct ggggggccgc aaatcctagg agagttgtgt 45180
ctgtatggcc ccttctggtg ctggttctgc tgggacctgc tgagaagtag ggcttgtcag 45240
agcgaagcta cagttgagaa attgtaggaa aggatctgct gggatctttc tgagaagagc 45300
catggtttcc atcagattct ccatggagtc tggacccagg tcttgttctg gagccacagc 45360
tgattggcat ggaagaagct ttcaaagcat gttcagagca tctgtgaaat taaatgaacg 45420
gcatgggcct gaaagtctca tgcagttcag gatttttaga atcttaagct cctagttcca 45480
ggttcctcat ggaggaggat gttctgttcc agtccctggg ctgggctttg gctcttcctt 45540
ctgtgcccca actgctgcca cctcatgata ggctgaaagg ttgggatggg gagcatcttt 45600
cacagagaaa ctgagtggat cacagggttc tgagggccgg cccacccatg ttgacctttg 45660
atttgcagct ccagaagtgc tgggtccaga gaagtatgac aagtcctgtg acatgtggtc 45720
cctgggtgtc atcatgtaca tcctgtgagt gtgctgggga gggggctggg tggggcaggg 45780
agtcagggcg gcctgtactt ctccaggaca agaggaagag gcagacgtta gcattcccct 45840
ttgtacaggg gagtcccctg cgtgcccctt ctctcagttc cgatagctaa gtggcgggtg 45900
taaaccagct catgggctgg aaagtgtgcg ccttagatac ttgctatgct ggattcccgg 45960
gccccgggct tgactttgta tattgtgcct gtgtcaataa gccctgattt ctctgtgacc 46020
tttacaagga gaagagcctg tttctcatcc tgttcctggt acagggccac taagtgacag 46080
ctgttctgtc tcccacttcc ttcctcctgt tgatgcaggc tgtgtgggta tccccccttc 46140
tactccaacc acggccttgc catctctccg ggcatgaaga ctcgcatccg aatgggccag 46200
tatgaatttc ccaacccaga atggtcagaa gtatcagagg aaggtaagaa cccaggcttt 46260
caggacaagg ggaagagccc gtgtgtgtgt gtgtgtgtgt atgtgtgtac acgcagacac 46320
atgtatgggc ctccatctca tgtgcgtggt gtaactgtgg gttagcacct atgcccacgc 46380
ctgcggggtg cgtcctgctt cattttgcct gtgtggaggg ctggaggcag ggccaaggct 46440
gtggggctgt gcagggcctc tcaagtggta cagccgtaat ggtccttggg gccagttgct 46500
ccggcagcct gcctccatgc accccctctt tgaacctggt ttccccatga aaactgggga 46560
aaggagcagg ccagggagag tgacccctga gctgtcactg ccccctgtcc caccccacag 46620
tgaagatgct cattcggaat ctgctgaaaa cagagcccac ccagagaatg accatcaccg 46680
agtttatgaa ccacccttgg atcatggtaa gctcggcagg ctggggagcc ttgggtctca 46740
cgggactatt ccacaggaca gagtcttagc caggacccta ccccaggctt tcactcggac 46800
cccttttctc tcttctcagc aatcaacaaa ggtccctcaa accccactgc acaccagccg 46860
ggtcctgaag gaggacaagg agcggtggga ggatgtcaag gtgaggggca ccactgggtg 46920
agaggggctc caggtggggt gggcggcttg cggggagtgc ccaggtgtga ggcgtggtgc 46980
tggtagggga gagcttgatt ctgcctctct catcccaggg gtgtcttcat gacaagaaca 47040
gcgaccaggc cacttggctg accaggttgt gagcagagga ttctgtgttc ctgtccaaac 47100
tcagtgctgt ttcttagaat ccttttattc cctgggtctc taatgggacc ttaaagacca 47160
tctggtatca tcttctcatt ttgcagaaga gaaactgagg cccagaggcg gagggcagtc 47220
tgctcaaggt cacgcagctg gtgactggtt ggggcagacc ggacccaggt ttcctgactc 47280
ctggcccaag tctcttcctc ctatcctgcg ggatcactgg ggggctctca gggaacagca 47340
gcagtgccat agccaggctc tctgctgccc agcgctgggg tgaggctgcc gttgtcagcg 47400
tggaccacta accagcccgt cttctctctc tgctcccacc cctgccgccc tcaccctgcc 47460
cttgttgtct ctgtctctca cgtctctctt ctgctgtctc tcctacctgt cttctggctc 47520
tctctgtacc cttcctggtg ctgccgtgcc cccaggagga gatgaccagt gccttggcca 47580
caatgcgcgt tgactacgag cagatcaaga taaaaaagat tgaagatgca tccaaccctc 47640
tgctgctgaa gaggcggaag aaagctcggg ccctggaggc tgcggctctg gcccactgag 47700
ccaccgcgcc ctcctgccca cgggaggaca agcaataact ctctacagga atatattttt 47760
taaacgaaga gacagaactg tccacatctg cctcctctcc tcctcagctg catggagcct 47820
ggaactgcat cagtgactga attctgcctt ggttctggcc accccagagt gggagaggct 47880
gggaggttgg gaggctgtgg agagaagtga gcaaggtgct cttgaacctg tgctcatttt 47940
gcaattttat cagtaatttg acttagagtt tttacgaaac ctcttttgtt gtccttgccc 48000
cactcctctc caccagacgc cttcctctct ggatactgca aaggcttgtg gtttgttaga 48060
gggtatttgt ggaaactgtc atagggattg tccctgtgtt gtcccatctg ccctccctgt 48120
ttctccacaa cagcctgggg ttgtccccgc tggctcacgc gttctgggag ctcaaggcca 48180
ccttggagga ggatgccacg cacttcctct ctcggagccc tcagacatct ccagtgtgcc 48240
agacaaatag gagtgagtgt atgtgtgtgt gtgtgtgtgt gtgtgtgcac acgtgtgtat 48300
gagtgcgcag atctgtgcct gggatcgtgc atttgagggg ccaggggcag gcagggctgc 48360
agagggagac ggccctgctg gggcttagga accttctccc ttcttgggtc tgccctgccc 48420
atactgagcc tgccaaagtg cctgggaagc ccacccagat tctgaaacag gccctctgtg 48480
gcctgtctct attagctggg ttccgggagg cagagaggag tgaccgggca ctggcactgc 48540
gatcaggaag actggacccc cagcccccag ggcccccctc cccccactta gtgctggtcc 48600
taggtcctct gaggcactca tctactgaat gacctctcta cttccccttc ttgccattat 48660
taacccattt ttgtttattt tccttaaatt tttagccatt tctccatggg ccaccgccca 48720
gctcatgtag gtgagcctgg gcagcttctg ttggcagagc ttttgcattt cctgtgtttg 48780
tcctgggttc tggggcatca gccagctacc ccttgtgggc aaaggcaggg ccacttttga 48840
agtcttccct cagatttcca ttgtgtggcc tggtgggtca gggggagtct ttgcaccaaa 48900
gatgtcctga ctttgccccc ttgcccatca gccatttgcc atcaccccaa acaactcagc 48960
ttcggggccg gtgaggggag gggcctcccc cagcacagat gaggagcagc tggggtaggc 49020
tgtctgtgcc atggcccccc actccccctt cccttggagg gagaggtggc aggaatactt 49080
cacctttcct ctccctcagg ggcaggtggt ggaggggcgc ccagggtcgt ctttgtgtat 49140
gggggaaggc gctgggtgcc tgcagcgcct cccttgtctc agatggtgtg tccagcactc 49200
gattgttgta aactgttgtt ttgtatgagc gaaattgtct ttactaaaca gatttaatag 49260
ttgaga 49266
<210> 2
<211> 400
<212> PRT
<213>People source
<400> 2
Met Leu Ser Asn Ser Gln Gly Gln Ser Pro Pro Val Pro Phe Pro Ala
1 5 10 15
Pro Ala Pro Pro Pro Gln Pro Pro Thr Pro Ala Leu Pro His Pro Pro
20 25 30
Ala Gln Pro Pro Pro Pro Pro Pro Gln Gln Phe Pro Gln Phe His Val
35 40 45
Lys Ser Gly Leu Gln Ile Lys Lys Asn Ala Ile Ile Asp Asp Tyr Lys
50 55 60
Val Thr Ser Gln Val Leu Gly Leu Gly Ile Asn Gly Lys Val Leu Gln
65 70 75 80
Ile Phe Asn Lys Arg Thr Gln Glu Lys Phe Ala Leu Lys Met Leu Gln
85 90 95
Asp Cys Pro Lys Ala Arg Arg Glu Val Glu Leu His Trp Arg Ala Ser
100 105 110
Gln Cys Pro His Ile Val Arg Ile Val Asp Val Tyr Glu Asn Leu Tyr
115 120 125
Ala Gly Arg Lys Cys Leu Leu Ile Val Met Glu Cys Leu Asp Gly Gly
130 135 140
Glu Leu Phe Ser Arg Ile Gln Asp Arg Gly Asp Gln Ala Phe Thr Glu
145 150 155 160
Arg Glu Ala Ser Glu Ile Met Lys Ser Ile Gly Glu Ala Ile Gln Tyr
165 170 175
Leu His Ser Ile Asn Ile Ala His Arg Asp Val Lys Pro Glu Asn Leu
180 185 190
Leu Tyr Thr Ser Lys Arg Pro Asn Ala Ile Leu Lys Leu Thr Asp Phe
195 200 205
Gly Phe Ala Lys Glu Thr Thr Ser His Asn Ser Leu Thr Thr Pro Cys
210 215 220
Tyr Thr Pro Tyr Tyr Val Ala Pro Glu Val Leu Gly Pro Glu Lys Tyr
225 230 235 240
Asp Lys Ser Cys Asp Met Trp Ser Leu Gly Val Ile Met Tyr Ile Leu
245 250 255
Leu Cys Gly Tyr Pro Pro Phe Tyr Ser Asn His Gly Leu Ala Ile Ser
260 265 270
Pro Gly Met Lys Thr Arg Ile Arg Met Gly Gln Tyr Glu Phe Pro Asn
275 280 285
Pro Glu Trp Ser Glu Val Ser Glu Glu Val Lys Met Leu Ile Arg Asn
290 295 300
Leu Leu Lys Thr Glu Pro Thr Gln Arg Met Thr Ile Thr Glu Phe Met
305 310 315 320
Asn His Pro Trp Ile Met Gln Ser Thr Lys Val Pro Gln Thr Pro Leu
325 330 335
His Thr Ser Arg Val Leu Lys Glu Asp Lys Glu Arg Trp Glu Asp Val
340 345 350
Lys Glu Glu Met Thr Ser Ala Leu Ala Thr Met Arg Val Asp Tyr Glu
355 360 365
Gln Ile Lys Ile Lys Lys Ile Glu Asp Ala Ser Asn Pro Leu Leu Leu
370 375 380
Lys Arg Arg Lys Lys Ala Arg Ala Leu Glu Ala Ala Ala Leu Ala His
385 390 395 400
<210> 3
<211> 21
<212> DNA
<213>Artificial sequence
<400> 3
tggggcgtta actgcatctg g 21
<210> 4
<211> 20
<212> DNA
<213>Artificial sequence
<400> 4
tgggcctgct gttcacagtt 20
<210> 5
<211> 20
<212> DNA
<213>Artificial sequence
<400> 5
gcaccgtcaa ggctgagaac 20
<210> 6
<211> 19
<212> DNA
<213>Artificial sequence
<400> 6
tggtgaagac gccagtgga 19

Claims (10)

1. molecular marker MK2 genes, it is characterised in that sequence is as shown in SEQ ID NO.1.
2. molecular marker MK2 albumen, it is characterised in that sequence is as shown in SEQ ID NO.2.
3. application of the molecular marker MK2 genes in the product for preparing diagnosis Huppert's disease.
4. application according to claim 3, it is characterised in that the product includes：Pass through real-time quantitative PCR, immune group Change, in situ hybridization, genetic chip or high-flux sequence detection of platform MK2 gene expression doses are to diagnose the production of Huppert's disease Product.
5. application according to claim 4, it is characterised in that the production that Huppert's disease is diagnosed with real-time quantitative PCR Product at least include the primer of a pair of specific amplified MK2 genes；The product of the use immunodiagnosis Huppert's disease includes： The antibody combined with MK2 protein-specifics；The product for diagnosing Huppert's disease with situ hybridization includes：With MK2 genes Nucleic acid array hybridizing probe；The product for diagnosing Huppert's disease with chip includes：Protein chip and genetic chip, Wherein protein chip includes the antibody combined with MK2 protein-specifics, and genetic chip includes the nucleic acid array hybridizing with MK2 genes Probe.
6. application according to claim 5, it is characterised in that the production that Huppert's disease is diagnosed with real-time quantitative PCR Product at least include the primer of a pair of specific amplified MK2 genes, and sequence is such as：Shown in SEQ ID NO.3 and SEQ ID NO.4.
7. a kind of instrument for being used to diagnose Huppert's disease, it is characterised in that the instrument can detect MK2 genes in sample Expression.
8. instrument according to claim 7, it is characterised in that the instrument includes chip, kit, test paper or high flux Microarray dataset.
9. instrument according to claim 8, it is characterised in that the chip includes genetic chip or protein-chip, described Genetic chip includes solid phase carrier and is fixed on the oligonucleotide probe of solid phase carrier, and the oligonucleotide probe includes being used for Detect the oligonucleotide probe for MK2 genes of MK2 gene transcription levels；The protein-chip include solid phase carrier and It is fixed on the specific antibody of the MK2 albumen of solid phase carrier；The kit includes gene detecting kit and protein immunization group Change kit；The gene detecting kit includes the reagent for being used to detect MK2 gene transcription levels；The protein immunization group Kit includes the specific antibody of MK2 albumen；The test paper includes the test paper for being used to detect MK2 gene transcription levels；It is described High-flux sequence platform includes the reagent for being used to detect MK2 gene transcription levels.
10. instrument according to claim 9, it is characterised in that the reagent of the detection MK2 gene transcription levels includes pin To the primer and/or probe of MK2 genes.
CN201710351805.4A 2017-05-18 2017-05-18 Molecular marker MK2 genes and its application Pending CN107058259A (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016044463A2 (en) * 2014-09-17 2016-03-24 Celgene Avilomics Research, Inc. Mk2 inhibitors and uses thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016044463A2 (en) * 2014-09-17 2016-03-24 Celgene Avilomics Research, Inc. Mk2 inhibitors and uses thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ANONYMOUS: "NG_029640.1-Homo sapiens mitogen-activated protein kinase-activated protein kinase 2 (MAPKAPK2),RefSeqGene on chromosome 1", 《GENBANK》 *
ANONYMOUS: "NP_116584.2-MAP kinase-activated protein kinase 2 isoform 2 [Homo sapiens]", 《GENBANK》 *

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Application publication date: 20170818