CN114438204B - Platelet molecular marker for lung cancer, kit and detection method - Google Patents

Abstract

The invention provides application of a platelet molecular marker for lung cancer in preparation of a lung cancer detection kit, wherein the base sequence of the molecular marker is shown as SEQ ID NO. 1. The molecular marker is a TEPs specific gene for diagnosing lung cancer, the detection mode is simple, and the rapid relative quantitative detection can be realized by adopting real-time fluorescent quantitative PCR.

Description

Platelet molecular marker for lung cancer, kit and detection method

Technical Field

The invention belongs to the technical field of biomedical detection, and particularly relates to a platelet molecular marker for lung cancer.

Background

Lung cancer has become one of the common tumors at home and abroad, and the morbidity and mortality rate of the lung cancer are high. However, due to the popularity of low-dose lung CT (LDCT) in physical examination in recent years, a large number of lung cancer patients can find and perform operations in early stages, and the mortality rate of the patients is remarkably reduced. In addition to lung cancer, many benign diseases can also be represented by small nodules (diameter 10mm or less), and it is difficult to determine whether they are benign or malignant by LDCT alone. For nodules that cannot be judged by LDCT, diagnosis is often confirmed by needle biopsy or pathological examination after surgical resection. Puncture biopsy is an invasive examination, and the puncture difficulty of a small nodule is high, so that false negative results are easy to occur. And the doctor judges that 20-50% of the nodes of the patient who performs the operation are benign through postoperative pathology, which not only brings unnecessary wounds to the patient, but also increases the economic burden of the patient to a certain extent. Therefore, liquid biopsy is used as a rapid and noninvasive diagnosis mode and becomes a research hot spot for accurately diagnosing lung cancer in early stage.

Liquid biopsy procedures currently under investigation for diagnosing lung cancer include Circulating Tumor Cells (CTCs), micrornas (miRNAs), cell free DNA (cfDNA), and the like. CTCs are tumor cells that metastasize from the primary tumor to the whole body in the blood circulation, which can help diagnose tumor and determine patient prognosis by detecting CTCs, but a multi-center study published in Lancet Respir Med indicated that CTCs have a sensitivity of only 26.3% for lung cancer (95% ci 11.8-48.8), thus suggesting CTCs are not suitable for screening for lung nodules; miRNA is a non-coding, short and single-stranded RNA, abnormal expression of miRNA participates in proliferation, metastasis, invasion and angiogenesis of tumor cells, and quantitative PCR is used for detecting the miRNA level in the blood of a patient to assist diagnosis of benign and malignant lung nodules, but because microRNAs are short in length and easy to degrade, separation and storage are relatively difficult, and the miRNA is not suitable for being developed in clinical routine; cfDNA is a DNA fragment released into the blood due to apoptosis or necrosis, and cfDNA in tumor patients has the same genetic and epigenetic changes as tumors, indicating that they originate from tumor cells, also called circulating tumor DNA (ctDNA), but ctDNA has a short half-life in the blood of about 30 minutes to 2 hours, and treatment and analysis methods are not standardized, and there are difficulties in clinical application.

It follows that although these hotspot indicators have a better role in differentiating lung nodules, there are difficulties in clinical application or lung nodule screening. Therefore, finding a method or biomarker which can accurately distinguish benign and malignant lung nodules and is convenient to develop clinically can realize a great breakthrough in accurate diagnosis of early lung cancer.

Disclosure of Invention

Aiming at the technical problem that the early lung cancer diagnosis is not suitable for clinical molecular indexes, the invention provides a platelet molecular marker for lung cancer.

When platelets come into contact with tumor cells or tumor-associated proteins, the platelets can be taught and change the RNA profile to tumor teaching platelets (TEPs). The invention discovers that mRNA with obvious differential expression exists in platelets of normal people and lung cancer patients through sequencing, and screens out that filamin A (FLNA) gene presents low expression in serum and relatively high expression in TEPs, thus being a TEPs specific gene for diagnosing lung cancer. The detection mode is simple, and the rapid relative quantitative detection can be realized by adopting real-time fluorescence quantitative PCR.

In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows:

the application of a platelet molecular marker for lung cancer in preparing a lung cancer detection kit is provided, wherein the base sequence of the molecular marker is shown as SEQ ID NO. 1.

The invention provides a lung cancer detection kit, which comprises an upstream primer and a downstream primer of a platelet molecular marker.

The base sequence of the upstream primer FLNA-F (5 'to 3'): TCATTCGTGCCCAAGGAGAC

The base sequence of the downstream primer FLNA-R (5 'to 3'): CTGACCAGAGACCCGAACAC

Preferably, the kit further comprises GAPDH internal reference gene primers;

base sequence of GAPDH-F (5 'to 3'): GGAGCGAGATCCCTCCAAAAT

Base sequence of GAPDH-R (5 'to 3'): GGCTGTTGTCATACTTCTCATGG.

The invention also provides a detection method of the lung cancer detection kit, which comprises the following steps:

1) Extracting total RNA of a platelet sample to be detected and normal tissues, and synthesizing cDNA by reverse transcription;

2) Using cDNA as a template, performing FLNA gene amplification by adopting fluorescent quantitative PCR under the guidance of the upstream primer and the downstream primer, and amplifying GAPDH reference genes, and simultaneously detecting fluorescent values and CT values of the FLNA gene and the GAPDH gene;

3) The relative quantification of GAPDH was used to calculate the expression level of the molecular markers.

Preferably, the fluorescent quantitative PCR reaction conditions are set: pre-denaturation at 95 ℃ for 30s, denaturation at 95 ℃ for 5s, annealing at 60 ℃ for 30s, 40 cycles total, and collecting fluorescent signals; a dissolution profile analysis was performed at 60-95℃to determine the identity of the amplified product.

The invention has the beneficial effects that:

according to the invention, through analyzing the platelet RNA sequencing data of the non-small cell lung cancer, the FLNA gene is finally screened out to have stable expression in platelets and low expression in serum. The FLNA gene is used as a molecular marker for screening lung nodules for diagnosing lung cancer for the first time, is used for distinguishing lung nodule groups with benign and malignant lung nodules, has higher sensitivity and specificity, and is a TEPs specific gene for diagnosing lung cancer; and the rapid relative quantitative detection can be realized through real-time fluorescence quantitative PCR, the detection mode is simple, and the clinical popularization and application are convenient.

Drawings

FIG. 1 is a volcanic chart of differential genes between lung cancer and normal populations.

FIG. 2 shows functional enrichment analysis (A, B, C, D) and protein interaction analysis (E) of FLNA gene.

FIG. 3 is a comparison of the expression of FLNA gene in platelets and serum.

FIG. 4 shows the expression of the FLNA gene among malignant nodules, benign nodules and normal populations.

FIG. 5 is a ROC curve of relative expression level of FLNA gene for diagnosing lung cancer.

FIG. 6 is a ROC curve of FLNA gene relative expression levels for distinguishing between benign and malignant nodule populations.

Detailed Description

The invention will be further described by the following examples for the purpose of more clearly and specifically describing the object of the invention. The following examples are only for specific illustration of the implementation method of the present invention and do not limit the protection scope of the present invention.

Differential gene screening

Analysis of TEPs RNA-Seq sequencing data of 55 normal persons and 60 NSCLC in GEO database resulted in total screening out 123 differentially expressed genes associated with non-small cell lung cancer (NSCLC), of which 66 up-regulated genes and 57 down-regulated genes were combined (fig. 1). Expressed according to PValue less than or equal to 0.05, |log2foldchange| more than or equal to 2-fold difference and intra-group variances RPLP2 (ribosomal protein lateral stalk subunit P2), RPL37 (ribosomal protein L), FLNA (filamin A). These 3 genes were searched and FLNA was found to play a role in the formation of platelet morphology while also involved in the adhesion aggregation between platelets and signaling.

RPLP2 and RPL37 are both ribosomal genes, not platelet-specific genes, and by functional enrichment analysis of FLNA gene (fig. 2), it was found that FLNA is highly relevant to cell adhesion, extracellular mechanism and structure formation, and platelet aggregation. The protein coded by the gene is subjected to protein interaction analysis with high-expression proteins of platelets and lung cancer in a string database, and FLNA is found to be related to GPIX and GPIb on the surface of the platelets and also related to a lung cancer related protein mTOR. Meanwhile, other molecules of the blood platelet such as P-selectin, VEGF and the like have certain interaction relation with common proteins of lung cancer. The present invention therefore selects FLNA for subsequent studies.

By detecting the expression levels of FLNA in 83 TEPs and 25 serum samples (fig. 3), it was confirmed that FLNA exhibited low expression in serum and relatively high expression in TEPs, so that FLNA was selected as a TEPs-specific gene for diagnosing lung cancer.

Verification of differential expression of FLNA in lung cancer, benign nodules, and normal populations

The study object is a patient with lung nodule which is suspected of lung nodule/tumor and is to be operated through CT screening, a whole blood sample is collected before treatment after patient is admitted, then the patient is followed up, and clinical basic information, imaging information and pathological results of the patient are collected and recorded. The patients were classified into lung cancer (malignant nodules) groups and benign control (benign nodules) groups according to their post-operative pathological outcome. In addition, the healthy physical examination group is taken as a control group. Inclusion exclusion criteria were as follows:

lung cancer (malignant nodule) group: primary lung cancer primary patients; is diagnosed by definite pathology (cytology, histopathology).

Benign control (benign nodule) group: lung cancer is highly suspected, and post-operative pathology is determined as a subject who is not lung cancer.

Healthy control group: follow-up with a 3-year continuous low-dose helical CT screen precluded subjects with possible lung cancer.

Exclusion criteria: patients with severe liver function injury and end stage renal disease and acute stage inflammation.

Based on inclusion exclusion criteria, 33 cases of lung cancer (malignant nodules) groups and 25 cases of benign control (benign nodules) groups were collected together; healthy control group 25 cases.

2 ml of EDTA anticoagulated venous blood from the patient is collected and the sample is processed within 24 hours after blood collection. After that, the sample is centrifuged at 120G for 20 minutes at room temperature, and relatively more platelets, namely Platelet Rich Plasma (PRP), are retained in the plasma at the upper layer of the tube. Separating PRP, centrifuging under 360G centrifugal force for 20 min, precipitating to obtain platelets, adding 30uL of RNAlater protectant from Life company, and storing the platelet sample in-80 deg.C refrigerator for long term storage. Followed by extraction of total RNA. The sample was taken out of the refrigerator and total RNA was extracted using mirVana RNA isolation kit from Life Corp. The expression of FLNA gene in each group of specimens was detected by reverse transcription real-time fluorescent quantitative PCR, and the relative expression level was calculated by expression with GAPDH.

1. Primer design of FLNA gene

Searching the sequence of the FLNA GENE in a GENE database of NCBI, introducing the sequence of the FLNA GENE into Primer-BLAST, setting the size of RANGE and PCR products, and then designing and comparing primers on line. One primer with a specificity is selected from the obtained primers, the band size is as single as possible, other gene sequences are not compared as much as possible, and the designed primer base sequence is delivered to the Optimago company for synthesis.

Primers for FLNA gene:

base sequence of FLNA-F (5 'to 3'): TCATTCGTGCCCAAGGAGAC

Base sequence of FLNA-R (5 'to 3'): CTGACCAGAGACCCGAACAC.

The expression level of the FLNA gene was calculated by relative quantification with GAPDH, and the GAPDH internal reference gene primers were as follows:

base sequence of GAPDH-F (5 'to 3'): GGAGCGAGATCCCTCCAAAAT

Base sequence of GAPDH-R (5 'to 3'): GGCTGTTGTCATACTTCTCATGG.

2. Real-time fluorescent quantitative PCR detection

1) Extracting total RNA of a platelet sample to be detected and normal tissues, and synthesizing cDNA by reverse transcription;

preparation of cDNA

mRNA was reverse transcribed into cDNA using TAKARA's PrimeScript RT Reagent Kit with gDNA Eraser (Perfect Real Time) reverse transcription kit, which was strictly according to the instructions, and the cDNA was stored at-80℃for use.

(1) Genomic DNA clearance

Incubate at 42℃for 2min or at room temperature for 5min.

(2) Reverse transcription reaction

Incubation at 37℃for 15min, 5s at 85℃and storage at 4 ℃.

2) Using cDNA as a template, performing FLNA gene amplification by adopting fluorescent quantitative PCR under the guidance of the upstream primer and the downstream primer, and amplifying GAPDH reference genes, and simultaneously detecting fluorescent values and CT values of the FLNA gene and the GAPDH gene; each sample was subjected to FLNA gene amplification by adding 3 reaction wells and further to the reference gene GAPDH amplification by adding three reaction wells according to the following reaction system.

(3) PCR reaction system:

fluorescent quantitative PCR reaction condition setting:

pre-denaturation at 95 ℃ for 30s, denaturation at 95 ℃ for 5s, annealing at 60 ℃ for 30s, 40 cycles total, and collecting fluorescent signals; a dissolution profile analysis was performed at 60-95℃to determine the identity of the amplified product.

3) The relative quantification of GAPDH was used to calculate the expression level of the molecular markers.

Detecting the amplified fluorescence intensities of the reference genes GAPDH and the FLNA by using a real-time fluorescence quantitative PCR instrument, setting a proper threshold and a proper base line to obtain CT values of the samples GAPDH and the FLNA, and carrying out relative quantitative analysis on the expression of the FLNA gene by using the CT values. And calculating the relative expression quantity of the FLNA gene by adopting a 2-delta ct method. Firstly, calculating Ct average values of 3 hole FLNA genes and internal reference genes GAPDH of each sample, respectively subtracting the Ct average values of the FLNA from the Ct average values of the 3 FLNA genes to obtain the delta Ct of the FLNA genes, obtaining the delta Ct of the GAPDH by the same method, obtaining the delta Ct average value of a benign group by calculating the delta Ct average value of the benign group, obtaining the delta Ct by subtracting the delta Ct average value of the benign group from the delta Ct of each sample of the two groups, and calculating the 2-delta Ct value of each sample to obtain the relative expression quantity relative to the control group.

Statistical analysis: all statistical analyses applied SPSS 20.0 (SPSS inc., chicago, IL) and R4.0.4 (Version 1.74). The FLNA gene was compared for expression between malignant nodules, benign nodules and normal populations using a nonparametric test wilcox. And drawing an ROC curve according to the FLNA gene expression quantity between the malignant nodule population and the normal person, and judging the diagnosis efficiency of the FLNA gene for distinguishing lung cancer from the normal person.

FIG. 4 shows the expression of FLNA gene between malignant and benign nodules and normal populations, showing that the expression of FLNA gene is significantly different between malignant and benign nodules (P < 0.01) and can be used to distinguish the small benign and malignant nodule populations.

And drawing an ROC curve by using the FLNA gene expression quantity between the malignant nodule population and the normal population, namely, the normal population is a control group, and judging the efficiency of diagnosing lung cancer by the FLNA gene. FIG. 5 is a ROC curve of FLNA gene relative expression amount for diagnosing lung cancer, with AUC of 0.74, sensitivity and specificity of 0.85 and 0.52, respectively, positive predictive value of 0.77, and negative predictive value of 0.64.

And drawing an ROC curve by using the FLNA gene expression amounts of the malignant nodule population and the benign nodule population, and judging the diagnosis efficiency of the FLNA gene for distinguishing the malignant nodule population from the benign nodule population. FIG. 6 is an ROC curve of the relative expression level of the FLNA gene for distinguishing benign and malignant nodule groups, wherein the AUC of the FLNA gene for distinguishing benign and malignant nodule groups is 0.73, the sensitivity and specificity are 0.70 and 0.72, respectively, the positive predictive value is 0.70, and the negative predictive value is 0.72.

Therefore, the detection of the platelet FLNA gene is used for diagnosing lung cancer and distinguishing benign and malignant nodules, has high sensitivity and specificity, and can be used as a molecular marker for screening lung nodules.

A lung cancer detection kit comprises an upstream primer and a downstream primer of a platelet FLNA gene.

The base sequence of the upstream primer FLNA-F (5 'to 3'): TCATTCGTGCCCAAGGAGAC

The base sequence of the downstream primer FLNA-R (5 'to 3'): CTGACCAGAGACCCGAACAC;

the kit also comprises GAPDH internal reference gene primers:

base sequence of GAPDH-F (5 'to 3'): GGAGCGAGATCCCTCCAAAAT

Base sequence of GAPDH-R (5 'to 3'): GGCTGTTGTCATACTTCTCATGG.

The detection method of the lung cancer detection kit comprises the following steps:

1) Extracting total RNA of a platelet sample to be detected and normal tissues, and synthesizing cDNA by reverse transcription;

2) Using cDNA as a template, performing FLNA gene amplification by adopting fluorescent quantitative PCR under the guidance of the upstream primer and the downstream primer, and amplifying GAPDH reference genes, and simultaneously detecting fluorescent values and CT values of the FLNA gene and the GAPDH gene;

3) The relative quantification of GAPDH was used to calculate the expression level of the molecular markers.

Fluorescent quantitative PCR reaction condition setting: pre-denaturation at 95 ℃ for 30s, denaturation at 95 ℃ for 5s, annealing at 60 ℃ for 30s, 40 cycles total, and collecting fluorescent signals; a dissolution profile analysis was performed at 60-95℃to determine the identity of the amplified product.

The foregoing examples merely illustrate specific embodiments of the invention, which are described in greater detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention.

SEQUENCE LISTING

<110> Sichuan province tumor Hospital

<120> platelet molecular marker for lung cancer, kit and detection method

<130> 2021

<160> 8

<170> PatentIn version 3.3

<210> 1

<211> 27311

<212> DNA

<213> filamin A (FLNA)

<400> 1

cgatcctgtt tctgctgccc accccaggcc agacctggca gcgcctgatc catggcagtc 60

aggttcatcc cagggctcgg agccctgcag tcccgtcccg gggccctgcc tgacgtaggc 120

gcccagccaa cacctgcaac gacctgttgc ccccacccca ggaaacaatc ccggcttggg 180

acagacacca ggggctgtga ctgtgggtgg ctccttggag aaggtggcct tgggcctcgg 240

cctttccacc tggggtgcgc ttggctcccc cagactcaga cacgcaggat ccttgtgatt 300

gggcctagac acgagccctg cagtccctgt gggcgctgga ggcgactccc ccacgaagcc 360

ctccctcccc cagggcctgg cggccctcgc cggcggtgag ctcatctcgg ggggcggcgt 420

cgcccggcgg cgggtcgctg gagctcacgg cgtgcccccg cacccccgcc ccccacgcac 480

ccgtcccggg ctcctgggca attttaccaa ttaaggcttt ctcgccgggc cgggccgggc 540

gcggggcggg cgcggggcgg gggtgcggcc aacaagaagg cgggcggcgg cggcccattc 600

gcgtggaggc gcgtcgcgcg cagcggacgc cgacagaatc cccgaggcgc ctggcgcggg 660

cgcgggcgcg aaggcgatcc gggcgccacc ccgcggtcat cggtcaccgg tcgctctcag 720

gaacagcagg tgaggtctcc gcggcccggc ttcgcgccgt agggtcgccg cgctcctcgt 780

cggccggggg cggggttgga gaaggcgggc agagaggccg gaaaacgcag gcgccagctc 840

gcgcccaggt ccgggccagg ttcagctggg atgcgtgagc cgatggaggc tcccctggca 900

tctgggagtg agtgtccaga gaatagcttc cctagttgac ccacgagcca tgtctgtgcc 960

cacgaatgtg ggagcctgag gccatggggg gtccccgaga gagagagggt gtctgggcac 1020

ttgcccatga acggaccact gcatgcctga gagagaaggg ccctgacacc aggaacttgt 1080

gtggactcag ggcgagtggc cacctggaag gacacttctc agccagtgtg cccactggga 1140

gggggacagc gtgtcccttg ctggctcgag tccacagggc accagctgct ttgggcacag 1200

gggagctggt tctggggctg ctctactcca tgggggccat ggtgaggatg agaccaggcc 1260

aggttccttg gttgggccct gggccctatg ccaggttggc tctgagttgg atgcgcactg 1320

ccagccagga tataaccaac aatgtcccca gattcccgtg ccacctgggg ccaggcaaaa 1380

taggttaaaa tccactggca gacccatttg taagtgctcc gagtgcaccg tcaaggtggg 1440

agcttgggaa tgggcgacct cacctgccgc acctggactt gccagcacag gtctcttctc 1500

accttgccag tctgcggctt ccttccctcc tgcgccccct ggctacgcag gcctggaaac 1560

agctccttag ggatcccagc tgaacagctc ctgggcttgg gctatacctc ccagtgggag 1620

ggtctggcct ggggtctgag ggccaggggt ggtgtggtgg gcggggtgtg cctaacatcc 1680

aagaatctac tccaaataag ggaaaatatg aaagagctgg attttggctt cccaggactg 1740

cccgatctgg tggctttggg atcgggcgac atctggcggt tgatactatg ttctagggac 1800

aagaaccctc tacacgccca ttccttgttt cctctaaata gggaaaagct agggctggaa 1860

gacacggcac ccacctcctg acactctttc tgctggaatt gaccactggt cactctgact 1920

cagtttccct gagctctgaa gattaaggaa tgaacccaga caacccagct caatggccct 1980

tagtggagag agagtcaatt attgatggaa ttccgtgcct gggaacttgc tttccagtgg 2040

ggcaggggga ccagaaggac aacttcaagc ccgtcttgaa tggggcccgt gcagggaggg 2100

gtctgtatgg gttccccctt cccagccctc cctcccacct ccaccccagc tcccagacgc 2160

ccaggcgcac ttggtgcagg tgtgtctcaa agtggtcagg agaggggaca atatacaggg 2220

gttggcctca gtactggaaa atgccatttg caccccctaa caccactact agtgggaaaa 2280

accagtgggg ccacaggctg ccccgaagtg aatgtgctgg gcggatcaca gccccaccac 2340

gtgtcatgta gacacccagg gctacaggag agtcaaccat atttgggcat gacgtgctgg 2400

ccaacccagg gcctccatct ctcactggtc tttagcagat aaattaatta ttactagaat 2460

tgaacaggag ggacagatac ctgccttcct agaaaaaaaa aaaaaacact cagaggagga 2520

aacattctgg agaggggagg taccgaggac tgagagggag gggagtgcac aatggggagg 2580

aagccatagg caggctggct tctgactgaa gatagtagca gtgagcgtcc ccagccgtcc 2640

cctggtccca gtctacatct cggcacagca gccactgcac catctccaga gttatctacc 2700

agaaaatttt gagtgaagag cccccacagc tctggggaca caacccccca ggcccctgat 2760

ccctgtcctc aggcctcctc tgcacctgcc accacacctg aagcccaccc agacccaggt 2820

gctcagagcc tgacaagtct tatgggaacc tctgagcagg cccctctggg tctggaatgc 2880

cactcccgtg tttgccccct gccctggcca cgctgggctg aggggacttt ccgacaggag 2940

aggacccccc gcttcagctc cccccagtgc cccgggtgaa ctggcatgaa gggttaggcg 3000

ggcacacgtc tctgcatgca tcgcacggcg ccctctgccc gcgcccactg gcgtgcccgt 3060

gggcatgccc ggaccccttc gaaggctgct tgctcctgga gtgccggcgg cctcctggcc 3120

cagtgcccca tatcctcccg ggtggaggga agcagagagg gaggcggtgg gaagcagcgg 3180

gcacaggtgc gcgggcgagg gccctacgga ctgggaaggc gggcccgtgg cagggggtct 3240

gcggggaccg cgtaggcgca ggccttgccc tgcagccgct tcccaggtga tgagcacaca 3300

aagcgccctc ggcagtgcct ggtccggggc tgccgctctc caggggttcg ctcttagggt 3360

tggccccttc ccgccgctgc cgggccggcg gacggggatg ccgccgggga accctaggcc 3420

ctgggggccc ggggaagggg gatgacggcg gggggtaggg atgtgcaggg acggggcaga 3480

gcccaagtga cttagggaag agagccaccc caccccccgc aggggccctg gggaaggaaa 3540

gtcccttgta aggcgcgccg gctgcaggcc aagcagtccc gcgccagaca ctgctcagcc 3600

tttcctcgct ctagggcgcg tttcctgcag cgccaacgcg aggctgcggg gctgggagca 3660

tccttgccca ccccactgcc cagcgaccca ccctggagcc cgggtttcgc gtctgatcag 3720

ccgaacccgg gggcctagtg ggggcattcc aacgggcggg gcggggtggg gcaggcgcgc 3780

cctcgaagcc ccaccccacg gcccctcccc gcaggaggcc cgccctctgg ccccgccctc 3840

ctcccgcatt taaagggctc gctctctccg cagcgcaacc tctgctccct gcctcgcctc 3900

ccgcgcgcct aggtgcctgc gactttaatt aaagggccgt cccctcgccg aggctgcagc 3960

accgcccccc cggcttctcg cgcctcaaaa tgagtagctc ccactctcgg gcgggccaga 4020

gcgcagcagg cgcggctccg ggcggcggcg tcgacacgcg ggacgccgag atgccggcca 4080

ccgagaagga cctggcggag gacgcgccgt ggaagaagat ccagcagaac actttcacgc 4140

gctggtgcaa cgagcacctg aagtgcgtga gcaagcgcat cgccaacctg cagacggacc 4200

tgagcgacgg gctgcggctt atcgcgctgt tggaggtgct cagccagaag aagatgcacc 4260

gcaagcacaa ccagcggccc actttccgcc aaatgcagct tgagaacgtg tcggtggcgc 4320

tcgagttcct ggaccgcgag agcatcaaac tggtgtccat cggtgaggga cgccccgaag 4380

cgccgggcgg gcgggggcgg ggactccgtg cgtccttcca tctccccggg cggacccctc 4440

caaaccaccc ccttccgggc cgcagcgcaa ctcccgaggc ctccggctgc tctgctccac 4500

gcgggcctgc agaacaaagg cgcgcgggct ggctggctgg ccggccgggc gtgcgaaccg 4560

ctacgcgccc ccgcccgctg cgccccggct ggccggcgcc caggctgaac tcacatcctc 4620

cgcaggccgc gcctccgcgg ctgtgctcgc ggtgggagtg ggcctggggg gtcgggcact 4680

gggcaggctc ctggccctgc gccccgtccc cgaggaggag gaggcccggg ctctcccccc 4740

acagctgttt agagagggcc ctctggcctt cagaggcccg ttggttctga agtgtgggtt 4800

ggagctcgtg cgtcccattg tctggggctc tgggctcttt gtgtccctgg aggatgggtc 4860

gcccatcgga gatggctcca accaccagtt tagatctacc cccccacaca cacacaggca 4920

acctgggggc cagaaagaag catagttagc aaaactgcac ccggggtgag ttgggcgtgg 4980

ttgactgaga gacctggctt gtagactact tggacgccaa cttttctctt catcttgggc 5040

caaactcttg cttccatgta ggccttgctt ttctgcgtgt agcacgcagg agttggttgg 5100

tagatgctcc agcgtgcatt gtgccagccc agtgtctgtg tcactggact ggagaggctg 5160

gcattgacca ggcctgctgc agcagcccaa ttcgttctca agataggggc tgcatgagag 5220

ggcaggcccc agaatgggtg actcgggggt gatgtggaac agggagagga gagggatgat 5280

gagtcttctc ttcttcagaa ttgagaggcc ttccagttac cctgcagccc tggaatggcc 5340

cttgctgaac ccaagaaagt gagggaccaa gacttgggtt cccccagcca ctcagagggc 5400

agatcccaag aaccaaagtt ctttctttgg aatgccagtt gaggcgaggg gcaggcccac 5460

ccccacaagt cccagcctca gtctagccaa gctgagtcat ctcagtcctg gatggtaggg 5520

ggggcgaccg gatgtgggga ggcgggccac accccagctc tttagggggg gtctcctccc 5580

tcctcccagc tcccctcctc cttgtgagct ggctggagca ggcctagttc ccagagcttt 5640

gccatataag gcaaaaaaat gttggaaagc agcagtgatt aggggaggga ggggttggct 5700

ggcccagggg ctggggaaag gggggtggga tggggcgggg ccatccagcc agacagtctc 5760

ccagccctgg gccctggtcc ttccctctct cttttgggag ggggatgggg acggagtagg 5820

ctggcctaac atggacctgg tgggcctggt tgctccccgt gtgggggtcg ttgaggggtc 5880

tgaggctgga ctggggtggg ggcctgatgg gaagccctgg ctggccttgt tccctggccc 5940

tgcacccttg gtcctctccg agtccccctc cctgctgcca tttggggaga gtgaaaggca 6000

gcctcgtggg gccaggacca acctgggaac tggggaggcc actctgagcg gctcacctgg 6060

ccctccaggt gctgcctgct gggctgggaa gcagctgcca tggacagccc agcccctcct 6120

cataggcccg ccccctagtt gactcaccag agaagtattt tgtctgatta tgggggaggc 6180

tgacagggct tctaaagatg agccatcctg ccttgagcct gagacccaga agtagccttg 6240

ggtggggccc agagcccagc agggttgcca tggtgatggg gggtgtcagg gacccgccct 6300

aaccacttcc cctgtggcga gcctgccgag gcccagaggc ccgttaggtg gcaagtgggt 6360

caggcagtgg gtggcaaggg ggttcccagt gtctcctgct gccaccccgg catctcccga 6420

tgggccccag tgagtcaggg ccatgtggcc caactgctgg ttcctttggg ctctgggccc 6480

ctcccctggc taggcctgtg gcctcccaac tccaagcttg agtcttcgag gtggtagagg 6540

aagtgggggt ggtggcgggg gtcacaatcc ctcacccaaa gggcgtttgc ccaactcccc 6600

cacttcccct gcctcacccc acccaccctg gaacaacagt tgtatttccc aaccacccag 6660

ggccacccac cttcctcttc ctcagcccca gccccactcc aggtcatggg ccctggattc 6720

tgggccagct ggagtcagcc tcctcctcct ggttccccat aggcccctga ggtgtgtggc 6780

ggacccctgc tctggcagct gtggacactg tccagctgtt ctccagagct gcccatctgc 6840

aggctgggtc tgccctctgc ccttcctctg ggcccagggc ttctggcagt aatcataccc 6900

tgctggcatc gtgtgtgtgt gtctgtgtgt atctgccctc accccaaagt ctccttcacc 6960

tctgaacacc ccctgggacc ctgggcagag agggcctcat ccacatccta ccactggcca 7020

agagggggtg tctacctcct ccttggcccc tgctaccccg tgctgccaga ccctgacccc 7080

aaagcctggg atcggcccct cccgcacagc cgtgtgccca gcgtggccac tcttctactc 7140

acagacagca aggccatcgt ggacgggaac ctgaagctga tcctgggcct catctggacc 7200

ctgatcctgc actactccat ctccatgccc atgtgggacg aggaggagga tgaggaggcc 7260

aagaagcaga cccccaagca gaggctcctg ggctggatcc agaacaagct gccgcagctg 7320

cccatcacca acttcagccg ggactggcag agcggccggg ccctgggcgc cctggtggac 7380

agctgtgccc cgggtgaggg ggcgcagagg caggagagac tgggctgggg tcacccatgg 7440

gtggcccttg agggggttct gcggccccca gactcacagc ctcccttgcg ccacaggcct 7500

gtgtcctgac tgggactctt gggacgccag caagcccgtt accaatgcgc gagaggccat 7560

gcagcaggcg gatgactggc tgggcatccc ccaggtacac ccgcccggcc tggctacagc 7620

tgtaggggac cggatcggca gttgggagag actcggaggc ccgatggctc ctgttttctg 7680

cctgtgacag gtgatcaccc ccgaggagat tgtggacccc aacgtggacg agcactctgt 7740

catgacctac ctgtcccagt tccccaaggc caagctgaag ccaggggctc ccttgcggcc 7800

caaactgaac ccgaagaaag cccgtgccta cgggccaggt gagggagccc caccaggggt 7860

gcacccgtgt gccaacgtct tccccataag acagtgtccc atctaaacgt ggcggtcccc 7920

aggggtcttg ggttacatgg cggctaggtg atcaagccac ccaagagtca gacaagagtg 7980

ggtttttggg actagtgaaa acgacaagag aatgctaata ggttcctcca gtcacagaag 8040

cctgctgcgt ggggtcctcg gggaggtagc atccagcggt ctttattata aatggagatg 8100

aaccaggtct cagtaacagg gacctaaaac ccagtcaagg ctcacacagg aacaagggcc 8160

cagtgcggtg agaatgaatg ttccagaaaa caggctgggc ctgtcccagc agcgagagca 8220

cagctgggtc ccgaggccag gctctgtgcc acagctcaca ctgaatgaac ttgcccagtt 8280

tggaatgtgc ctgacttagg ggccagcagg gagggctgaa gggtggaggg gtgaggctgt 8340

ggccttaaca gctggcccgt ggttggctcg ccttcccctg ccaggcatcg agcccacagg 8400

caacatggtg aagaagcggg cagagttcac tgtggagacc agaagtgctg gccagggaga 8460

ggtgctggtg tacgtggagg acccggccgg acaccaggag gaggtagggc cagctgctgg 8520

cagcagaggc cccgcagcgc tccctttcag tggggctgct cttagcaaag gctcacaggc 8580

tccttcccac tgcaggcaaa agtgaccgcc aataacgaca agaaccgcac cttctccgtc 8640

tggtacgtcc ccgaggtgac ggggactcat aaggtgagcc cttggccagg ggggaggctt 8700

gtgacctcag gcagtggctg gaggccccca gccctaccct cacggcccac ccttctgggg 8760

acaggttact gtgctctttg ctggccagca catcgccaag agccccttcg aggtgtacgt 8820

ggataagtca cagggtgacg ccagcaaagt gacagcccaa ggtcccggcc tggagcccag 8880

tggcaacatc gccaacaaga ccacctactt tgagatcttt acggcaggtg aggggaggct 8940

gccaaggccc agctggtctg tggggagagc cgcactgggg cctgggagct gagcaggtgc 9000

ctcgtggcag gagctggcac gggcgaggtc gaggttgtga tccaggaccc catgggacag 9060

aagggcacgg tagagcctca gctggaggcc cggggcgaca gcacataccg ctgcagctac 9120

cagcccacca tggagggcgt ccacaccgtg cacgtcacgt ttgccggcgt gcccatccct 9180

cgcagcccct acactgtcac tgttggccaa ggtaggcccg gccctgactg ccctctgctg 9240

tggcactgca gtctgggcct cccacaggag ggggaccccc tgggccagtc ccgctgcagc 9300

ccgccccggg gcccctactc tttgagcttc tactgtctgg gcacatgctg tttttcctcc 9360

cctgctgggg cccttcctcc tctcctctcc tcgcccctct ccatctctct cctcacgctt 9420

tcaccttcgg aagagctcgc tccagctgtc tagaaacctg ctgctctctg ctctgcccac 9480

agggtcaacg gtacttggat ctgtcttctt cttggaaggg ggagggtggg gggctctggg 9540

ctggtgtgga gcaggtggga ggaggcctgc catgtcctgg cccaggccac agttcctggg 9600

gtcacagtcc cttccagttc tgtcttgcat gtccccctcc agccaagctc tgagggaccc 9660

accaatcctg acagcccggc cctgtgggag gcaagggagg ggtcgcccag ccgagctgct 9720

ggtgctcagc tggcccctct gcctgcagcc tgtaacccga gtgcctgccg ggcggttggc 9780

cggggcctcc agcccaaggg tgtgcgggtg aaggagacag ctgacttcaa ggtgtacaca 9840

aagggcgctg gcagtgggga gctgaaggtc accgtgaagg gccccagtaa gttggcctgg 9900

agccaggcca ggatgggtgg cggcagcccc cgggttcact gctgggcagg cctgaggccc 9960

tccttgtctt ggcagaggga gaggagcgcg tgaagcagaa ggacctgggg gatggcgtgt 10020

atggcttcga gtattacccc atggtccctg gaacctatat cgtcaccatc acgtggggtg 10080

gtcagaacat cgggcgcagg tgaggccccc aggcatccct ctcccagctc tgcacactct 10140

gcctcttctc ttccttctgc ctccccacct gatgatgagg ggccacatgg gctcttcctg 10200

cctggccgcc acagtggggc ccacgctggc tcatgtggtg atcctcggtg ttccctgtgt 10260

ggctctcgca tttgcagtcc cttcgaagtg aaggtgggca ccgagtgtgg caatcagaag 10320

gtacgggcct ggggccctgg gctggagggc ggcgtcgttg gcaagtcagc agactttgtg 10380

gtggaggcta tcggggacga cgtgggcacg ctgggtaagt tggaggctgc agcatgggca 10440

cctggggaca gacgatggca aggacggccc accctgaggc tccagggcac tgaggggact 10500

ggtggctgtt gtcaggcttc tcggtggaag ggccatcgca ggctaagatc gaatgtgacg 10560

acaagggcga cggctcctgt gatgtgcgct actggccgca ggaggctggc gagtatgccg 10620

ttcacgtgct gtgcaacagc gaagacatcc gcctcagccc cttcatggct gacatccgtg 10680

acgcgcccca ggacttccac ccagacaggg taaagagcag gctgtgatgg cctaagtctc 10740

gccctgctgc tcctgcttgg ggtctggtgg ggatggcact ctgtgtccct ggccaccctg 10800

cctgggttgt agctctggca gcctggacct ggccccctga cagctgggtg gtctcccgct 10860

aggtgaaggc acgtgggcct ggattggaga agacaggtgt ggccgtcaac aagccagcag 10920

agttcacagt ggatgccaag cacggtggca aggccccact tcgggtccaa gtccaggtag 10980

agcacccacg ggtgttgggg gcagggcagg tgtgggcacc caggcctggg cactgaccag 11040

caggccacct gctcctatct gcctgacagg acaatgaagg ctgccctgtg gaggcgttgg 11100

tcaaggacaa cggcaatggc acttacagct gctcctacgt gcccaggaag ccggtgaagc 11160

acacagccat ggtgtcctgg ggaggcgtca gcatccccaa cagccccttc agggtgagcc 11220

aacctccacc ggcctttagt ccatctcgat agcattcacg tagcacacga ttcgtccatt 11280

tcaagtggtt gtggcacacc atcctgactc ccctgtccca cagctcctgg tgaccactaa 11340

cctgctttct gctgtggctt tgcctatcct ggacatgttc tatagatgga actgtgtgcc 11400

atgtggcctt ttgtgtctgg cttctttcgc tcaccacgaa gttttccgtg ttttcgttca 11460

tgtttggcat gtcttggcac tttgttcctt tataattatt ttgtttattt atttatttat 11520

ttgagatgga gtctcacact gtcgcccagg ctggagtgca atggcgcgat ctcggctcac 11580

tgcaggctct ttctcccagg ttcacaccat tgtcttgcct cagcctcccg ggtagctggg 11640

actacaggcg cccgccaccc ccccggctaa ttttttgtat ttttagtaga gacaaggttt 11700

cactgtgtta gccaggatgg tgtcgatctc ctgaccttgt gatctgccca cctcggcctc 11760

ccaaagtgct gggatgacag gcatgagcca ccgcgcccat cctggttttt tttttttgag 11820

ccggagtttt gctcttattg ccaggctgga gtgcaatgac gtgatctcag ctcactgcaa 11880

cctctgcctg ccagattcaa gcaattctct cctgcctcag cctcctgagt agctgggatt 11940

gcaggcatac gccaccattc ccagctaatt ttgtattttt agtagagacg gggtttctcc 12000

atgttggtca gactggtctc aaccgcctga ccccaggtgg tccgcccacc ttggccttcc 12060

aaagtgctgg gattacaggc atgagccacc acgtccggcc gcttcattta tttttatggc 12120

cgcataatac tccattgtgt ggccagacca cattccattt ttccgttcac ttgctaacag 12180

atattgagga tgagcttgcc tttggtgatc atgaacagtg ctgttaacag tgagtccagg 12240

tgtttgcgtg gacatttgct ttcatttctc ttgggtagtt gcctcggagt gaaccggagc 12300

atcacacagt ccctctgtgg tcaactgagg aactgccaga ctcttcccaa ggcagctgga 12360

ccattttgtg tgcctgccag tgtagctgag ggagcttctg agtctcctcc aggctgctca 12420

tgcccttgcc cttgcccctg tgccctgcag gtgaatgtgg gagctggcag ccaccccaac 12480

aaggtcaaag tatacggccc cggagtagcc aagacagggc tcaaggccca cgagcccacc 12540

tacttcactg tggactgcgc cgaggctggc cagggtaagg cctggctgtg ggtgggaggg 12600

caggtggctg gggtgtccct gcgaggtctc agcctccgct cctctccccg ccgcagggga 12660

cgtcagcatc ggcatcaagt gtgcccctgg agtggtaggc cccgccgaag ctgacatcga 12720

cttcgacatc atccgcaatg acaatgacac cttcacggtc aagtacacgc cccggggggc 12780

tggcagctac accattatgg tcctctttgc tgaccaggtg ggtgctgcgc tcctggccgc 12840

taagatgtca ttgcggatga cgtctgccct cctctaaggc cttctcctcc cactgcctgc 12900

aggccacgcc caccagcccc atccgagtca aggtggagcc ctctcatgac gccagtaagg 12960

tgaaggccga gggccctggc ctcagtcgca ctggtgagga caggtacccc atggcaggtt 13020

gcggggcatc aagggtagga gggcttgggg cagggtgccc ctacatggtc cctgtgtgtt 13080

cctcaggtgt cgagcttggc aagcccaccc acttcacagt aaatgccaaa gctgctggca 13140

aaggcaagct ggacgtccag ttctcaggac tcaccaaggg ggatgcagtg cgagatgtgg 13200

acatcatcga ccaccatgac aacacctaca cagtcaagta cacgcctgtc cagcaggtag 13260

ccacaccctc cactacagtc accgagtccc cagcctcctc atgcagcagc ctaggggaca 13320

gctccgaggg actttctgcc cactctcatt ccttacctgg gagaggacag gcatggaggt 13380

ccaggagggg gttggggagc aattctggtg tctctaaata cccccttccc ttctgcaccc 13440

ttcccagggt ccagtaggcg tcaatgtcac ttatggaggg gatcccatcc ctaagagccc 13500

tttctcagtg gcagtatctc caagcctgga cctcagcaag atcaaggtgt ctggcctggg 13560

agagagtaag tagttggggc ccttgtcgca aaggcctttg tcacatccag ggattggctg 13620

agctgggtgt catgtctctc tccttccttt catcgtttcc acagaggtgg acgttggcaa 13680

agaccaggag ttcacagtca aatcaaaggg tgctggtggt caaggcaaag tggcatccaa 13740

gattgtgggc ccctcgggtg cagcggtgcc ctgcaaggtg gagccaggcc tgggggctga 13800

caacagtgtg gtgcgcttcc tgccccgtga ggaagggccc tatgaggtgg aggtgaccta 13860

tgacggcgtg cccgtgcctg gcagcccctt tcctctggaa gctgtggccc ccaccaagcc 13920

tagcaaggta attggggtat gggaggccct aagggtagaa cccactgtgc tctctgaggg 13980

ccccaaaggt accccatctt ctccagacct gccctaaatc cttttttttt tttttttttc 14040

ctgagacaga ctgtcaacct tgaccctgtt taagtgagag taaaggggag gagggaaata 14100

gaaacccaca aaaagtcatc agggttgaag aatttctttt tttttttttt ctttcttttt 14160

tttttttttt tttttttttt ttttttttga gaaagagtct cactctgtcg cccaggctgg 14220

agtgcagtgg cgcgatattg gctcacggca ccctccgcct cctggcttca agcaatactc 14280

ctgcctcagc ctcccaagta gctgggatta caggtgcccg ccaccatgcc cagctaattt 14340

tggtaatttt agtagagacg agatttcacc atgttggcca ggctgttctc aaactcctga 14400

cctcaggtga tccacccacc ttggcctccc aaagggctga gattacaggc gtgagtgacc 14460

gcgccctgcc ctcctttttg tccttatgac tgatggatgg cccatctggc aggcagcctg 14520

ggcccgcccg tgtccctgtt tggtgagggc aggaagccag ggcaagggga ggcaggtgga 14580

aagtcaagca ggaggtctgg gatcggggca tagtgtggcc ttggctcctg accccttctt 14640

ccctcaggtg aaggcgtttg ggccggggct gcagggaggc agtgcgggct cccccgcccg 14700

cttcaccatc gacaccaagg gcgccggcac aggtggcctg ggcctgacgg tggagggccc 14760

ctgtgaggcg cagctcgagt gcttggacaa tggggatggc acatgttccg tgtcctacgt 14820

gcccaccgag cccggggact acaacatcaa catcctcttc gctgacaccc acatccctgg 14880

ctccccattc aaggcccacg tggttccctg ctttgacgca tccaaagtca agtgctcagg 14940

ccccgggctg gagcgggcca ccgctgggga ggtgggccaa ttccaagtgg actgctcgag 15000

cgcgggcagc gcggagctga ccattgagat ctgctcggag gcggggcttc cggccgaggt 15060

gtacatccag gaccacggtg atggcacgca caccattacc tacattcccc tctgccccgg 15120

ggcctacacc gtcaccatca agtacggcgg ccagcccgtg cccaacttcc ccagcaagct 15180

gcaggtggaa cctgcggtgg acacttccgg tgtccagtgc tatgggcctg gtattgaggg 15240

ccagggtgag ttgccctgcc gtggggtatg tgacggggga cagggaccag aacccccgag 15300

ccccaagcct cctctctgtg cctttgcagg tgtcttccgt gaggccacca ctgagttcag 15360

tgtggacgcc cgggctctga cacagaccgg agggccgcac gtcaaggccc gtgtggccaa 15420

cccctcaggc aacctgacgg agacctacgt tcaggaccgt ggcgatggca tgtacaaagt 15480

ggagtacacg ccttacgagg agggtgcgtg ctggggactc ccacaggctg gcagacgggg 15540

tgggtggggt ggcccgggct cctcctgacc tggcctgcac tcctccagga ctgcactccg 15600

tggacgtgac ctatgacggc agtcccgtgc ccagcagccc cttccaggtg cccgtgaccg 15660

agggctgcga cccctcccgg gtgcgtgtcc acgggccagg catccaaagt ggcaccacca 15720

acaagcccaa caagttcact gtggagacca ggtaagggag cccccaggag cccgtgtggc 15780

tggtgggcct gggaacccca ggaatgaccg gctgtctgtt tggaccaggg gagctggcac 15840

gggcggcctg ggcctggctg tagagggccc ctccgaggcc aagatgtcct gcatggataa 15900

caaggacggc agctgctcgg tcgagtacat cccttatgag gctggcacct acagcctcaa 15960

cgtcacctat ggtggccatc aagtgccagg tgaggaggtg gcggccaggg cagggctggg 16020

actgtctgct cgtccccacc ctgtctcata cagccgccat ccctttgccc caaccctcag 16080

gcagtccttt caaggtccct gtgcatgatg tgacagatgc gtccaaggtc aagtgctctg 16140

ggcccggcct gagcccaggc atggttcgtg ccaacctccc tcagtccttc caggtggaca 16200

caagcaaggc tggtgtggcc ccattgcagg tcaaagtgca agggcccaaa ggtgagtgtt 16260

tgcatgcctg gagccagggg tcaggagggc agtgtcctgt ttgagggccg ggaaaggaaa 16320

ctggaggccc aaagtgagga ccagagtgca gaggtggtgc aagtgtgtag gtgtttttgg 16380

aagagcctca ggtgacagtg cagccaggtg ggggacttgg aggaggtaga gcagatggga 16440

ctggtcactg agcagatgtg gcaggaaggc aggcaggcaa gggtcccagc tggcacccag 16500

atgggtgtct ttcgtgccag agggggtcaa ggccattccc agggtcgggt ggatgaggat 16560

aaagcgaggt ctaggcctgg ggctgggtcc tggccgtcca gtgtggtggg gtaggaatgg 16620

aggcccagga gactagctga tgctctgtcc ctggggctgg ggccaggcct ggtggagcca 16680

gtggacgtgg tagacaacgc tgatggcacc cagaccgtca attatgtgcc cagccgagaa 16740

gggccctaca gcatctcagt actgtatgga gatgaagagg taccccggag gtaagaggca 16800

gggcctgctg cctgtgggga gtggcccagg ctggatgctg agaacctgtc tgactgctca 16860

caacaccaat ccctcacagc cccttcaagg tcaaggtgct gcctactcat gatgccagca 16920

aggtgaaggc cagtggcccc gggctcaaca ccactggcgt gcctgccagc ctgcccgtgg 16980

agttcaccat cgatgcaaag gacgccgggg agggcctgct ggctgtccag atcacggtga 17040

gctccgggca caggcagggg aggcaggggg gcctgggcgg ctggagtgtg ggctgcgggt 17100

ggctggtgtc tgatctccct gcggagctgc accaaggagg gcagggaagg acagggcccc 17160

tgtctttggt ttgctaaggt tgtgactacc ttaggatggt ggtgacaggc acaggcttcc 17220

ccagacgtgc acttgctgct aactttgagc acgtcaccaa agccttctgt gcctcagttt 17280

ccccacctgt aatattctca tggggcaggt ataggataga agcttgagat caccccggtg 17340

agctcctcct agtgctggcc aggccctggg gctgaggatg ggtgccaagc tggctggccg 17400

cgcctgttgt catgtgggaa gtgtgggatg ttttagtctc cattctggac tctgccaggt 17460

gcaggcagcc cttttgggcc atagcagtta agatgcctga ggcccagaga ctgtccctgt 17520

agcagcagcg ctgggcactc tgggagcgcc tgaggcaggc ccggggctac tcgggcttgg 17580

gccaactgcc tcccctgcct ctgccacaca ggatcccgaa ggcaagccga agaagacaca 17640

catccaagac aaccatgacg gcacgtatac agtggcctac gtgccagacg tgacaggtcg 17700

ctacaccatc ctcatcaagt acggtggtga cgagatcccc ttctccccgt accgcgtgcg 17760

tgccgtgccc accggggacg ccagcaagtg cactgtcaca ggtgagcccg cccgctgttg 17820

gccgctgcgg cgctcggcac ggggcttggg ttgtgcctgt gtgcaagggc tcgctgcttg 17880

gggcggcgtg tgggagtggg ggccgccccc ttgagtgact cacctgacct ttctccttgc 17940

tctctgccct ctcacctcag tgtcaatcgg aggtcacggg ctaggtaagc tgcttgctgg 18000

ggccactccc aatgcccctc ctgcccagag gggcagcttc ctttcctctt ggggccgatt 18060

cccaggacag ttccagctgc agctctaggc agggacagag ttgggtgggg tgcagaagat 18120

gaggccgtgg gctggagggc gagggcccag ggaggcttcc ggagctgagc atgaccgctc 18180

cagctcttcc cctctctggg cctttatttc cccatctgag aactcgggtt gcagctgaga 18240

gctttggggt gaggtgcatc ctgagaggca ccccactggg gactttgggg tctcgggatc 18300

ttggcaccca gggaggcaca agggctcctg cgtgggctga gggaacaagg ggtctcccca 18360

actcaatggg agccccagcc ctggccagat ccacagaccc ccatccccac cttccagggg 18420

cctctgcgtc ccgggccggg ggaggtgcat gagcgggtgg ggctggctgg tagtgcagta 18480

gcgccagtgt cgactgcttg ggcggccagc atggcggggc ccggcccagc ctcagagccc 18540

caaagtgcat tctgtggccc gtctccctcc ttcccccttc ccctatcccc cagcagtgac 18600

gttattcaga cattccccaa caggcatgga gggtcacctg ccccagccag ggacacccag 18660

ggatgctgat gagaggctac aggcctggca cttgggcgag gtggctaagg aaagggtgag 18720

agggtggcta gagttttgcg gcaggagcga gatggcttta tagagcaggc cgtaggatcc 18780

gatgtgtgtg tgacaggcgt gtgtgcgcag ggcgggggag gctggggagt actgattgca 18840

gggccttggg ggctttgagg gtgggagcaa gactcagtaa cctcagtttc cctggctact 18900

gtttgcagaa tggagacagg gagcccgggg tggggaaggg ggcgctgaag ttgctctctg 18960

gcaagcttga cggccacggg gcttcagtga gagccaaatt aggaggcata ctttgaggat 19020

ggagtggctg agacctgcgg ttggctggga gccgggcagg gaaagtgggg gcagtcgggt 19080

acctgtgagg ttggggccca agccactaac tagatggcat ggggtgcaca tgagtgaagc 19140

ctcccagggc agcgtgggca gccggcagcc aggtctggag cctgggagag ctgcctggcc 19200

gcggcaggcc agcatggagc ctagtgcagc aggagccctc ggccgatagt gggacttggt 19260

ggcagggccc ctcgagcttg gccttcaagt aggtttcgtg ctcctggctg cttccctggc 19320

aggaggccag gctgcagggg gcactctcac tgccaggcct cacagcccac agccccccaa 19380

gcctccacgg cggagaccca gatcataacg tcactgtctc tcccctttcc tcgggcactc 19440

tggggccctg gagcctgctc agctgctctg ccctcgcaac ccagcagtgg tggctgtagc 19500

ttgggcctcc tgttccctgt ctccctgggt gctatccccg ctctgccaag cctgctgcgc 19560

tgcgctactc gggtgctaac cccgctgcct cccgcttccc cgctcccact cctgtctgca 19620

ccgggctgct tttcaccgcg agccttctca gttgtcagga aactccggtt tcaccatcag 19680

gcctgggggg tggggaggca agtgtcacat ccccccacgc tgcagcagga ctccgcctca 19740

ccgcgtccac acacagcaat gggggccggg gccaggccag aagggcgcag gcagccgtgt 19800

gcccagccag atgctggcgt tcagcagtgt gggccatggt gctggacgag gctgagtagg 19860

caagggagtg gctgtcaaca cttaggctgg agtgggccct tccttttcct cccaggcacc 19920

tgggtgctac tgtcgggcct ctcccacatg ggcacttgag gtggtcagga gaaaccttcc 19980

tgccttctga gaaaccactc gccatcccag cttggagggg cagggcgggg caggaggcct 20040

ggcggcctgg ctgtgcttgg tggtggcaat gagaggcctg tgtgggcggc aggcggacgg 20100

gaacaacctg aacccgagct cacacgctgg cccccctttg gggacccacc ctgccccacc 20160

aggtgctggc atcggcccca ccattcagat tggggaggag acggtgatca ctgtggacac 20220

taaggcggca ggcaaaggca aagtgacgtg caccgtgtgc acgcctgatg gctcagaggt 20280

ggatgtggac gtggtggaga atgaggacgg cactttcgac atcttctaca cggcccccca 20340

gccgggcaaa tacgtcatct gtgtgcgctt tggtggcgag cacgtgccca acagcccctt 20400

ccaagtgacg gtgaggaggg gtggggggta ggtcagcagg ggtgtctggc aggtcccggc 20460

ctgttcccct ggggcccctc tgtgacaaca gactctccag cagctctctg ctttgccctg 20520

caggctctgg ctggggacca gccctcggtg cagccccctc tacggtctca gcagctggcc 20580

ccacagtaca cctacgccca gggcggccag cagacttggg tacggcctgg ccagctaggg 20640

acactggggc tagccagctg ggtgttctgt gagcccctag tttaccatgt gtgaggaggg 20700

accccagatc ctcccactgt ccctcaccca tgccctgtgt ctccactgca ggccccggag 20760

aggcccctgg tgggtgtcaa tgggctggat gtgaccagcc tgaggccctt tgaccttgtc 20820

atccccttca ccatcaagaa gggcgagatc acaggtgagt ggggacttgg gaaggagctc 20880

gggagccaag gaggccagac tgtgccaatg agctgccctg acctcagccc cactgcccca 20940

caggggaggt tcggatgccc tcaggcaagg tggcgcagcc caccatcact gacaacaaag 21000

acggcaccgt gaccgtgcgg tatgcaccca gcgaggctgg cctgcacgag atggacatcc 21060

gctatgacaa catgcacatc ccaggtgggc ctgccctgcc tctgcccact ccaccgccac 21120

cacctcacag agagatgggg ctgggggaca cacgaggctg ccattccaca aggcccttct 21180

tcctgcctca ggaagcccct tgcagttcta tgtggattac gtcaactgtg gccatgtcac 21240

tgcctatggg cctggcctca cccatggagt agtgaacaag cctgccacct tcaccgtcaa 21300

caccaaggat gcaggagagg gtgagcaata gctctggtct tgacctgctc tgtgcccggg 21360

atgccctcac cggggtaagg gctggactca ggagatactc ctgaatgggg ctccctgccc 21420

tgctgccctg gccatcggag ctcctcagtg caggccaact ggagtgtccc cagcatagtt 21480

cccatgctca cccacggctc tctccagggg gcctgtctct ggccattgag ggcccgtcca 21540

aagcagaaat cagctgcact gacaaccagg atgggacatg cagcgtgtcc tacctgcctg 21600

tgctgccggg ggactacagc attctagtca agtacaatga acagcacgtc ccaggcagcc 21660

ccttcactgc tcgggtcaca ggtgggtggc actgggagca gtgacagaaa cgggtggcag 21720

ggtgggcatg gtctcccatg gctggacgca cactgatggc tggcctgtcc ccaccaggtg 21780

acgactccat gcgtatgtcc cacctaaagg tcggctctgc tgccgacatc cccatcaaca 21840

tctcagagac ggatctcagc ctgctgacgg ccactgtggt cccgccctcg ggccgggagg 21900

agccctgttt gctgaagcgg ctgcgtaatg gccacgtggg taagcggctg aagggtccag 21960

ggggttcaga aaggaggcag cctgtgggga gccatgccct ccctgactga cagccatgcc 22020

ctgtgtccag ggatttcatt cgtgcccaag gagacggggg agcacctggt gcatgtgaag 22080

aaaaatggcc agcacgtggc cagcagcccc atcccggtgg tgatcagcca gtcggaaatt 22140

ggggatgcca gtcgtgttcg ggtctctggt cagggccttc acgaaggcca cacctttgag 22200

cctgcagagt ttatcattga tacccgcgat gcaggtaggc agtggctgcc caccagctgg 22260

ggcgggagca ctgtgcatag caccgaggct caggggtatc catcccctag gctatggtgg 22320

gctcagcctg tccattgagg gccccagcaa ggtggacatc aacacagagg acctggagga 22380

cgggacgtgc agggtcacct actgccccac agagccaggc aactacatca tcaacatcaa 22440

gtttgccgac cagcacgtgc ctggtgagtg cagcagtgcc tcggcagccc ccctcactac 22500

accagggccc acaggcagta tgtgactgga ggggcgtggg ccgtgctttc ttcctgcagg 22560

cagccccttc tctgtgaagg tgacaggcga gggccgggtg aaagagagca tcacccgcag 22620

gcgtcgggct ccttcagtgg ccaacgttgg tagtcattgt gacctcagcc tgaaaatccc 22680

tggtaggggc tgtgggaagc ctggggaggg gtcctggggc tcaagcagcc ccaagaggag 22740

gggtggagcc cagggctgct gctcactagc ccatccccac cctgcagaaa ttagcatcca 22800

ggatatgaca gcccaggtga ccagcccatc gggcaagacc catgaggccg agatcgtgga 22860

aggggagaac cacacctact gcatccgctt tgttcccgct gagatgggca cacacacagt 22920

cagcgtgaag tacaagggcc agcacgtgcc tgggagcccc ttccagttca ccgtggggcc 22980

cctaggggaa gggggagccc acaaggtccg agctgggggc cctggcctgg agagagctga 23040

agctggagtg ccaggtaggc ctgctcacac cccgagtgct cgctctcctg cgttggccag 23100

ggtggggttg ggggtggagg aaagagccgg caggcatgtc taccttggct atgcctggag 23160

ggggccgagg ctggtgaagc agccttcagt gaggacaaac tgttctccca cagccgaatt 23220

cagtatctgg acccgggaag ctggtgctgg aggcctggcc attgctgtcg agggccccag 23280

caaggctgag atctcttttg aggaccgcaa ggacggctcc tgtggtgtgg cttatgtggt 23340

ccaggagcca ggtactggga acccggctgg ggttggagga ggtggggcct gagtgtgggt 23400

attggaccca gggctggcac agatagccct gcctggcact cactcttcac tttgaggtca 23460

cagggccacc caggctgttc gtgggaggtg ggaggcccaa aggctgatga gccggtctta 23520

cactctttcc ctgcccaggt gactacgaag tctcagtcaa gttcaacgag gaacacattc 23580

ccgacagccc cttcgtggtg cctgtggctt ctccgtctgg cgacgcccgc cgcctcactg 23640

tttctagcct tcaggtgagg caccgagaga aaccgcccac ctggggctcc tgggcccgga 23700

cagaccagag ccaccgcaga gaccaggcct tggccctgtg ttcagtgacc cccctggccc 23760

agctctgggt tccaggcctc tgctctggcc gggtgcagcc cacgcttggg gtcggcagga 23820

aatatcctgc ccaccacgtg gagtttttct cgtgtctcag gtctaaaggc tttgaacaga 23880

agcctgtgcc ccttgagcac cagggctgcg gtttaaagag ggacagcctg ccaaggctgg 23940

gtgctgccca cagggtgggg cagccctccc ttcctgccac ctgggtgtgt gcgacggcag 24000

agtgtggggg caggggcggg gatgtgcgcc tgtggcttgc tgccccttgc aaatcagtgg 24060

ctctccctct ttaaaccaag ttggacgcca gatgggtaag tgcgacccgc gggcctcttg 24120

ctcctggggc ctttccgtcc tcccttgccc tggctcaagc acccccatct aaccatgtct 24180

gctgtgcttc caggagtcag ggctaaaggt caaccagcca gcctcttttg cagtcagcct 24240

gaacggggcc aagggggcga tcgatgccaa ggtgcacagc ccctcaggag ccctggagga 24300

gtgctatgtc acagaaattg accaaggtga ggccctgtcc ctgcccggcc gccctgccct 24360

gctggctgga agcaggctga gagtacaaaa ggaaggtacc gcttctgtaa gcagccaaga 24420

cggggcccca actcctgggc cccctaagcc aggcaaccct ttcccaattg gtgacccaag 24480

ccgctgggga tgtggctcgc cacccacagc tattaggaag gccttctacc ctgcagtacc 24540

atgccatctc cacagggaaa gactcaaagt ccagcctcag tgctgcccgg cccttcagcc 24600

acggccatgg tacttgtgca caggcctggg gcagccttgt gggtaggagg agcccagtgg 24660

gcttcatttc acggcctagt cctgcccttc ccagctgcat gacctgaagc aagttcctgc 24720

ccgtctctga gcctgttaac atgcagggga tatggtgttc acggtaacct ttcttaggag 24780

gcagcaaggc ctgggtttgg cttgtaaaag atttccgtcc caatgcaccc tgacggtggg 24840

cagcttgagt gtctaaacct tcggccctca ggaggtctgc tgcggggtgc aggtgcccag 24900

cgtggctggg ctcacactga tggaccggca aaatggggag ggccagggtt gaggctgttc 24960

ctcacagaca ccatctgctg gtttgaggag gggccccagg cccacagcat gactccctgc 25020

tcctcagata agtatgctgt gcgcttcatc cctcgggaga atggcgttta cctgattgac 25080

gtcaagttca acggcaccca catccctgga agccccttca agatccgagt tggggagcct 25140

gggcatggag gggacccagg cttggtgtct gcttacggag caggtctgga aggcggtgtc 25200

acaggtaagt ctgtcggtgc ggctgcacgt gtgcacacag ctgctaggcc cttgcctcca 25260

agctcttggt gacaacagga ggcacctgga ggtgacaagc ctgtgctggg tggccgagga 25320

cagggaggca ggccagtctg gctctgcctg acctcctgtg ctcccaggga acccagctga 25380

gttcgtcgtg aacacgagca atgcgggagc tggtgccctg tcggtgacca ttgacggccc 25440

ctccaaggtg aagatggatt gccaggagtg ccctgagggc taccgcgtca cctatacccc 25500

catggcacct ggcagctacc tcatctccat caagtacggc ggcccctacc acattggggg 25560

cagccccttc aaggccaaag tcacaggtga gcctggggcc aagctattgg catctgccca 25620

acgcccggca ccacagccac ctcttagccc cacccactct gccttgcagg cccccgtctc 25680

gtcagcaacc acagcctcca cgagacatca tcagtgtttg tagactctct gaccaaggcc 25740

acctgtgccc cccagcatgg ggccccgggt cctgggcctg ctgacgccag caaggtggtg 25800

gccaagggcc tggggctgag caaggcctac gtaggccaga agagcagctt cacagtagac 25860

tgcagcaaag caggtgggca acctgggccc ccggcccacc ttcccaccaa aatgaggcca 25920

gaaatcacag gatggccagt gtctgggaga gcagggaccg cctttggggc gtggctttca 25980

cttctagagc cacctccctg ccacctcctg gacccctcaa acaaagacca gggaggagct 26040

ggaatctgct aagctggcct gggttgggga ggtgctggca gctctttagg aagagagaac 26100

tgtcacccca gaactggctt agggacagga gaggagagca ggacaccagc gggaggaagg 26160

gggctgggac ctgggactga ggacccctct tgcctcaggc aacaacatgc tgctggtggg 26220

ggttcatggc ccaaggaccc cctgcgagga gatcctggtg aagcacgtgg gcagccggct 26280

ctacagcgtg tcctacctgc tcaaggacaa gggggagtac acactggtgg tcaaatgggg 26340

ggacgagcac atcccaggca gcccctaccg cgttgtggtg ccctgagtct ggggcccgtg 26400

ccagccggca gcccccaagc ctgccccgct acccaagcag ccccgccctc ttcccctcaa 26460

ccccggccca ggccgccctg gccgcccgcc tgtcactgca gccgcccctg ccctgtgccg 26520

tgctgcgctc acctgcctcc ccagccagcc gctgacctct cggctttcac ttgggcagag 26580

ggagccattt ggtggcgctg cttgtcttct ttggttctgg gaggggtgag ggatgggggt 26640

cctgtacaca accacccact agttctcttc tccagccaag aggaataaag ttttgcttcc 26700

attctccttt gcgtggctgt agctttgttg gggttggggg accagtcaag gccaacctga 26760

caggggtgcc aatgagcaga tcaaagcatg ggctgagcac aggtcacctc cctccagggc 26820

tcctgcttca ccccaccctg actccagaag gcaccatcta tctttgcttc ctctgcctcc 26880

ctgagttcaa gccaacgtaa ggagaggaac atgtagcaac ctggaatgga gcccaggcag 26940

caaggaggca aagagctggg caggagagcc cattactggg tagggtggtg tcaggaaggc 27000

tccctggtgg aggtggccag ataatcaaca gcaggcccgg ccgggtgtgg tggctcacgc 27060

ctgtaatccc agcacttcgg gaggccgagg caggcagatc acaaggtcag gagatcaaga 27120

ccatcctggc taacacggtg aaaccccgtc tctactaaaa atacaaaaaa ttagccaggt 27180

gtggtggcgg gcgcctgtag tcccagctac tcgggaggct gaggcaggag aatggtgcga 27240

acccaggaga cagagcttgc agtgagccga gatcgcgcca ctgccctcca gcctgggcga 27300

cagagcgaga c 27311

<210> 2

<211> 20

<212> DNA

<213> FLNA-F

<400> 2

tcattcgtgc ccaaggagac 20

<210> 3

<211> 20

<212> DNA

<213> FLNA-R

<400> 3

ctgaccagag acccgaacac 20

<210> 4

<211> 3855

<212> DNA

<213> GAPDH reference Gene

<400> 4

gctctctgct cctcctgttc gacagtcagc cgcatcttct tttgcgtcgc caggtgaaga 60

cgggcggaga gaaacccggg aggctaggga cggcctgaag gcggcagggg cgggcgcagg 120

ccggatgtgt tcgcgccgct gcggggtggg cccgggcggc ctccgcattg caggggcggg 180

cggaggacgt gatgcggcgc gggctgggca tggaggcctg gtgggggagg ggaggggagg 240

cgtgtgtgtc ggccggggcc actaggcgct cactgttctc tccctccgcg cagccgagcc 300

acatcgctca gacaccatgg ggaaggtgaa ggtcggagtc aacgggtgag ttcgcgggtg 360

gctggggggc cctgggctgc gaccgccccc gaaccgcgtc tacgagcctt gcgggctccg 420

ggtctttgca gtcgtatggg ggcagggtag ctgttccccg caaggagagc tcaaggtcag 480

cgctcggacc tggcggagcc ccgcacccag gctgtggcgc cctgtgcagc tccgcccttg 540

cggcgccatc tgcccggagc ctccttcccc tagtccccag aaacaggagg tccctactcc 600

cgcccgagat cccgacccgg acccctaggt gggggacgct ttctttcctt tcgcgctctg 660

cggggtcacg tgtcgcagag gagcccctcc cccacggcct ccggcaccgc aggccccggg 720

atgctagtgc gcagcgggtg catccctgtc cggatgctgc gcctgcggta gagcggccgc 780

catgttgcaa ccgggaagga aatgaatggg cagccgttag gaaagcctgc cggtgactaa 840

ccctgcgctc ctgcctcgat gggtggagtc gcgtgtggcg gggaagtcag gtggagcgag 900

gctagctggc ccgatttctc ctccgggtga tgcttttcct agattattct ctggtaaatc 960

aaagaagtgg gtttatggag gtcctcttgt gtcccctccc cgcagaggtg tggtggctgt 1020

ggcatggtgc caagccggga gaagctgagt catgggtagt tggaaaagga catttccacc 1080

gcaaaatggc ccctctggtg gtggcccctt cctgcagcgc cggctcacct cacggccccg 1140

cccttcccct gccagcctag cgttgacccg accccaaagg ccaggctgta aatgtcaccg 1200

ggaggattgg gtgtctgggc gcctcgggga acctgccctt ctccccattc cgtcttccgg 1260

aaaccagatc tcccaccgca ccctggtctg aggttaaata tagctgctga cctttctgta 1320

gctgggggcc tgggctgggg ctctctccca tcccttctcc ccacacacat gcacttacct 1380

gtgctcccac tcctgatttc tggaaaagag ctaggaagga caggcaactt ggcaaatcaa 1440

agccctggga ctagggggtt aaaatacagc ttcccctctt cccacccgcc ccagtctctg 1500

tcccttttgt aggagggact tagagaaggg gtgggcttgc cctgtccagt taatttctga 1560

cctttactcc tgccctttga gtttgatgat gctgagtgta caagcgtttt ctccctaaag 1620

ggtgcagctg agctaggcag cagcaagcat tcctggggtg gcatagtggg gtggtgaata 1680

ccatgtacaa agcttgtgcc cagactgtgg gtggcagtgc cccacatggc cgcttctcct 1740

ggaagggctt cgtatgactg ggggtgttgg gcagccctgg agccttcagt tgcagccatg 1800

ccttaagcca ggccagcctg gcagggaagc tcaagggaga taaaattcaa cctcttgggc 1860

cctcctgggg gtaaggagat gctgcattcg ccctcttaat ggggaggtgg cctagggctg 1920

ctcacatatt ctggaggagc ctcccctcct catgccttct tgcctcttgt ctcttagatt 1980

tggtcgtatt gggcgcctgg tcaccagggc tgcttttaac tctggtaaag tggatattgt 2040

tgccatcaat gaccccttca ttgacctcaa ctacatggtg agtgctacat ggtgagcccc 2100

aaagctggtg tgggaggagc cacctggctg atgggcagcc ccttcatacc ctcacgtatt 2160

cccccaggtt tacatgttcc aatatgattc cacccatggc aaattccatg gcaccgtcaa 2220

ggctgagaac gggaagcttg tcatcaatgg aaatcccatc accatcttcc aggagtgagt 2280

ggaagacaga atggaagaaa tgtgctttgg ggaggcaact aggatggtgt ggctcccttg 2340

ggtatatggt aaccttgtgt ccctcaatat ggtcctgtcc ccatctcccc cccaccccca 2400

taggcgagat ccctccaaaa tcaagtgggg cgatgctggc gctgagtacg tcgtggagtc 2460

cactggcgtc ttcaccacca tggagaaggc tggggtgagt gcaggagggc ccgcgggagg 2520

ggaagctgac tcagccctgc aaaggcagga cccgggttca taactgtctg cttctctgct 2580

gtaggctcat ttgcaggggg gagccaaaag ggtcatcatc tctgccccct ctgctgatgc 2640

ccccatgttc gtcatgggtg tgaaccatga gaagtatgac aacagcctca agatcatcag 2700

gtgaggaagg cagggcccgt ggagaagcgg ccagcctggc accctatgga cacgctcccc 2760

tgacttgcgc cccgctccct ctttctttgc agcaatgcct cctgcaccac caactgctta 2820

gcacccctgg ccaaggtcat ccatgacaac tttggtatcg tggaaggact catggtatga 2880

gagctgggga atgggactga ggctcccacc tttctcatcc aagactggct cctccctgcc 2940

ggggctgcgt gcaaccctgg ggttgggggt tctggggact ggctttccca taatttcctt 3000

tcaaggtggg gagggaggta gaggggtgat gtggggagta cgctgcaggg cctcactcct 3060

tttgcagacc acagtccatg ccatcactgc cacccagaag actgtggatg gcccctccgg 3120

gaaactgtgg cgtgatggcc gcggggctct ccagaacatc atccctgcct ctactggcgc 3180

tgccaaggct gtgggcaagg tcatccctga gctgaacggg aagctcactg gcatggcctt 3240

ccgtgtcccc actgccaacg tgtcagtggt ggacctgacc tgccgtctag aaaaacctgc 3300

caaatatgat gacatcaaga aggtggtgaa gcaggcgtcg gagggccccc tcaagggcat 3360

cctgggctac actgagcacc aggtggtctc ctctgacttc aacagcgaca cccactcctc 3420

cacctttgac gctggggctg gcattgccct caacgaccac tttgtcaagc tcatttcctg 3480

gtatgtggct ggggccagag actggctctt aaaaagtgca gggtctggcg ccctctggtg 3540

gctggctcag aaaaagggcc ctgacaactc ttttcatctt ctaggtatga caacgaattt 3600

ggctacagca acagggtggt ggacctcatg gcccacatgg cctccaagga gtaagacccc 3660

tggaccacca gccccagcaa gagcacaaga ggaagagaga gaccctcact gctggggagt 3720

ccctgccaca ctcagtcccc caccacactg aatctcccct cctcacagtt gccatgtaga 3780

ccccttgaag aggggagggg cctagggagc cgcaccttgt catgtaccat caataaagta 3840

ccctgtgctc aacca 3855

<210> 5

<211> 21

<212> DNA

<213> GAPDH-F

<400> 5

ggagcgagat ccctccaaaa t 21

<210> 6

<211> 23

<212> DNA

<213> GAPDH-R

<400> 6

ggctgttgtc atacttctca tgg 23

<210> 7

<211> 348

<212> DNA

<213> RPLP2

<400> 7

atgcgctacg tcgcctccta cctgctggct gccctagggg gcaactcctc ccccagcgcc 60

aaggacatca agaagatctt ggacagcgtg ggtatcgagg cggacgacga ccggctcaac 120

aaggttatca gtgagctgaa tggaaaaaac attgaagacg tcattgccca gggtattggc 180

aagcttgcca gtgtacctgc tggtggggct gtagccgtct ctgctgcccc aggctctgca 240

gcccctgctg ctggttctgc ccctgctgca gcagaggaga agaaagatga gaagaaggag 300

gagtctgaag agtcagatga tgacatggga tttggccttt ttgattaa 348

<210> 8

<211> 294

<212> DNA

<213> RPL37

<400> 8

atgacgaagg gaacgtcatc gtttggaaag cgtcgcaata agacgcacac gttgtgccgc 60

cgctgtggct ctaaggccta ccaccttcag aagtcgacct gtggcaaatg tggctaccct 120

gccaagcgca agagaaagta taactggagt gccaaggcta aaagacgaaa taccaccgga 180

actggtcgaa tgaggcacct aaaaattgta taccgcagat tcaggcatgg attccgtgaa 240

ggaacaacac ctaaacccaa gagggcagct gttgcagcat ccagttcatc ttaa 294

Claims (3)

1. The application of the platelet molecular marker for lung cancer in preparing a lung cancer detection kit is characterized in that the base sequence of the platelet molecular marker is shown as SEQ ID NO. 1;

a lung cancer detection kit comprising an upstream primer and a downstream primer of the platelet molecular marker;

base sequence of FLNA-F (5 'to 3'): TCATTCGTGCCCAAGGAGAC

Base sequence of FLNA-R (5 'to 3'): CTGACCAGAGACCCGAACAC;

according to the application of the lung cancer detection kit in preparing a diagnosis kit for diagnosing lung cancer and/or distinguishing benign and malignant nodules, the method comprises the following steps:

1) Extracting total RNA of a platelet sample to be detected and normal tissues, and synthesizing cDNA by reverse transcription;

2) Using cDNA as a template, performing FLNA gene amplification by adopting fluorescent quantitative PCR under the guidance of the upstream primer and the downstream primer, and amplifying GAPDH reference genes, and simultaneously detecting fluorescent values and CT values of the FLNA gene and the GAPDH gene;

3) The relative quantification of GAPDH was used to calculate the expression level of the molecular markers.

2. The use of a platelet molecular marker for lung cancer according to claim 1 in the preparation of a lung cancer detection kit, wherein the kit further comprises GAPDH internal reference gene primers;

base sequence of GAPDH-F (5 'to 3'): GGAGCGAGATCCCTCCAAAAT

Base sequence of GAPDH-R (5 'to 3'): GGCTGTTGTCATACTTCTCATGG.

3. The use of the platelet molecular marker for lung cancer according to claim 1 in preparing a lung cancer detection kit, wherein the fluorescent quantitative PCR reaction conditions are set: pre-denaturation at 95 ℃ for 30s, denaturation at 95 ℃ for 5s, annealing at 60 ℃ for 30s, 40 cycles total, and collecting fluorescent signals; a dissolution profile analysis was performed at 60-95℃to determine the identity of the amplified product.