CN108315430B - Application of PDL1SNP genotype as marker for predicting neoadjuvant chemotherapy curative effect of breast cancer - Google Patents

Abstract

The invention provides application of PDL1SNP genotype in preparing a marker for diagnosing and predicting the new auxiliary chemotherapy curative effect of breast cancer. Also provides application of PDL1SNP genotype as a marker in preparing a reagent for diagnosing and predicting the curative effect of breast cancer neoadjuvant chemotherapy. Also provides a kit for detecting PDL1SNP genotype, which contains primers shown in SEQ ID NO. 1-18. The invention finds that after the PDL1 gene non-coding region SNP rs4143815C is changed from G in all breast cancer populations and HR positive populations, a GC genotype patient can more easily reach pCR than a GG and CC genotype patient. Through the detection of SNP of a PDL1 gene non-coding region in the blood of a patient, a GC genotype patient is discovered as early as possible, and neoadjuvant chemotherapy is carried out, so that the complete remission of pathology is achieved, and the survival time is prolonged.

Description

Application of PDL1SNP genotype as marker for predicting neoadjuvant chemotherapy curative effect of breast cancer

Technical Field

The invention belongs to the field of medical detection, and relates to a marker for predicting the neoadjuvant chemotherapy curative effect of breast cancer, in particular to application of PDL1SNP genotype as a marker for predicting the neoadjuvant chemotherapy curative effect of breast cancer.

Background

Breast cancer is one of the most common malignancies in women worldwide. According to the latest global epidemiological data (GLOBOCAN), there are 167 thousands of new breast cancer cases worldwide and 52.2 thousands of people die from breast cancer in 2012 only. The age-standardized incidence rate of the breast cancer in China in the same year reaches 21.6 ten-thousandth. The incidence of breast cancer in China is still lower than that in developed countries (up to 100 out of ten thousand in countries such as North America and North Europe), but has been on the rise from the eighties of the last century, and the growth rate is more than 2 times of the average level in the world. In economically developed areas such as the southeast coast of China, breast cancer has become the first malignant tumor of female morbidity.

The high incidence of the disease attracts the attention of medical workers on breast cancer all over the world. For years, the treatment mode of breast cancer has gradually changed from single surgical treatment in the nineteenth century to the comprehensive treatment mode combining the prior surgery, chemotherapy, radiotherapy and targeted therapy. Among them, Neoadjuvant chemotherapy (NAT) for breast cancer has also become a routine item for breast cancer treatment. Neoadjuvant chemotherapy can not only reduce tumor burden and provide surgical and breast preservation opportunities for patients with locally advanced stages, but also can evaluate the responsiveness of patients to treatment at an early stage, so as to adjust subsequent treatment and predict the long-term prognosis of patients. Complete remission of pathology (pCR) of neoadjuvant chemotherapy refers to the primary foci of the post-treatment surgical specimen, or primary foci plus axillary lymph nodes, with no tumor cell or only intraductal cancer components remaining. Numerous large clinical trials have demonstrated that the prognosis for patients with pCR in NAT is much better than for patients without pCR. However, not all patients can achieve pCR results on the same NAT treatment, and therefore, finding a predictor of pCR becomes a focus and focus of the current phase of research on how to predict early patient response to NAT.

Programmed death-ligand 1 (PDL 1) is a transmembrane glycoprotein of cell membranes. The protein is mainly expressed on the cell membrane surfaces of T cells, NK cells, dendritic cells and tumor cells, and the functions of the protein are mainly related to immunosuppression. Programmed death 1 (PD 1) is mainly expressed on the surface of T cell membrane, and can induce T cell apoptosis and induce T cell differentiation to inhibit T cell after being combined with its ligand PDL 1. Congenital PDL1 expression deficiency is associated with autoimmune diseases, such as systemic lupus erythematosus. Acquired alterations in PDL1 expression correlate with immune escape from the tumor.

Basic experiments suggest that PDL1 is regulated and affected by a variety of cellular pathways, such as the MAPK and PI3K/AKT pathways. In epigenetic aspect, PDL1 is also regulated by many mirnas (Micro-Ribonucleic acids), and thus participates in tumor development processes such as Epithelial-Mesenchymal Transition (EMT). In addition, in the research of gastric cancer and lung cancer, Single Nucleotide Polymorphism (SNP) of PDL1 non-coding region has been reported to be related to tumor onset risk and prognosis.

Unlike gene mutation, a certain mutant base at a site needs to reach a certain frequency of occurrence (greater than 1/1000) in a population to be called as SNP. SNP changes in people are often related to disease onset, drug metabolism and curative effect. SNP sites can be classified into coding region SNPs and non-coding region SNPs according to their distribution on the genome. Coding region SNPs can be divided into synonymous substitutions and non-synonymous substitutions. Both can ultimately affect protein function by either directly altering the protein product or affecting the translation process, respectively. While SNPs in non-coding regions are not involved in protein synthesis, they can affect cellular functions by affecting various biological processes such as gene splicing, transcription factor binding, and mRNA degradation. Non-coding region SNP mutations are often reported to be associated with tumor pathogenesis. The clinical value of PDL1SNP in gastric cancer and lung cancer is just represented by non-coding region SNP.

Preliminary results have been obtained in our new adjuvant clinical trials (SHPD001 and SHPD002) with paclitaxel in combination with cisplatin peritherapy, showing higher pCR rates. Among them, the pCR rate in the total population is 34.4%, and is more than 64.7% and 52.4% in the positive type and triple negative type of Human epidermal growth factor receptor 2 (HER 2) breast cancers, and the therapeutic effect is generally considered to be related to metronomic chemotherapy and immunomodulation. However, in Hormone Receptor (HR) positive patients, pCR rate is less than ideal, so we would prefer to find a predictor of pCR in this part of patients, thereby improving the therapeutic effect. The literature suggests that cisplatin can increase the expression level of PDL1 in tumor cells, and the medicine is also one of the chemotherapy medicines adopted by patients in clinical trials of NAT in the family I.

Disclosure of Invention

The invention aims to provide application of PDL1SNP genotype as a marker for predicting the efficacy of neoadjuvant chemotherapy for breast cancer, and the application aims to solve the technical problem that no appropriate biomarker is available in the prior art for predicting the efficacy of neoadjuvant chemotherapy for breast cancer patients.

The invention provides application of PDL1SNP genotype in preparing a marker for diagnosing and predicting the new auxiliary chemotherapy curative effect of breast cancer.

Further, rs4143815C > G in the PDL1SNP genotype.

Furthermore, the breast cancer is hormone receptor positive.

The invention also provides application of the PDL1SNP genotype as a marker in preparing a reagent for diagnosing and predicting the curative effect of breast cancer neoadjuvant chemotherapy.

The invention also provides application of the PDL1SNP gene as a marker in preparing a reagent for diagnosing and predicting the curative effect of breast cancer neoadjuvant chemotherapy, wherein the sequence of the PDL1SNP gene is shown as SEQ ID NO. 20.

The invention also provides a kit for detecting the PDL1SNP genotype, which contains the primers shown in SEQ ID NO. 2-19.

According to the invention, by detecting the SNP genotype in the whole blood sample before the patient newly assists chemotherapy, if the change of the SNP rs4143815C to G of the non-coding region of the PDL1 gene is found, the patient is more easily to reach pCR; thereby predicting the patient's sensitivity and tolerance to treatment. GC genotype patients are more accessible to pCR than GG and CC genotype patients; however, patients with GG genotype are more prone to DFS events and have poorer survival than those with GC and CC genotypes.

Compared with the prior art, the invention has remarkable technical progress. The invention discovers that after the SNP rs 4143815C-G of the PDL1 gene non-coding region is changed in all people and people with positive HR, a GC genotype patient can more easily reach pCR than GG and CC genotype patients; however, patients with GG genotype are more prone to DFS events and have poorer survival than those with GC and CC genotypes. Through the detection of SNP of a non-coding region of PDL1 gene in the blood of a patient, a GC genotype patient is discovered as soon as possible, so that new adjuvant chemotherapy can be carried out, so that the complete relief of pathology can be achieved, and the survival time is prolonged. If the new adjuvant chemotherapy is found to have poor effect, other means such as operation and the like are adopted, so that excessive treatment is avoided, and the operation time is not delayed.

Drawings

FIG. 1 shows disease-free survival time for patients with different PDL1SNP genotypes; log-rank P value is 0.01.

Fig. 2 shows the disease-free survival time, Log-rank p 0.003, for patients with different SNP genotypes in the HR positive subgroup.

Detailed Description

Example 1

In order to clarify the prediction effect of the SNP genotype of the non-coding region of the PDL1 gene (shown as SEQ ID NO. 1) on the neoadjuvant chemotherapy sensitivity of the breast cancer, in particular to clarify the prediction effect of the SNP genotype in HR positive breast cancer; at the same time, patients of different genotypes were observed for tolerance and safety to treatment. We hypothesized that PDL1SNP genotype can predict patient sensitivity and tolerance to treatment in all populations and HR positive breast cancers, and sensitivity analysis is performed by detecting SNP genotype in whole blood samples of patients before neoadjuvant chemotherapy.

1 materials and methods

1.1 Main Instrument

1.2 major kit consumables

1.3 grouping patients and specimen Collection

All enrolled cases were from patients enrolled in two clinical trials of the mammary gland surgery of the affiliated economic hospital of the Shanghai university of medical school. Two clinical trials were registered as SHPD001(NCT02199418), SHPD002(NCT02221999) on the clinical trials. gov website, respectively. Both groups received a one-week neoadjuvant chemotherapy regimen of paclitaxel in combination with cisplatin, with SHPD002 patients randomized to endocrine therapy.

And (5) reserving a whole blood sample of the patient before treatment for subsequent SNP detection. And (3) finishing and counting the basic information and tumor data of the patient, including the age, the menstrual state, the family history, the primary tumor size before the operation, an estrogen and progestogen receptor of the puncture specimen, an HER-2 receptor of the puncture specimen, adverse reactions during chemotherapy and PCR after chemotherapy. Both clinical trials were approved by the ethical committee of the affiliated renji hospital of the medical college of Shanghai university of transportation, and all patients signed informed consent to voluntarily enter the group and provided specimens and clinical data for relevant medical studies.

1.4SNP site selection

The SNP sites detected were selected by the public database (NCBI/TargetScan) according to the criteria of A. rare allele Frequency (MAF) >0.05 and B. located in the 3' UTR region of PDL1 gene, or in other non-coding regions of PDL1 gene but reported to have predictive or prognostic value in other tumors. A total of 9 SNP sites were selected. The DNA extraction and SNP genotype detection of the specimen are all completed by Shanghai deep Zhi Biotech company.

1.5DNA extraction: the method was carried out using a TIANAmp Genomic DNA Kit.

A. Mu.l of the protease K solution was added to the sample blood (200. mu.l) and mixed well.

B. Add 200. mu.l buffer GB to the mixture, 10min at 70 ℃.

C. Add 200. mu.l 100% ethanol into the mixture obtained in the above step, shake for 15 s.

D. The solution was put into an adsorption column CB3, centrifuged at 12000rpm for 30s, discarded, and the adsorption column CB3 was placed in a collection tube.

E. To adsorption column CB3, 500. mu.l of buffer solution was added, centrifuged at 12000rpm for 30 seconds, and the waste liquid was discarded, and adsorption column CB3 was put in a collection tube.

F. 600. mu.l of rinsing solution PW was added to adsorption column CB3, and the mixture was separated at 12000rpm for 30 seconds, and the waste liquid was discarded, and adsorption column CB3 was placed in a collection tube.

G. And F, repeating the operation step.

H. The adsorption column CB3 was put back into the collection tube, centrifuged at 12000rpm for 2min, and the waste liquid was discarded. The adsorption column CB3 is placed at room temperature for a plurality of minutes and dried.

I. Placing the adsorption column CB3 in another clean centrifuge tube, suspending and dripping 100 μ l of elution buffer TE into the middle of the adsorption membrane, standing at room temperature for 2-5min, centrifuging at 12000rpm for 2min, and collecting the solution in the centrifuge tube.

1.6PCR reaction

The primers described below were designed on-line by Assay Design Suite V2.0 Online.

The primer sequence is as follows:

A. in the case of 384-well plates, 1. mu.l of DNA sample was added to each well and centrifuged for use.

B. PCR was carried out using Qiagen kit, and the reaction system was as follows:

C. add 4. mu.l of reaction system to each well of DNA sample, put 384 well plate into PCR instrument, the reaction program is as follows:

after the PCR was completed, 384 well plates were ready for use.

1.7SAP treatment

A. SAP reaction liquid preparation is carried out by using a SEQUENOM kit, and the system is as follows:

B. mu.l of SAP reaction was added per well after the PCR reaction in 384 well plates.

C. The SAP procedure was performed as follows:

after completion of SAP, 384 well plates were ready for use.

1.8 elongation reaction

A. An iPLex reaction system is prepared by using a kit of SEQUENOM company, and the reaction system is as follows:

B. mu.l of extension reaction was added per well in the 384 well plate SAP post reaction system.

C. The extension procedure was performed as follows:

D. after the extension is completed, the 384-well plate is ready for use.

1.9 purification of the product

A. Placing a little resin on 384-hole resin scraper, scraping off excessive resin to uniformly cover, and standing at room temperature for 20 min.

B. The extended 384 well plates were centrifuged at 1000rpm for 1min, 25. mu.l of deionized water was added to each well, and the plates were inverted on a resin plate. The resin plate was reversed, and the resin was dropped into a 384-well plate to seal the membrane. C. The 384-well plate is turned over for 20min by taking the long axis of the 384-well plate as the axis, and centrifuged for 5min at 3500rpm for standby.

1.10SNP genotype detection

A, spotting a nanodispenser SpectroCHIP chip;

the test samples were transferred from 384-well reaction plates to MassARRAY SpectroCHIP chips on a surface-coated substrate.

Mass spectrometric detection of MassARRAY Analyzer Compac;

and transferring the sample to a SpectroCHIP chip, and then putting the chip into a mass spectrometer for detection, wherein each detection point only needs 3-5 seconds, and full-automatic analysis is carried out.

And C, analyzing an experiment result by TYPER software to obtain typing data.

1.11 statistical analysis

The hardy-weinberg balance of SNP genotypes was done by the chi-square test. Linkage disequilibrium analysis between different SNPs was done by HaploView software (http:// broad. mit. edu/mpg/HaploView). When SNP genotypes are analyzed, four models are built in total: assuming that "T" is a common allele and "a" is a rare/mutant allele at a certain SNP site, the dominant model compares the differences of TT vs TA + AA; comparing the differences of TT + TA vs AA by the recessive model; the co-dominant model compares the differences of TT + AA vs TA; the additive model compares the differences in TT vs TA vs AA.

Calculating the correlation between PDL1SNP position and side reaction by Spearman correlation analysis; the correlation between different genotypes and pCRs of PDL1SNP was analyzed by logistic regression model for single and multifactorial analysis. The present invention uses Disease Free Survival (DFS) for survival assessment, defined as the surgical removal of a lesion to the occurrence of any of the following events: 1. distant metastasis; 2. local recurrence in the ipsilateral region; 3. contralateral breast cancer; 4. and death. The survival curves between different groups are compared and drawn by a Kaplan-Meier method, the survival rate difference is compared by log-rank test, and the COX risk model is subjected to multi-factor adjustment and HR and 95% CI calculation.

The data are analyzed through STATA 12.0 and SPSS 23.0, and the statistical difference of p <0.05 on two sides exists.

2 results of the experiment

2.1 predictive value of PDL1SNP expression

The invention totally detects 9 SNP sites of the non-coding region of the PDL1 gene, and the position and the genotype frequency of each site are shown in Table 1. Two sites, rs7864231 and rs10217310, have not been analyzed subsequently in the text because no mutants can be detected. The remaining 7 sites were tested and were in Hardy-Weinberg equilibrium and were included in subsequent analyses. By multifactorial logistic regression analysis, we found that additive models (OR1 ═ 9.284, p1 ═ 0.049; OR2 ═ 13.501, p2 ═ 0.023) and co-dominant models (OR ═ 2.800, p ═ 0.042) of PDL1 at a non-coding region SNP site, rs4143815C to G change (rs4143815C > G), were associated with the patient's new adjuvant therapy pCR (table 1). By further calculation, the dominant model of this change was also associated with PR expression (p ═ 0.043).

TABLE 1 relationship between different SNP genotypes and pCR

a: p <0.05, with statistical differences.

b: multifactorial analysis adjusted age, tumor size, ER, PR, HER2, and ki 67.

2.2 prognostic value of PDL1SNP expression

At 18 months of median follow-up, patients presenting rs4143815C > G (shown in SEQ ID NO. 20) exhibited different DFS. Among them, Kaplan-Meier plot shows that the patients with GG genotype survived worse than those with GC + CC genotype (Log-rank p ═ 0.01; FIG. 1). The COX multi-factor risk model also suggests that the risk of relapse is higher in rs4143815GG genotype patients (HR 20.098, 95% CI 2.774-145.596; p 0.003; see table 2 for details), an independent predictor of DFS. In addition, the patient's age (HR ═ 0.125, 95% CI 0.024-0.656; p ═ 0.014), ki67 status (HR ═ 1.050, 95% CI 1.015-1.088; p ═ 0.005), and post-operative lymph node status (HR ═ 9.750, 95% 1.894-50.194; p ═ 0.006) are also independent predictors of DFS.

TABLE 2 PDL1SNP genotype vs. survival relationship (COX multifactor risk model)

a: p <0.05, with statistical differences.

2.3 analysis of side reactions

Side reaction analysis was performed on patients with different rs4143815 genotypes, and it was found that neutropenia (rho-0.183, p-0.032) and edema (rho-0.179, p-0.036) were more likely to occur in CC + CG compared to GG patients. CC + GG is more likely to cause elevation of aspartate aminotransferase than CG patients (rho-0.186, p-0.030). The specific spectrum of adverse reactions is shown in Table 3.

TABLE 3 adverse reactions in patients with different rs4143815 genotypes

Table 3Adverse events according to different genotype of rs4143815

a: p <0.05, with statistical differences.

2.4 subgroup analysis

In a subgroup analysis, we observed similar conclusions as the total population in HR positive breast cancer.

Multifactorial logistic regression analysis in HR positive breast cancer showed that patients with a GC genotype of rs4143815C > G are more susceptible to pCR than GG + CC (OR ═ 2.362, p ═ 0.022; table 4) in predicting pCR.

In terms of survival, Kaplan-Meier plots in HR positive breast cancers show that GG genotype survival is inferior to GC + CC group patients (Log-rank p 0.003; fig. 2). The COX multifactorial risk model suggests that patients with rs4143815C > G have a higher risk of relapse for GG genotype than GC + CC (HR 10.781, p 0.008; table 4). No predictive value of PDL1 was observed in HER2 positive BC, and no effective subgroup analysis was performed due to fewer TNBC breast cancer patient cases.

TABLE 4 relationship of PDL1SNP genotype to pCR and DFS in HR positive subgroups

a: p <0.05, with statistical differences.

3 conclusion

We discuss in the present invention the predictive value of different genotypes of the patient PDL1SNP for neoadjuvant chemotherapy pCR and the prognostic value of the long term. The method is the prediction and prognosis value of the novel adjuvant chemotherapy for reporting the PDL1SNP genotype in breast cancer patients for the first time, and is also the prediction and prognosis value of the PDL1SNP genotype in HR positive breast cancer for the first time. The relation between the PDL1SNP genotype and the side effect of the new adjuvant chemotherapy is also observed for the first time.

We found that in all people and the people with positive HR, after the SNP rs4143815C to G of the non-coding region of the PDL1 gene is changed, the GC genotype patients can more easily reach pCR than the GG and CC genotype patients; however, patients with GG genotype are more prone to DFS events and have poorer survival than those with GC and CC genotypes.

Rs4143815 is located in the non-coding 3' UTR region of the PDL1 gene. SNPs in non-coding regions, although not involved in protein synthesis, can affect cellular functions by affecting a variety of biological processes such as gene splicing, transcription factor binding, and mRNA degradation. The invention finds the clinical prediction value of the locus rs 4143815. In one aspect, a genetic polymorphism change at this site causes a change in the expression of PDL1 protein, thereby affecting cell function. Under the induction of IFN gamma, the expression of PDL1 protein on the surfaces of macrophages/monocytes in an rs4143815CC genotype carrier is far higher than that of an individual with a GG genotype.

Sequence listing

<110> Shanghai university of traffic medical college affiliated renji hospital

Application of PDL1SNP genotype as marker for predicting breast cancer neoadjuvant chemotherapy curative effect

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ccctacttca acataaactg caaaaggaga gtgacttttc tttcactctg acttccgtat 4800

tcctcatgct taaaatagtg cctagcacag aagaggtgct caatcagtgt ttgctaaacg 4860

aaataattag tcacatttca agcaggatga ctaaatgaag aatagaatct aggcagatac 4920

tctggaagag tggctgtgag tcattcatat cttagtatga attagtcaaa tccaactctc 4980

tccccttccc actccccact gttagtagaa gaatctgttt attgagagaa tagatttata 5040

atttagaata agtgagaggg gcagaagagg agattttgaa ggatggcacc tgaaggagga 5100

ctagcatggc tgagacagtg aagtggaagc cttgaatagc taaagggtaa gatgaaagta 5160

tttagctgta gggggaaaaa gcattgacag gttggaaaag taaaagtcag attctccttg 5220

ctctgaaatt ttgtacaggg caggttctac taggtatgtt acaatgcaga aaaaacatga 5280

aataattgag aggaatttgg tgcaatatta tcttcttggc ttcttttgag tgggcagatt 5340

tttttcacgg cctgtaacta taataaattt gaaacttctc atcttttagt aacttttttc 5400

acttaagttt atgtggctgt gggcaatgga atgaagatat tgaacttcca attccctgtt 5460

gggtttccac aattacaagt caatcatgac tggttattag aagactattt cagttagaac 5520

caccaagtcc catattgtca tattgtatgt ttaattatta agtgaagcag tcttcttttc 5580

gtgttttcca taattagggc attccagaaa gatgaggata tttgctgtct ttatattcat 5640

gacctactgg catttgctga acggtaagac accaaatcct tccattaggt tctatatttt 5700

aaatatttta accatgagtt taaaactaaa atgatcattt aaaatgcatg caattttctt 5760

atagagagaa cattctattc tttcttctac tttacacaat ggcaaagtct tctttctact 5820

ttacgcaatg ataaagttac ctgtgtcatt ttgtaaaaat atagagaata tagacaaatt 5880

gaaagacaca aaataatcta ttacccattt cccagggtta actactgaaa atatctgggg 5940

aaatggcctg tatgtataca tttatttgtt tgctttcaac aaggccaaga tcctttgatc 6000

tttcagtctt ggttgctctg tgacatgcct ttcctgatga ggatacttta aggaagaatt 6060

gtaagataca tggaaaatgt caggctaaca cagtactggc atcaccctgt gctctttcct 6120

gaactccata ccaatgtact tcttgccaga aaactgatca aaagtttagg gaagtaaaaa 6180

gagatgactg ttagaatcta ccattccctc tatgtaggaa gcaaataggt gtcctgtcaa 6240

aggacattct ggggatgtct acatgaaacc aagtctccct ggttgtaagg actccatctc 6300

catataatat ttatacagta atatatgttt ataaattgtg ggggcaactt gtttagctaa 6360

ttttattatt ctgctattgg gacactgtgt ctcagcatga gatatagtgt cccaaaacat 6420

atttcaagcc cattggataa aatatgtgtt tagcaagttc ttaaatataa tgataacata 6480

accgaccaga taaagtgatt tataaacgct gtgccaattt tgtaaatgtt tcgaggaatt 6540

ttcccttttc tgaagattgt ccttctttct ttttagcatt tactgtcacg gttcccaagg 6600

acctatatgt ggtagagtat ggtagcaata tgacaattga atgcaaattc ccagtagaaa 6660

aacaattaga cctggctgca ctaattgtct attgggaaat ggaggataag aacattattc 6720

aatttgtgca tggagaggaa gacctgaagg ttcagcatag tagctacaga cagagggccc 6780

ggctgttgaa ggaccagctc tccctgggaa atgctgcact tcagatcaca gatgtgaaat 6840

tgcaggatgc aggggtgtac cgctgcatga tcagctatgg tggtgccgac tacaagcgaa 6900

ttactgtgaa agtcaatggt aagaattatt atagatgaga ggcctgatct ttattgaaaa 6960

catattccaa gtgttgaaga cttttcattc ttgtaagtcc atacttattt tcaaacagaa 7020

cagcatagtc tgttcattca ttcattcaat tcatgaattc attcacataa ttatccaatt 7080

tcttgagcac ctatttgata gtcactggaa atccagagac aaacaacaca gagccatgtt 7140

ctacagtatg tacagttttc caaaaagaat ttctagtctt tactttttta ttacaaatgg 7200

aatacgtata cttgcaaata attcagatac tgtggaagag atcaaatgaa ttgcaaaagt 7260

gtccctcctc ccttcaccac tatctcccat ggcatgcaga gagagtaacc attatttgtg 7320

tgtccctcca gaaatttttt tattcaacta ctattttttt attttattag gtccgtcagt 7380

tttccttttt tgagcctctc tatatcaaat gcaaataaat atattcagaa caaaccccac 7440

tgtaaggttc acattaaaaa agacttgaag tcaccctatg aagacaaaaa ataatcacat 7500

taagtgtgaa agaacctatt cttccagtac aggataagcc atacttactg ggcatatatt 7560

catcttgaaa atctatactg atgttgtctt ggggaattga aaaggaacta ggagtgttag 7620

ttcctcggta ttgacccaca gttatgttat caggtcactt gagttcaaag ttttgtgttg 7680

gcactagcta agtaaaggaa aacacctctg ctttcattgt tgagtttcac agaattgaga 7740

gctgaaagga tcccaggcag gagcagctaa tccaaactcc cacaaagaac aaaaatcccc 7800

cagaggatct tctgttctta tatttcctgc aatggcgtcc ctgtcatatc ccacaatggc 7860

ctccctgcca tttggatatc ccttccatat cctgttgaaa ttactcccta atagtaagct 7920

gaaatctgcc cctctagttg tagtcttggg attatttcat ttacatgatg accttttaat 7980

atttgactag aattaaatca tctccccttg gtctttccat tcctgggcta actaccatca 8040

atctgagggc taacaataca agtagaaaaa gtatacattt gtcactgatc actgatcaat 8100

tattaatcaa tgatcactga taactataaa ctcaaaaaca aaatcatgtg gggattaaga 8160

gaaatgtatc agttttatgt tgtatttctg gtccctgata ctggctcagg taatgccact 8220

attgtcaaga agataccact tgtaaagtag atttaatttt cattatattt taccatatgc 8280

ttctccattc atgacatctc ttgagatgtt gtggtttata ctttcagttt ttctccagtc 8340

catccgcaaa tatcaggcat ctactgtgtt ccaagatatt aaagaaatca tcatgactta 8400

gcctcatcaa cagcattgct agatctggga tggaaaggaa gagtataatc ctggcagtca 8460

ggaagaaggc agcataaagt ataagtttct gcttccaaaa aaggtctctc atcagcctgt 8520

agggagtgtg tagggaaggg acagctgtcc ttgtagtagg gaagggtttt attcaggtcg 8580

tctgggctcc ataatatccc ttgtgtatct gcagtctcct ttgccatgga tcaacacaat 8640

aggaaatctt ccggcactga tggtttttcc aagggggagt tcttcctgga gcaaagcaaa 8700

tgaccaacca ggtttgagga cctgatttgt ttgacaattc cattttgtat tgtaaattac 8760

ttaattggca ttctactccc aatccatctt gtcatttgca tacagtggtt ttgggattga 8820

gttcagctat accaaaagtc tgaaccttct gcacttagaa caaggcaacc accaagcttc 8880

acttgcactg aggccgtgtc tccaatggaa atgaggcagc tggcttgcag gagcttccca 8940

actcagggaa gtagaactcc tgagtcacct ccatatgcaa atgatttcac agtaatgctg 9000

ttgaacttca cttcccatca cagcaaatgt gtggtaacat agcttcccca caggagttta 9060

ctcaccatgg tattttaaag gtgaaacatt tcaaaactga aatttgaaag aatttagttt 9120

tggattcact caattatcac tatcacttcg ggtgttattg cacctttctt gtttgtgagt 9180

ttaaatgcca gactctcagg ccactaactt tcaattaaaa gtgtttttct ttaatcgctg 9240

aacctaacag cagggaaaac gaaatgttca ttcagacttt cagaaccttc aatgagatta 9300

ggcagctgaa agatcaaagt gttgcatagt tgtcccgata aagctatttg gatcatatgg 9360

accaaatcga ctgctgtcat tccccaccaa ccccatctct ccccaaaatt cccagccctg 9420

tttaagtgtt ctctgtagca tttatctcta tctagtatat tgtgtagcat atcatatcat 9480

acttttctgt tttgtttatt gtctctctcc tcctagaata taaactccac aagcacaaag 9540

atttgggcct gttttataat attgttgcat ccccagggcc tgatatacag cagagtggtg 9600

gtacgaaaag agcacacaaa aaaatatttg ttgagtcaat gaatgaatga tttcctcaaa 9660

taggattagc ctaaaatttt ggaaacatga acagatttgg atatgtgaaa atttatttcc 9720

agactgttca tcaggaactg ttagcagctt ctaaagggta cactggagca gcagtagtaa 9780

aaggaggaag aggagcagct ctgctactgc tactatcgag tactactaca attagcactt 9840

gcttattctg tgtgttaggc cctgtactga acactctgtc taaattagtt catttcctcc 9900

tggaaatgac tctagggggt aagtgcttca tcatgtaaga tgagtatttt tcacattttg 9960

ttgtgtctga aatctgagtg tgtctttcaa tgatggaatc tttgattcca tgataagtgg 10020

tattattccc attttaagga tgaggaaact gaggtccaaa gaaattaagt aatttgccca 10080

aattcaccca gcctagaaaa tgataaagct agttctaaac ccaagcagat tagctctgaa 10140

gtctgggccc ttaataacca ctttttattg cctatatttg tacctctggt gtacgtatca 10200

agttatatgt tgacttcaaa actatcatga ccttttcttg gttttgattg tccaacatta 10260

gtatagtgtt ctgggtctgc aaaaattttg attactcatc tcatctgtaa aacattttga 10320

actcgtgtgt ttgtgcatgc acatttgtgt gtaattataa aaattttact ttctgttaat 10380

atataagttg tatcataaga aactgccgtt tttgaagagc aaaaaaaggt tgaatgttac 10440

cagttacatc tggttcaacc taatagacat ttgtacaaaa acagacattt taagaggttg 10500

aaataaaaat ttaataaaca atattttcag tttttactaa ttgtgatgct tcactatcat 10560

tagctaatat gtcaaggcat aatatacctt agggtgaact ttatcattaa caaaggtgga 10620

tggtgtcaat aatcttgagg tttgtgtttt tttatataac actgcgaggt ctaattaagt 10680

acttactgtt taccacctca tacagtggcc gataaaaagt gtcacttctg ctgtttcctc 10740

tgggttgtgc ttgaattatt agtattatct tcagtcctca gtttctttgt gggaaacttt 10800

ttaattagtt gtttaatttt gtaagatggt tagtttagtc aaaattagat aagagaattt 10860

gaaaatccgt agctacccca aagcaaccta cacataagaa ctattatttt tgtgttttga 10920

aatcataatt ttattgattt ccagtgtttc cactggtagt ggtttcattg atataggagt 10980

atcaaaacat cactcattat ttatttcagt ttcatttgat cctagccgtt ttgtattaac 11040

tctctgtgaa gaaattacct cacaaatcta ttgctgtcct tggtaaagga atggagaatt 11100

aaggctctag atcattagtg gttacactat agtattagaa gtaaaaaaaa gattatacca 11160

acaaaataag aacatgttaa tgtacttgta atgaataaac atgaataaag ctcttatgct 11220

atataggtgc actaaacaat ctactagaat tgtcagcaaa ctacgtatct taatcctgaa 11280

agggtcccaa accaatgatc taaaattgaa tcaaactttc ttccttgagc ataattactt 11340

aaatgattta ttaaaatagc cagcatttaa aagcttaaaa tgtaaatatc ataatgtggt 11400

atcctagata gcatcccaga acagaaaaag gatattaggg aaaaactgga ggaatggaat 11460

aaattatgca gtttagttat taataatgta ctaacgtcct tagttatgac gattgtacca 11520

tggtaatgta agatactaac aatagaggaa accgggtaag gagtatacag taactctata 11580

ctatctttgc aacttttttg taaatttaaa acttctaaaa taaagaacaa atttaaacat 11640

taaaaagtat caccaggaac atatatcact gtttacagat gaaatactat gtattttcat 11700

atctaatttc tgatcattga cttcaaatca gaaaagtgaa tgacacctca aaatcaggtt 11760

ttctgtttac tgaagtctaa gaaaagaaag cataccagct ggagagattc atgtttataa 11820

agacagattt ataacaacaa aaataaaata tccaagaata aatttaagaa gaagcacttt 11880

actgagaaac atatgaaaac ctgaacaaat ggagagggat attttgtatt tgaatagaaa 11940

gacttctggt ttaaagataa ttctctttaa attatttttt gtagaaattt aaggggtaca 12000

agagcagtgt tgtcacatgg atatattaca tagtggtgaa gtctggggtt ttagtgtaaa 12060

ttaatcttta cattttgttt gagcccaata aatgtaccaa catgattttt atagaaagat 12120

agtcattcct attaatccaa acttgtccca actttgaatt gaattgaggc agagctagca 12180

ggtgttcccc acggctgagg catctgaaca ttaagcatat ccctctgaga accagcctgc 12240

attgatactc tttctaatgt ggacagcatc aagctatgta cgtagttctg tgctcagcaa 12300

aagccctgac ttctttttgt ttatgtccta gccccataca acaaaatcaa ccaaagaatt 12360

ttggttgtgg atccagtcac ctctgaacat gaactgacat gtcaggctga gggctacccc 12420

aaggccgaag tcatctggac aagcagtgac catcaagtcc tgagtggtaa gaccaccacc 12480

accaattcca agagagagga gaagcttttc aatgtgacca gcacactgag aatcaacaca 12540

acaactaatg agattttcta ctgcactttt aggagattag atcctgagga aaaccataca 12600

gctgaattgg tcatcccagg taatattctg aatgtgtcca ttaaaatatg tctaacactg 12660

tcccctagca cctagcatga tgtctgccta tcatagtcat tcagtgattg ttgaataaat 12720

gaatgaatga ataacactat gtttacaaaa tatatcctaa ttcctcacct ccattcatcc 12780

aaaccatatt gttacttaat aaacattcag cagatattta tggaatatac cttttgttcc 12840

atgcattgta gtactcattg gatacacata gaataataag actcagttca cactcttcag 12900

gaaacagata aaaaactaag aaacaaacaa aaaacaggca atccaacacc atgtgggaaa 12960

tgctttcata gccgggaaac ctggggaata cctgagagga atactcaatt caggccttgt 13020

ttcaggaatc caaatcctgg cacatcagag ctgcttccct ctttccaggg tggcaggaaa 13080

taaatggaac atatttttct atcttatgcc aaacatgagg gaccctttct ccccggtgcc 13140

tctcccaagg tagtctacaa tatttcaact ctagcagtct gcttagtgca tagaacatga 13200

ggctgtgtgt ccctgggcaa attactagac ttctgtgtgc ttcactttcc ctgtaggatt 13260

ataatctact gagcaagctt attgtaaggg tcagattagc aacagtgtat gaaaatgatt 13320

tgagaccatt gcctgcacaa attcaactat ttttttttat ctcactactc tacagaagta 13380

ggtagggtgg gagacagagt ctgatgagag gctcagaatg tgaaagaaag tgaggcgagt 13440

gagcatgata tttaatataa acacaaagat attctgagaa gagctgctca ctgccccctc 13500

ccccaataca tgttgatagg aaaatgccac gtacttcagc aaaaacaact gaaaaattag 13560

atagaaaagt caatcaatag gaaaagataa tccaggacgg tgttgtgaac agaaagaggg 13620

ggaaaaaact ttagaaaatg atggggatgc tcttactggg gtacgagtcc tcaggtattg 13680

aactggcttt cagtaaaagc tagattagtg ggttcctgcc atttacaagc tgttttatga 13740

caacttactt gttgggtggc ctacagtaac tcacctaact gcactgagtc tgtttcctca 13800

tctgtaaatt ggggattttt ttttaaatac ctggcatgcc taactcataa agttgttctg 13860

aaactgaaat aaaacatacg tgaacaggca ttgtaaactg taagttacgg aaaaagctgg 13920

ctgttgttgt gtctttaaag tttcacctgg gtagtcaaag atggatcatg ggtctcagtg 13980

gagagctgag ccaggcagga gctgactaag ggtgagaggt gggagttagc agcctctgaa 14040

catctgtgta ccatgggacc ccctttcctc ctgcatggta ccccagacaa ggagcctagt 14100

aagagatact aatggcttgt tgtccagaga tgttcaaact gcagagaaag ataagacaac 14160

aagcattggc ctccaatcat gatgacagat aggaggaggt gggagctcct tagcagtgct 14220

ggttggcctt ccatgttcta ctgtgggcca tctctgccat gtactgtagg ctactagctt 14280

ctatattaaa gaatgcaaga ggggccagga gcggaggctc atgcctgtaa tctcagcact 14340

ttgggaggcc aaggtgggca gatcacttga ggtcaggagt ttgtgaccag cctggccaac 14400

atggtgaaac tctgccttta ctaaaaatat aaaaattagc tgggtgtggt ggtgtgcacc 14460

tgtaatccca gctactcggg agactgaggc acaagaattg cttgaacctg ggaggcggaa 14520

gttgcagtga gcccagattg cgccactgca ctccaccctg ggcaacagag aaagactctg 14580

cctcaaaaaa aaaaaaaaaa agcaagagga agtgaaataa tcaaggccgc catttaatag 14640

tgagcagcca ctccatgtgg tactgtgcaa gcacattata aatattagcc tcacaagaaa 14700

tgtattagca tttgtatttt gtacactggt taagtatctt gcccaagacc tcaaaactgg 14760

ttaagggcag cagaatttag ccccagcacc accttttcaa agcctgggct tctcacactt 14820

ctccatgctg ttcccatttt aacacaggta tctcgccatt ccagccactc aaactttggc 14880

atttaagaaa attatcctaa agctaaacta aacttcaagg atgaccattc tcctgacccc 14940

ttcccatcaa aattttatct ttagtcagtt tgttttcgtt ttgttttgtt tttcagaact 15000

acctctggca catcctccaa atgaaaggac tcacttggta attctgggag ccatcttatt 15060

atgccttggt gtagcactga cattcatctt ccgtttaaga aaaggtagta tttccttaat 15120

tgcagtggtc tccactgggg gtgaggaagg ggtgagaatt ggatcatggc tgcaaggaaa 15180

cccgacttaa cctctgcaag gtggtgcaaa ggcattccac tgttcaacag caattatatt 15240

gaagctgagt gggatcactg ggtgaagatg aagcgtaagg ggtgaggggc aggagaatgg 15300

gtatggatgg aggtagaaga tgcagtgtca tacagttttt ttctatcatg aaaataacca 15360

cagacttaca gaagagaaag agctaaaatg cccgtcattt tcagttgcat tttagtcttg 15420

cattagttgc aaccagctgg tttctgggta ccctaagtaa taaaaatagt tcctctgtag 15480

aactgtagta tgtttaccat agagtatttt gcaaaatttt tggtagagga tgttacataa 15540

tttgcatgtg ttcatttctc catttacctg tgggaacaat taaaatccag gaaaatgagt 15600

atattcaaat aatttcctcc catttaagat gagtcagagt aaataattcc tccaatactt 15660

agagaagtat accaagagat ccagtgatgg tatagagttg tctgatgtta aatagggaag 15720

tagaatatgg aaggggattc caatagtcgt tgaaaaattc cccataaccc cttacatggg 15780

ggaaagtagt gttaactgag agagtagaga taagctgttt ccaaaaatta tattcttaac 15840

aggactgaga tagccagaat ataaggatca agtttcaatg acagtaagat cctgagatgg 15900

agttgatttg cacaaagaaa taattgttgc cagcatgcat tttgaatatt tctctggaaa 15960

aaaagattag ttggcagtag aaatggatag aaatcaatag atattaaaat acctcagaat 16020

ttggttcatc tctgggaaaa gatgaaaaat aaaagtgtat actcctcaag aacatctagg 16080

atcaaaagca tgtgccctac actattgaat taattaacct cataagttgg gacctgtgga 16140

ataaggatgt ccaccagact tcctagggat tacaaatgtt tcacagaact tgaaatttaa 16200

acttgggtca ctgtatggga tgtagagctg tgctatatgg aaataaaaat gatttctttt 16260

tctcaaggga gaatgatgga tgtgaaaaaa tgtggcatcc aagatacaaa ctcaaagaag 16320

caaagtggta agaatatcag aaggaattgg gaagtaaaag tcaaaggaaa caaaaagcta 16380

aagcaataac aaagagaaat ccatcagtca taatctcctc tccttttaaa gaatgctggt 16440

tcccctttgc ctcacagcta acacaagaac tcctccaccg tctgaggagg tttaggagca 16500

gggaagggga aggagtcagc ttcatttgct aatcttctgt tgccctgcac cctagcagct 16560

ccttgcagca ggggacaagg atgacttagg tggatggata attaattgat tctaaaatat 16620

tgtgtgtcag tattgtaata ctatgttaat tgcaccatgc acggtatctc atttaatccc 16680

ccaccccttg ccattaccaa agagagagag agagagagag agagaaatac tagaatttat 16740

cctcatttta cagtagagaa aacagagggt caagaagata atgtaaagtg cccaagaaca 16800

cacagctgat cacaaaaatc aagcttgggg gccattagcc taaccacaga cccttactct 16860

taacccatct gcttcaatcc attttgctac aaatgtttac atttataagc agggcagaaa 16920

aacctcatcc aggttattga actaagaaga aagttatatt aaggtttcta atttttttaa 16980

tgtagttaga aaccaaactt aacaatgagc ccaagtttaa agcagtctaa ttaacctgga 17040

caagctcagg caagtttcat tctgtggccc atagcatcat ctgtgttgta aagctaagta 17100

gcaaatgttg tttgggtcat gctgggggac aagccatccc aatttgctca ggactgaggg 17160

gttttccagg atatcatgta aggataattg ggtacaaata taacctgctg ctttctctca 17220

tttcaaattt atcatttatc atatcagcaa ctatgagtta tgttttttat tagatttctt 17280

gttacttttt ccccagacca cttcccatga aattaatata ctattatcac tctccagata 17340

cacatttgga ggagacgtaa tccagcattg gaacttctga tcttcaagca gggattctca 17400

acctgtggtt taggggttca tcggggctga gcgtgacaag aggaaggaat gggcccgtgg 17460

gatgcaggca atgtgggact taaaaggccc aagcactgaa aatggaacct ggcgaaagca 17520

gaggaggaga atgaagaaag atggagtcaa acagggagcc tggagggaga ccttgatact 17580

ttcaaatgcc tgaggggctc atcgacgcct gtgacaggga gaaaggatac ttctgaacaa 17640

ggagcctcca agcaaatcat ccattgctca tcctaggaag acgggttgag aatccctaat 17700

ttgagggtca gttcctgcag aagtgccctt tgcctccact caatgcctca atttcttttc 17760

tgcatgactg agagtctcag tgttggaacg ggacagtatt tatgtatgag tttttcctat 17820

ttattttgag tctgtgaggt cttcttgtca tgtgagtgtg gttgtgaatg atttcttttg 17880

aagatatatt gtagtagatg ttacaatttt gtcgccaaac taaacttgct gcttaatgat 17940

ttgctcacat ctagtaaaac atggagtatt tgtaaggtgc ttggtctcct ctataactac 18000

aagtatacat tggaagcata aagatcaaac cgttggttgc ataggatgtc acctttattt 18060

aacccattaa tactctggtt gacctaatct tattctcaga cctcaagtgt ctgtgcagta 18120

tctgttccat ttaaatatca gctttacaat tatgtggtag cctacacaca taatctcatt 18180

tcatcgctgt aaccaccctg ttgtgataac cactattatt ttacccatcg tacagctgag 18240

gaagcaaaca gattaagtaa cttgcccaaa ccagtaaata gcagacctca gactgccacc 18300

cactgtcctt ttataataca atttacagct atattttact ttaagcaatt cttttattca 18360

aaaaccattt attaagtgcc cttgcaatat caatcgctgt gccaggcatt gaatctacag 18420

atgtgagcaa gacaaagtac ctgtcctcaa ggagctcata gtataatgag gagattaaca 18480

agaaaatgta ttattacaat ttagtccagt gtcatagcat aaggatgatg cgaggggaaa 18540

acccgagcag tgttgccaag aggaggaaat aggccaatgt ggtctgggac ggttggatat 18600

acttaaacat cttaataatc agagtaattt tcatttacaa agagaggtcg gtacttaaaa 18660

taaccctgaa aaataacact ggaattcctt ttctagcatt atatttattc ctgatttgcc 18720

tttgccatat aatctaatgc ttgtttatat agtgtctggt attgtttaac agttctgtct 18780

tttctattta aatgccacta aattttaaat tcataccttt ccatgattca aaattcaaaa 18840

gatcccatgg gagatggttg gaaaatctcc acttcatcct ccaagccatt caagtttcct 18900

ttccagaagc aactgctact gcctttcatt catatgttct tctaaagata gtctacattt 18960

ggaaatgtat gttaaaagca cgtattttta aaattttttt cctaaatagt aacacattgt 19020

atgtctgctg tgtactttgc tatttttatt tattttagtg tttcttatat agcagatgga 19080

atgaatttga agttcccagg gctgaggatc catgccttct ttgtttctaa gttatctttc 19140

ccatagcttt tcattatctt tcatatgatc cagtatatgt taaatatgtc ctacatatac 19200

atttagacaa ccaccatttg ttaagtattt gctctaggac agagtttgga tttgtttatg 19260

tttgctcaaa aggagaccca tgggctctcc agggtgcact gagtcaatct agtcctaaaa 19320

agcaatctta ttattaactc tgtatgacag aatcatgtct ggaacttttg ttttctgctt 19380

tctgtcaagt ataaacttca ctttgatgct gtacttgcaa aatcacattt tctttctgga 19440

aattccggca gtgtaccttg actgctagct accctgtgcc agaaaagcct cattcgttgt 19500

gcttgaaccc ttgaatgcca ccagctgtca tcactacaca gccctcctaa gaggcttcct 19560

ggaggtttcg agattcagat gccctgggag atcccagagt ttcctttccc tcttggccat 19620

attctggtgt caatgacaag gagtaccttg gctttgccac atgtcaaggc tgaagaaaca 19680

gtgtctccaa cagagctcct tgtgttatct gtttgtacat gtgcatttgt acagtaattg 19740

gtgtgacagt gttctttgtg tgaattacag gcaagaattg tggctgagca aggcacatag 19800

tctactcagt ctattcctaa gtcctaactc ctccttgtgg tgttggattt gtaaggcact 19860

ttatcccttt tgtctcatgt ttcatcgtaa atggcatagg cagagatgat acctaattct 19920

gcatttgatt gtcacttttt gtacctgcat taatttaata aaatattctt atttattttg 19980

ttacttggta caccagcatg tccattttct tgtttatttt gtgtttaata aaatgttcag 20040

tttaacatcc cagtggagaa agtta 20065

<210> 2

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

acgttggatg atttgagggt cagttcctgc 30

<210> 3

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

acgttggatg actgagactc tcagtcatgc 30

<210> 4

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

acgttggatg tccactggga tgttaaactg 30

<210> 5

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

acgttggatg ttgtcacttt ttgtacctgc 30

<210> 6

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

acgttggatg acagagctcc ttgtgttatc 30

<210> 7

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

acgttggatg tgctcagcca caattcttgc 30

<210> 8

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

acgttggatg gaggatccat gccttctttg 30

<210> 9

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

acgttggatg cttaacaaat ggtggttgtc 30

<210> 10

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

acgttggatg ttaagtccca cattgcctgc 30

<210> 11

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

acgttggatg tctcaacctg tggtttaggg 30

<210> 12

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

acgttggatg atacctagta gaacctgccc 30

<210> 13

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

acgttggatg gcattgacag gttggaaaag 30

<210> 14

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

acgttggatg atttgctgcc ttgggcagag 30

<210> 15

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

acgttggatg tcgggaagct gcgcagaact 30

<210> 16

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

acgttggatg aacaggtggg aaagatgaac 30

<210> 17

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

acgttggatg gttgctgatg ggaattgagg 30

<210> 18

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

acgttggatg actttggtga ctgtgacctc 30

<210> 19

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

acgttggatg catgtgtgtg tcatacacag 30

<210> 20

<211> 20065

<212> DNA

<213> PDL1 Gene (Homo sapiens)

<400> 20

ggcgcaacgc tgagcagctg gcgcgtcccg cgcggcccca gttctgcgca gcttcccgag 60

gctccgcacc agccgcgctt ctgtccgcct gcaggtaggg agcgttgttc ctccgcgggt 120

gcccacggcc cagtatctct ggctagctcg ctgggcactt taggacggag ggtctctaca 180

ccctttcttt gggatggaga gaggagaagg gaaagggaac gcgatggtct agggggcagt 240

agagccaatt acctgttggg gttaataaga acaggcaatg catctggcct tcctccaggc 300

gcgattcagt tttgctctaa aaataattta tacctctaaa aataaataag ataggtagta 360

taggataggt agtcattctt atgcgactgt gtgttcagaa tatagctctg atgctaggct 420

ggaggtctgg acacgggtcc aagtccaccg ccagctgctt gctagtaaca tgacttgtgt 480

aagttatccc agctgcagca tctaagtaag tctcttcctg cgctaagcag gtccaggatc 540

cctgaacgga atttatttgc tctgtccatt ctgagaaccc aaaggagtcc taaaagagga 600

atggaggagc ctaagaataa aaatagtata ataaaacatt tcttagacac attgaccttg 660

gcctatgtca aagttcagtc tgggtttgtc ttataacaca aggagtaaaa gtaccattgt 720

tctacctctt tttttaatac ttgaaaaaaa tttactgtgg atgcttttct atgaattaaa 780

taaccttcta aaaaatgttt tcattgctgc attcgattag attgggtaac taaatgaaat 840

taattcctca ctgttgggta taaaggttat ttacagtggt tctgtcttag ccattcactg 900

aactcattgc atatatatct ctggaatatt gctgattgtt tccttcaagt aaacttagaa 960

gtgtaactac ttagtcaaag agcctgaata ttttaaaggc cttttgaaga aaactgaaaa 1020

tgctttccag aaaggatgta tcagttgaca atgacagtcg tcaacagtat ttaaggagaa 1080

ctatgatact ctgaagaaaa acttagcctt tctcagtaaa agtaggtagg cagaggccac 1140

atgacagcag ttagagtgtg gtcttcaagg aagtcacaga aatactgtgg ggaattgaaa 1200

ccccatgtgg aaaatgtaca agagtgtctc agtgtgactg agaaggaggt tgggcatggg 1260

gtttcatgga gtttaataaa gtttggtcac ttagtagagg tttaataaat caactgtctt 1320

aatctttgat cctacttaag aatttttttt ttgtttttgt agagatgggg ctcttgttat 1380

gttgcccagg ctgttctcga actcctagcc tcaggcgatc ctccctcctc aggctccaga 1440

agtcctggga ttactggcgg gagccaccat gcaggcctct tgctcctact tttgagaaag 1500

gaagtttaac cggttttttt tgtctttttt tttttttttt tgagacagag tctcactctg 1560

ttgcccatgc tggagtgcag tggtgcaatc tcagctcact gcctcccggg ttcaagtgat 1620

tctcctgcct cagcctcccg agtagctggg actacaggca cctgccacca cgcccagcta 1680

atttttgtat ttttagtaga aatggggttt caccatattg gccaggctga tctcgaactc 1740

ctgacctcag gtgatccgcc tgcctcggcc tcccaaagtg ctgggattac aggcatgagc 1800

cactgctcct ggctgcttaa ctttttctct atctcatcct cctacccatc ctacccttgg 1860

aagatagaga agtagtatta gttccatagt gttatactgg gcttccccca gggacaaacc 1920

cacttcccca acctgaatga gccatcactt cttccccagt ttacatttca ttgctcttta 1980

aatgtctcca ttcggatatg ggaattcaca tatggtcata attcttacct gaagaagatg 2040

tcagtcttct tctcttagac caactgccct gatatgaggt ttagaggtta aagaacatgt 2100

gtgtatttac atgatctttg tattctgcct tttcgtccct cactaatgac agctgcaccc 2160

caaggaaatg gagctgtgga agagagggtt tgataagaaa ttaagtaaat attggatcta 2220

atccatcacc ctccaggaag cctttattac tcctaaaaat ttcaaccaaa ttcattaaag 2280

gacaagaact ccaccagagt aggccataaa cattggcaaa attagttgta atccatgact 2340

agatttaatg tccctttgtt ttattcccat atggttataa tgctttgctt ggcattaggg 2400

gtattttaag ttttcttctg cctagtaagt gaatttgtgt ttataataca ataatcataa 2460

aatatcacat taatatttta taactgtaca gttataaaat attttataag taatatttat 2520

attttataag taatatttta taactgtaca gttaactctg gcccaaggaa aagatagtct 2580

gatagatgct gcagccccat tttagcaaat gtgacctcac aggcctgaat gccatcgcta 2640

ttccacatct acaggataga cggaaaggaa agaaataaaa aaataggtac ctaacactgg 2700

caagaggatg atgactcatg ttatttcact taaccttttt atcttttaac atgaaggact 2760

catacaggtt gataagaaac cagtgacata aacagaccaa aaaatgatca gatctttcaa 2820

attagcaaaa aaataatatt ttttaaacaa tgggtgaaaa tacagtgtaa cagtaccaat 2880

tatcaacatg tgttgagaac cagaaaaatg ttctttttct ttgatcagca acactatttg 2940

ggaaaatcta tcctcagggc ctagcctggg gccctggcac acagtaggca ctcaacgaat 3000

atttgctgaa cacacaaata cttatgatat tttaaaaaat tggcaacaat ctgataccta 3060

acaatagagg gattaaatat tatggaactg ttaaataaga tgcttatgaa taccatgcag 3120

taagatgggc aatatttatg ccataagctt taatgaaaca aatgggtatt aaatgtatga 3180

taaggttata aattactttt taaaagatta cagggaaaaa aattgaaaga tatacactga 3240

aatgtttttt gctcacagtg gtgacaaggt ttctcagcac tggcactgtt gacgttttag 3300

gctgtatgtc tttgctgtgg gaggctggcc tgtgcactgc agggtgtttg gcagcactct 3360

tggcctctgc ccctagatag caatagcagt cctccctcaa ccagcccaat tttgacaacc 3420

aaaaatgttt ccaggcatca ccagatgctc cctgggtgag agtgatgaaa tagtagggga 3480

ttttcccctt cttttcttat tttctgtaat tccattatat tactttaata ataaagaaaa 3540

aaacataaaa aataaacgaa tgttattatt ctacgtcagt ttggatgttt ggactccatt 3600

ttggggttct ttccattata tcacttggtc tgctaaacat tctacggttt ggtaaggtga 3660

agtgattcat gaaattttgg ttttattttt ttcctgatac taaaaataaa acattctttc 3720

acttggaaat ttggacacag aacaccaaaa aaaatccata atctcatctc tctttttctg 3780

tcttttcctt ccttttttcc ctttaaaaac aataaagagt gaaacctacc tgttctccct 3840

ctaatttaat tcctaaatat aatcactgtc aatatcttgg acatttcctg tgtctaaaca 3900

cacacacaca cttttttttt tcagcaaaag tggatttctg ctacatgtag tgttctgcaa 3960

cttactttct atgtgtttac aaaatcagta catgtacata tgctgaattc agtccttaat 4020

ggtattatat tttgtgaata taccaaaatt tgtttaacca cttagacaat ctaggatatt 4080

ctcagtttgc tgttatgagc aatgctcttc ctttacatat acagacatat atatatatat 4140

gtgtgtgtgt gtgtttttgt tttagtagga tagatttcta ggagagggtg aaaggtctta 4200

tgacatccgc atttacgatt gtaataggaa gtatcaaagt gccccctaaa gaaaaaaatc 4260

ctcccattag tgggtaagaa agcctatttg ttcatatctt cacaaacact aaatattaga 4320

aatatttaca attgtggtca agctcataag tgaaaatggt atttcatatc ttatattttt 4380

tattgtgaga ttgaacatct ttcatatgtt tacatgtcac ctgtatttct tattctctga 4440

actatatgtt atgacctttc actttttttc ctcatgggtt atgtgtagtt tgtatagttg 4500

tcttattgat tgttaggagc tatttatata ttaggaacat taatctcctg tcttatatat 4560

acgtggcatc gattagttga tcatttgtga gttcatgtct gtatacaaag attggagagg 4620

cactaagagg gaaaacttac ctctttctta tcaaagtttg taaatatatg tataacagaa 4680

gagggagaaa atattaataa atgcacagat tggctgaaat agagtataaa tcttttactc 4740

ccctacttca acataaactg caaaaggaga gtgacttttc tttcactctg acttccgtat 4800

tcctcatgct taaaatagtg cctagcacag aagaggtgct caatcagtgt ttgctaaacg 4860

aaataattag tcacatttca agcaggatga ctaaatgaag aatagaatct aggcagatac 4920

tctggaagag tggctgtgag tcattcatat cttagtatga attagtcaaa tccaactctc 4980

tccccttccc actccccact gttagtagaa gaatctgttt attgagagaa tagatttata 5040

atttagaata agtgagaggg gcagaagagg agattttgaa ggatggcacc tgaaggagga 5100

ctagcatggc tgagacagtg aagtggaagc cttgaatagc taaagggtaa gatgaaagta 5160

tttagctgta gggggaaaaa gcattgacag gttggaaaag taaaagtcag attctccttg 5220

ctctgaaatt ttgtacaggg caggttctac taggtatgtt acaatgcaga aaaaacatga 5280

aataattgag aggaatttgg tgcaatatta tcttcttggc ttcttttgag tgggcagatt 5340

tttttcacgg cctgtaacta taataaattt gaaacttctc atcttttagt aacttttttc 5400

acttaagttt atgtggctgt gggcaatgga atgaagatat tgaacttcca attccctgtt 5460

gggtttccac aattacaagt caatcatgac tggttattag aagactattt cagttagaac 5520

caccaagtcc catattgtca tattgtatgt ttaattatta agtgaagcag tcttcttttc 5580

gtgttttcca taattagggc attccagaaa gatgaggata tttgctgtct ttatattcat 5640

gacctactgg catttgctga acggtaagac accaaatcct tccattaggt tctatatttt 5700

aaatatttta accatgagtt taaaactaaa atgatcattt aaaatgcatg caattttctt 5760

atagagagaa cattctattc tttcttctac tttacacaat ggcaaagtct tctttctact 5820

ttacgcaatg ataaagttac ctgtgtcatt ttgtaaaaat atagagaata tagacaaatt 5880

gaaagacaca aaataatcta ttacccattt cccagggtta actactgaaa atatctgggg 5940

aaatggcctg tatgtataca tttatttgtt tgctttcaac aaggccaaga tcctttgatc 6000

tttcagtctt ggttgctctg tgacatgcct ttcctgatga ggatacttta aggaagaatt 6060

gtaagataca tggaaaatgt caggctaaca cagtactggc atcaccctgt gctctttcct 6120

gaactccata ccaatgtact tcttgccaga aaactgatca aaagtttagg gaagtaaaaa 6180

gagatgactg ttagaatcta ccattccctc tatgtaggaa gcaaataggt gtcctgtcaa 6240

aggacattct ggggatgtct acatgaaacc aagtctccct ggttgtaagg actccatctc 6300

catataatat ttatacagta atatatgttt ataaattgtg ggggcaactt gtttagctaa 6360

ttttattatt ctgctattgg gacactgtgt ctcagcatga gatatagtgt cccaaaacat 6420

atttcaagcc cattggataa aatatgtgtt tagcaagttc ttaaatataa tgataacata 6480

accgaccaga taaagtgatt tataaacgct gtgccaattt tgtaaatgtt tcgaggaatt 6540

ttcccttttc tgaagattgt ccttctttct ttttagcatt tactgtcacg gttcccaagg 6600

acctatatgt ggtagagtat ggtagcaata tgacaattga atgcaaattc ccagtagaaa 6660

aacaattaga cctggctgca ctaattgtct attgggaaat ggaggataag aacattattc 6720

aatttgtgca tggagaggaa gacctgaagg ttcagcatag tagctacaga cagagggccc 6780

ggctgttgaa ggaccagctc tccctgggaa atgctgcact tcagatcaca gatgtgaaat 6840

tgcaggatgc aggggtgtac cgctgcatga tcagctatgg tggtgccgac tacaagcgaa 6900

ttactgtgaa agtcaatggt aagaattatt atagatgaga ggcctgatct ttattgaaaa 6960

catattccaa gtgttgaaga cttttcattc ttgtaagtcc atacttattt tcaaacagaa 7020

cagcatagtc tgttcattca ttcattcaat tcatgaattc attcacataa ttatccaatt 7080

tcttgagcac ctatttgata gtcactggaa atccagagac aaacaacaca gagccatgtt 7140

ctacagtatg tacagttttc caaaaagaat ttctagtctt tactttttta ttacaaatgg 7200

aatacgtata cttgcaaata attcagatac tgtggaagag atcaaatgaa ttgcaaaagt 7260

gtccctcctc ccttcaccac tatctcccat ggcatgcaga gagagtaacc attatttgtg 7320

tgtccctcca gaaatttttt tattcaacta ctattttttt attttattag gtccgtcagt 7380

tttccttttt tgagcctctc tatatcaaat gcaaataaat atattcagaa caaaccccac 7440

tgtaaggttc acattaaaaa agacttgaag tcaccctatg aagacaaaaa ataatcacat 7500

taagtgtgaa agaacctatt cttccagtac aggataagcc atacttactg ggcatatatt 7560

catcttgaaa atctatactg atgttgtctt ggggaattga aaaggaacta ggagtgttag 7620

ttcctcggta ttgacccaca gttatgttat caggtcactt gagttcaaag ttttgtgttg 7680

gcactagcta agtaaaggaa aacacctctg ctttcattgt tgagtttcac agaattgaga 7740

gctgaaagga tcccaggcag gagcagctaa tccaaactcc cacaaagaac aaaaatcccc 7800

cagaggatct tctgttctta tatttcctgc aatggcgtcc ctgtcatatc ccacaatggc 7860

ctccctgcca tttggatatc ccttccatat cctgttgaaa ttactcccta atagtaagct 7920

gaaatctgcc cctctagttg tagtcttggg attatttcat ttacatgatg accttttaat 7980

atttgactag aattaaatca tctccccttg gtctttccat tcctgggcta actaccatca 8040

atctgagggc taacaataca agtagaaaaa gtatacattt gtcactgatc actgatcaat 8100

tattaatcaa tgatcactga taactataaa ctcaaaaaca aaatcatgtg gggattaaga 8160

gaaatgtatc agttttatgt tgtatttctg gtccctgata ctggctcagg taatgccact 8220

attgtcaaga agataccact tgtaaagtag atttaatttt cattatattt taccatatgc 8280

ttctccattc atgacatctc ttgagatgtt gtggtttata ctttcagttt ttctccagtc 8340

catccgcaaa tatcaggcat ctactgtgtt ccaagatatt aaagaaatca tcatgactta 8400

gcctcatcaa cagcattgct agatctggga tggaaaggaa gagtataatc ctggcagtca 8460

ggaagaaggc agcataaagt ataagtttct gcttccaaaa aaggtctctc atcagcctgt 8520

agggagtgtg tagggaaggg acagctgtcc ttgtagtagg gaagggtttt attcaggtcg 8580

tctgggctcc ataatatccc ttgtgtatct gcagtctcct ttgccatgga tcaacacaat 8640

aggaaatctt ccggcactga tggtttttcc aagggggagt tcttcctgga gcaaagcaaa 8700

tgaccaacca ggtttgagga cctgatttgt ttgacaattc cattttgtat tgtaaattac 8760

ttaattggca ttctactccc aatccatctt gtcatttgca tacagtggtt ttgggattga 8820

gttcagctat accaaaagtc tgaaccttct gcacttagaa caaggcaacc accaagcttc 8880

acttgcactg aggccgtgtc tccaatggaa atgaggcagc tggcttgcag gagcttccca 8940

actcagggaa gtagaactcc tgagtcacct ccatatgcaa atgatttcac agtaatgctg 9000

ttgaacttca cttcccatca cagcaaatgt gtggtaacat agcttcccca caggagttta 9060

ctcaccatgg tattttaaag gtgaaacatt tcaaaactga aatttgaaag aatttagttt 9120

tggattcact caattatcac tatcacttcg ggtgttattg cacctttctt gtttgtgagt 9180

ttaaatgcca gactctcagg ccactaactt tcaattaaaa gtgtttttct ttaatcgctg 9240

aacctaacag cagggaaaac gaaatgttca ttcagacttt cagaaccttc aatgagatta 9300

ggcagctgaa agatcaaagt gttgcatagt tgtcccgata aagctatttg gatcatatgg 9360

accaaatcga ctgctgtcat tccccaccaa ccccatctct ccccaaaatt cccagccctg 9420

tttaagtgtt ctctgtagca tttatctcta tctagtatat tgtgtagcat atcatatcat 9480

acttttctgt tttgtttatt gtctctctcc tcctagaata taaactccac aagcacaaag 9540

atttgggcct gttttataat attgttgcat ccccagggcc tgatatacag cagagtggtg 9600

gtacgaaaag agcacacaaa aaaatatttg ttgagtcaat gaatgaatga tttcctcaaa 9660

taggattagc ctaaaatttt ggaaacatga acagatttgg atatgtgaaa atttatttcc 9720

agactgttca tcaggaactg ttagcagctt ctaaagggta cactggagca gcagtagtaa 9780

aaggaggaag aggagcagct ctgctactgc tactatcgag tactactaca attagcactt 9840

gcttattctg tgtgttaggc cctgtactga acactctgtc taaattagtt catttcctcc 9900

tggaaatgac tctagggggt aagtgcttca tcatgtaaga tgagtatttt tcacattttg 9960

ttgtgtctga aatctgagtg tgtctttcaa tgatggaatc tttgattcca tgataagtgg 10020

tattattccc attttaagga tgaggaaact gaggtccaaa gaaattaagt aatttgccca 10080

aattcaccca gcctagaaaa tgataaagct agttctaaac ccaagcagat tagctctgaa 10140

gtctgggccc ttaataacca ctttttattg cctatatttg tacctctggt gtacgtatca 10200

agttatatgt tgacttcaaa actatcatga ccttttcttg gttttgattg tccaacatta 10260

gtatagtgtt ctgggtctgc aaaaattttg attactcatc tcatctgtaa aacattttga 10320

actcgtgtgt ttgtgcatgc acatttgtgt gtaattataa aaattttact ttctgttaat 10380

atataagttg tatcataaga aactgccgtt tttgaagagc aaaaaaaggt tgaatgttac 10440

cagttacatc tggttcaacc taatagacat ttgtacaaaa acagacattt taagaggttg 10500

aaataaaaat ttaataaaca atattttcag tttttactaa ttgtgatgct tcactatcat 10560

tagctaatat gtcaaggcat aatatacctt agggtgaact ttatcattaa caaaggtgga 10620

tggtgtcaat aatcttgagg tttgtgtttt tttatataac actgcgaggt ctaattaagt 10680

acttactgtt taccacctca tacagtggcc gataaaaagt gtcacttctg ctgtttcctc 10740

tgggttgtgc ttgaattatt agtattatct tcagtcctca gtttctttgt gggaaacttt 10800

ttaattagtt gtttaatttt gtaagatggt tagtttagtc aaaattagat aagagaattt 10860

gaaaatccgt agctacccca aagcaaccta cacataagaa ctattatttt tgtgttttga 10920

aatcataatt ttattgattt ccagtgtttc cactggtagt ggtttcattg atataggagt 10980

atcaaaacat cactcattat ttatttcagt ttcatttgat cctagccgtt ttgtattaac 11040

tctctgtgaa gaaattacct cacaaatcta ttgctgtcct tggtaaagga atggagaatt 11100

aaggctctag atcattagtg gttacactat agtattagaa gtaaaaaaaa gattatacca 11160

acaaaataag aacatgttaa tgtacttgta atgaataaac atgaataaag ctcttatgct 11220

atataggtgc actaaacaat ctactagaat tgtcagcaaa ctacgtatct taatcctgaa 11280

agggtcccaa accaatgatc taaaattgaa tcaaactttc ttccttgagc ataattactt 11340

aaatgattta ttaaaatagc cagcatttaa aagcttaaaa tgtaaatatc ataatgtggt 11400

atcctagata gcatcccaga acagaaaaag gatattaggg aaaaactgga ggaatggaat 11460

aaattatgca gtttagttat taataatgta ctaacgtcct tagttatgac gattgtacca 11520

tggtaatgta agatactaac aatagaggaa accgggtaag gagtatacag taactctata 11580

ctatctttgc aacttttttg taaatttaaa acttctaaaa taaagaacaa atttaaacat 11640

taaaaagtat caccaggaac atatatcact gtttacagat gaaatactat gtattttcat 11700

atctaatttc tgatcattga cttcaaatca gaaaagtgaa tgacacctca aaatcaggtt 11760

ttctgtttac tgaagtctaa gaaaagaaag cataccagct ggagagattc atgtttataa 11820

agacagattt ataacaacaa aaataaaata tccaagaata aatttaagaa gaagcacttt 11880

actgagaaac atatgaaaac ctgaacaaat ggagagggat attttgtatt tgaatagaaa 11940

gacttctggt ttaaagataa ttctctttaa attatttttt gtagaaattt aaggggtaca 12000

agagcagtgt tgtcacatgg atatattaca tagtggtgaa gtctggggtt ttagtgtaaa 12060

ttaatcttta cattttgttt gagcccaata aatgtaccaa catgattttt atagaaagat 12120

agtcattcct attaatccaa acttgtccca actttgaatt gaattgaggc agagctagca 12180

ggtgttcccc acggctgagg catctgaaca ttaagcatat ccctctgaga accagcctgc 12240

attgatactc tttctaatgt ggacagcatc aagctatgta cgtagttctg tgctcagcaa 12300

aagccctgac ttctttttgt ttatgtccta gccccataca acaaaatcaa ccaaagaatt 12360

ttggttgtgg atccagtcac ctctgaacat gaactgacat gtcaggctga gggctacccc 12420

aaggccgaag tcatctggac aagcagtgac catcaagtcc tgagtggtaa gaccaccacc 12480

accaattcca agagagagga gaagcttttc aatgtgacca gcacactgag aatcaacaca 12540

acaactaatg agattttcta ctgcactttt aggagattag atcctgagga aaaccataca 12600

gctgaattgg tcatcccagg taatattctg aatgtgtcca ttaaaatatg tctaacactg 12660

tcccctagca cctagcatga tgtctgccta tcatagtcat tcagtgattg ttgaataaat 12720

gaatgaatga ataacactat gtttacaaaa tatatcctaa ttcctcacct ccattcatcc 12780

aaaccatatt gttacttaat aaacattcag cagatattta tggaatatac cttttgttcc 12840

atgcattgta gtactcattg gatacacata gaataataag actcagttca cactcttcag 12900

gaaacagata aaaaactaag aaacaaacaa aaaacaggca atccaacacc atgtgggaaa 12960

tgctttcata gccgggaaac ctggggaata cctgagagga atactcaatt caggccttgt 13020

ttcaggaatc caaatcctgg cacatcagag ctgcttccct ctttccaggg tggcaggaaa 13080

taaatggaac atatttttct atcttatgcc aaacatgagg gaccctttct ccccggtgcc 13140

tctcccaagg tagtctacaa tatttcaact ctagcagtct gcttagtgca tagaacatga 13200

ggctgtgtgt ccctgggcaa attactagac ttctgtgtgc ttcactttcc ctgtaggatt 13260

ataatctact gagcaagctt attgtaaggg tcagattagc aacagtgtat gaaaatgatt 13320

tgagaccatt gcctgcacaa attcaactat ttttttttat ctcactactc tacagaagta 13380

ggtagggtgg gagacagagt ctgatgagag gctcagaatg tgaaagaaag tgaggcgagt 13440

gagcatgata tttaatataa acacaaagat attctgagaa gagctgctca ctgccccctc 13500

ccccaataca tgttgatagg aaaatgccac gtacttcagc aaaaacaact gaaaaattag 13560

atagaaaagt caatcaatag gaaaagataa tccaggacgg tgttgtgaac agaaagaggg 13620

ggaaaaaact ttagaaaatg atggggatgc tcttactggg gtacgagtcc tcaggtattg 13680

aactggcttt cagtaaaagc tagattagtg ggttcctgcc atttacaagc tgttttatga 13740

caacttactt gttgggtggc ctacagtaac tcacctaact gcactgagtc tgtttcctca 13800

tctgtaaatt ggggattttt ttttaaatac ctggcatgcc taactcataa agttgttctg 13860

aaactgaaat aaaacatacg tgaacaggca ttgtaaactg taagttacgg aaaaagctgg 13920

ctgttgttgt gtctttaaag tttcacctgg gtagtcaaag atggatcatg ggtctcagtg 13980

gagagctgag ccaggcagga gctgactaag ggtgagaggt gggagttagc agcctctgaa 14040

catctgtgta ccatgggacc ccctttcctc ctgcatggta ccccagacaa ggagcctagt 14100

aagagatact aatggcttgt tgtccagaga tgttcaaact gcagagaaag ataagacaac 14160

aagcattggc ctccaatcat gatgacagat aggaggaggt gggagctcct tagcagtgct 14220

ggttggcctt ccatgttcta ctgtgggcca tctctgccat gtactgtagg ctactagctt 14280

ctatattaaa gaatgcaaga ggggccagga gcggaggctc atgcctgtaa tctcagcact 14340

ttgggaggcc aaggtgggca gatcacttga ggtcaggagt ttgtgaccag cctggccaac 14400

atggtgaaac tctgccttta ctaaaaatat aaaaattagc tgggtgtggt ggtgtgcacc 14460

tgtaatccca gctactcggg agactgaggc acaagaattg cttgaacctg ggaggcggaa 14520

gttgcagtga gcccagattg cgccactgca ctccaccctg ggcaacagag aaagactctg 14580

cctcaaaaaa aaaaaaaaaa agcaagagga agtgaaataa tcaaggccgc catttaatag 14640

tgagcagcca ctccatgtgg tactgtgcaa gcacattata aatattagcc tcacaagaaa 14700

tgtattagca tttgtatttt gtacactggt taagtatctt gcccaagacc tcaaaactgg 14760

ttaagggcag cagaatttag ccccagcacc accttttcaa agcctgggct tctcacactt 14820

ctccatgctg ttcccatttt aacacaggta tctcgccatt ccagccactc aaactttggc 14880

atttaagaaa attatcctaa agctaaacta aacttcaagg atgaccattc tcctgacccc 14940

ttcccatcaa aattttatct ttagtcagtt tgttttcgtt ttgttttgtt tttcagaact 15000

acctctggca catcctccaa atgaaaggac tcacttggta attctgggag ccatcttatt 15060

atgccttggt gtagcactga cattcatctt ccgtttaaga aaaggtagta tttccttaat 15120

tgcagtggtc tccactgggg gtgaggaagg ggtgagaatt ggatcatggc tgcaaggaaa 15180

cccgacttaa cctctgcaag gtggtgcaaa ggcattccac tgttcaacag caattatatt 15240

gaagctgagt gggatcactg ggtgaagatg aagcgtaagg ggtgaggggc aggagaatgg 15300

gtatggatgg aggtagaaga tgcagtgtca tacagttttt ttctatcatg aaaataacca 15360

cagacttaca gaagagaaag agctaaaatg cccgtcattt tcagttgcat tttagtcttg 15420

cattagttgc aaccagctgg tttctgggta ccctaagtaa taaaaatagt tcctctgtag 15480

aactgtagta tgtttaccat agagtatttt gcaaaatttt tggtagagga tgttacataa 15540

tttgcatgtg ttcatttctc catttacctg tgggaacaat taaaatccag gaaaatgagt 15600

atattcaaat aatttcctcc catttaagat gagtcagagt aaataattcc tccaatactt 15660

agagaagtat accaagagat ccagtgatgg tatagagttg tctgatgtta aatagggaag 15720

tagaatatgg aaggggattc caatagtcgt tgaaaaattc cccataaccc cttacatggg 15780

ggaaagtagt gttaactgag agagtagaga taagctgttt ccaaaaatta tattcttaac 15840

aggactgaga tagccagaat ataaggatca agtttcaatg acagtaagat cctgagatgg 15900

agttgatttg cacaaagaaa taattgttgc cagcatgcat tttgaatatt tctctggaaa 15960

aaaagattag ttggcagtag aaatggatag aaatcaatag atattaaaat acctcagaat 16020

ttggttcatc tctgggaaaa gatgaaaaat aaaagtgtat actcctcaag aacatctagg 16080

atcaaaagca tgtgccctac actattgaat taattaacct cataagttgg gacctgtgga 16140

ataaggatgt ccaccagact tcctagggat tacaaatgtt tcacagaact tgaaatttaa 16200

acttgggtca ctgtatggga tgtagagctg tgctatatgg aaataaaaat gatttctttt 16260

tctcaaggga gaatgatgga tgtgaaaaaa tgtggcatcc aagatacaaa ctcaaagaag 16320

caaagtggta agaatatcag aaggaattgg gaagtaaaag tcaaaggaaa caaaaagcta 16380

aagcaataac aaagagaaat ccatcagtca taatctcctc tccttttaaa gaatgctggt 16440

tcccctttgc ctcacagcta acacaagaac tcctccaccg tctgaggagg tttaggagca 16500

gggaagggga aggagtcagc ttcatttgct aatcttctgt tgccctgcac cctagcagct 16560

ccttgcagca ggggacaagg atgacttagg tggatggata attaattgat tctaaaatat 16620

tgtgtgtcag tattgtaata ctatgttaat tgcaccatgc acggtatctc atttaatccc 16680

ccaccccttg ccattaccaa agagagagag agagagagag agagaaatac tagaatttat 16740

cctcatttta cagtagagaa aacagagggt caagaagata atgtaaagtg cccaagaaca 16800

cacagctgat cacaaaaatc aagcttgggg gccattagcc taaccacaga cccttactct 16860

taacccatct gcttcaatcc attttgctac aaatgtttac atttataagc agggcagaaa 16920

aacctcatcc aggttattga actaagaaga aagttatatt aaggtttcta atttttttaa 16980

tgtagttaga aaccaaactt aacaatgagc ccaagtttaa agcagtctaa ttaacctgga 17040

caagctcagg caagtttcat tctgtggccc atagcatcat ctgtgttgta aagctaagta 17100

gcaaatgttg tttgggtcat gctgggggac aagccatccc aatttgctca ggactgaggg 17160

gttttccagg atatcatgta aggataattg ggtacaaata taacctgctg ctttctctca 17220

tttcaaattt atcatttatc atatcagcaa ctatgagtta tgttttttat tagatttctt 17280

gttacttttt ccccagacca cttcccatga aattaatata ctattatcac tctccagata 17340

cacatttgga ggagacgtaa tccagcattg gaacttctga tcttcaagca gggattctca 17400

acctgtggtt taggggttca tcggggctga gcgtgacaag aggaaggaat gggcccgtgg 17460

gatgcaggca atgtgggact taaaaggccc aagcactgaa aatggaacct ggcgaaagca 17520

gaggaggaga atgaagaaag atggagtcaa acagggagcc tggagggaga ccttgatact 17580

ttcaaatgcc tgaggggctc atcgacgcct gtgacaggga gaaaggatac ttctgaacaa 17640

ggagcctcca agcaaatcat ccattgctca tcctaggaag acgggttgag aatccctaat 17700

ttgagggtca gttcctgcag aagtgccctt tgcctccact caatgcctca atttgttttc 17760

tgcatgactg agagtctcag tgttggaacg ggacagtatt tatgtatgag tttttcctat 17820

ttattttgag tctgtgaggt cttcttgtca tgtgagtgtg gttgtgaatg atttcttttg 17880

aagatatatt gtagtagatg ttacaatttt gtcgccaaac taaacttgct gcttaatgat 17940

ttgctcacat ctagtaaaac atggagtatt tgtaaggtgc ttggtctcct ctataactac 18000

aagtatacat tggaagcata aagatcaaac cgttggttgc ataggatgtc acctttattt 18060

aacccattaa tactctggtt gacctaatct tattctcaga cctcaagtgt ctgtgcagta 18120

tctgttccat ttaaatatca gctttacaat tatgtggtag cctacacaca taatctcatt 18180

tcatcgctgt aaccaccctg ttgtgataac cactattatt ttacccatcg tacagctgag 18240

gaagcaaaca gattaagtaa cttgcccaaa ccagtaaata gcagacctca gactgccacc 18300

cactgtcctt ttataataca atttacagct atattttact ttaagcaatt cttttattca 18360

aaaaccattt attaagtgcc cttgcaatat caatcgctgt gccaggcatt gaatctacag 18420

atgtgagcaa gacaaagtac ctgtcctcaa ggagctcata gtataatgag gagattaaca 18480

agaaaatgta ttattacaat ttagtccagt gtcatagcat aaggatgatg cgaggggaaa 18540

acccgagcag tgttgccaag aggaggaaat aggccaatgt ggtctgggac ggttggatat 18600

acttaaacat cttaataatc agagtaattt tcatttacaa agagaggtcg gtacttaaaa 18660

taaccctgaa aaataacact ggaattcctt ttctagcatt atatttattc ctgatttgcc 18720

tttgccatat aatctaatgc ttgtttatat agtgtctggt attgtttaac agttctgtct 18780

tttctattta aatgccacta aattttaaat tcataccttt ccatgattca aaattcaaaa 18840

gatcccatgg gagatggttg gaaaatctcc acttcatcct ccaagccatt caagtttcct 18900

ttccagaagc aactgctact gcctttcatt catatgttct tctaaagata gtctacattt 18960

ggaaatgtat gttaaaagca cgtattttta aaattttttt cctaaatagt aacacattgt 19020

atgtctgctg tgtactttgc tatttttatt tattttagtg tttcttatat agcagatgga 19080

atgaatttga agttcccagg gctgaggatc catgccttct ttgtttctaa gttatctttc 19140

ccatagcttt tcattatctt tcatatgatc cagtatatgt taaatatgtc ctacatatac 19200

atttagacaa ccaccatttg ttaagtattt gctctaggac agagtttgga tttgtttatg 19260

tttgctcaaa aggagaccca tgggctctcc agggtgcact gagtcaatct agtcctaaaa 19320

agcaatctta ttattaactc tgtatgacag aatcatgtct ggaacttttg ttttctgctt 19380

tctgtcaagt ataaacttca ctttgatgct gtacttgcaa aatcacattt tctttctgga 19440

aattccggca gtgtaccttg actgctagct accctgtgcc agaaaagcct cattcgttgt 19500

gcttgaaccc ttgaatgcca ccagctgtca tcactacaca gccctcctaa gaggcttcct 19560

ggaggtttcg agattcagat gccctgggag atcccagagt ttcctttccc tcttggccat 19620

attctggtgt caatgacaag gagtaccttg gctttgccac atgtcaaggc tgaagaaaca 19680

gtgtctccaa cagagctcct tgtgttatct gtttgtacat gtgcatttgt acagtaattg 19740

gtgtgacagt gttctttgtg tgaattacag gcaagaattg tggctgagca aggcacatag 19800

tctactcagt ctattcctaa gtcctaactc ctccttgtgg tgttggattt gtaaggcact 19860

ttatcccttt tgtctcatgt ttcatcgtaa atggcatagg cagagatgat acctaattct 19920

gcatttgatt gtcacttttt gtacctgcat taatttaata aaatattctt atttattttg 19980

ttacttggta caccagcatg tccattttct tgtttatttt gtgtttaata aaatgttcag 20040

tttaacatcc cagtggagaa agtta 20065

Claims (3)

1. The application of the rs4143815 genotype in preparing a marker for diagnosing and predicting the curative effect of breast cancer neoadjuvant chemotherapy, wherein the rs4143815 genotype is CC, CG or GG, and the neoadjuvant chemotherapy is a single-week neoadjuvant chemotherapy scheme for receiving paclitaxel combined cisplatin; the curative effect of the neoadjuvant chemotherapy is as follows: compared with a patient with the genotype of GG, the patient with the genotype of CC and CG is more likely to have neutropenia and is less likely to have adverse reaction of edema, and compared with a patient with the genotype of CC + GG, the patient with the genotype of CC + GG is more likely to have the rise of glutamic-oxaloacetic transaminase; patients with genotype GC are more susceptible to pCR than patients with genotypes GG and CC in predicting complete remission of pathology (pCR); in terms of survival, patients with genotype GG survived worse than patients with genotypes GC and CC; patients with genotype GG have a higher risk of relapse than patients with genotypes GC and CC.
2. 2. Use according to claim 1, characterized in that: the breast cancer is hormone receptor positive.
3. The application of the rs4143815 genotype in preparing a reagent for diagnosing and predicting the curative effect of breast cancer neoadjuvant chemotherapy, wherein the rs4143815 genotype is CC, CG or GG, and the neoadjuvant chemotherapy is a single-week neoadjuvant chemotherapy scheme for receiving paclitaxel combined cisplatin; the curative effect of the neoadjuvant chemotherapy is as follows: compared with a patient with the genotype of GG, the patient with the genotype of CC and CG is more likely to have neutropenia and is less likely to have adverse reaction of edema, and compared with a patient with the genotype of CC + GG, the patient with the genotype of CC + GG is more likely to have the rise of glutamic-oxaloacetic transaminase; patients with genotype GC are more susceptible to pCR than patients with genotypes GG and CC in predicting complete remission of pathology (pCR); in terms of survival, patients with genotype GG survived worse than patients with genotypes GC and CC; patients with genotype GG have a higher risk of relapse than patients with genotypes GC and CC.

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