RU2642315C1 - RECOMBINANT PLASMID pAL2-T-WBMAG DNA, USED FOR CREATION OF DNA-CALIBRATORS AT THERAPY EFFICIENCY ASSESSMENT FOR PATIENTS WITH ACUTE MYELOID LEUKEMIA - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to recombinant plasmid DNA. The recombinant plasmid pAL2-T-WBMAG DNA is developed, including fragments of WT1, BAALC, MN1, ABL1 and GUSB genes. The invention is used as calibrators in RT-PCR and real-time detection of genespecific products in order to compare the expression of these genes in patients with acute myeloid leukemia (AML). WT1, BAALC and MN1 genes act as tumour progression markers, and ABL1 and GUSB genes - as expression normalizers.

EFFECT: invention allows to increase the assessment accuracy of the relative expression of these genes due to the DNA calibrators design that combine sequences of all markers, complex analysis of the WT1, BAALC and MN1 genes allows to evaluate therapy effectiveness in most cases of acute myeloid leukemia, including in patients without detected chromosomal aberrations.

4 dwg

Description

The invention relates to biotechnology and medicine, in particular to oncohematology, and relates to recombinant plasmid DNA for use as calibrators when performing reverse transcription PCR (RT-PCR) and real-time detection of gene-specific products in order to evaluate the effectiveness of the therapy in patients with acute myeloid leukemia (AML).

The use of molecular genetic research methods, due to their specificity and sensitivity, is a key approach in controlling the patient's complete cure. However, in half of patients with AML, it is not possible to identify an informative genetic marker. It is for this group of patients that the introduction of alternative elimination control approaches seems to be most relevant in assessing the effectiveness of therapy.

Overexpression of the WT1 gene was detected in a number of blood diseases of a tumor nature, including AML. High expression of WT1 occurs in more than half of patients with AML and is a factor in the poor prognosis of the disease and a high risk of relapse [Liu-Yin, J. et al. Predictive value of minimal residual disease (MRD) monitoring by RQ-PCR in WT1 positive patients entered in the UK MRC AML-15 Trial. Blood 2008 112, 259]. Since RT-PCR can show clear differences in the level of WT1 transcript in normal and leukemic cells, WT1 expression is an effective marker in assessing the minimum residual disease in patients in the absence of major chromosomal abnormalities or other mutations characteristic of tumor clone cells.

The expression of BAALC in mature bone marrow cells and in the peripheral blood of healthy people is not detected. According to the literature, in AML, overexpression of BAALC occurs in more than 50% of patients with normal karyotype [Damiani D, Tiribelli M, Franzoni A et al. BAALC overexpression retains its negative prognostic role across all cytogenetic risk groups in acute myeloid leukemia patients. Am J Hematol. 2013 Oct; 88 (10): 848-52]. Excessive BAALC expression is associated with low overall survival rates and an increased cumulative risk of disease recurrence during therapy.

, von Neuhoff N, Schlegelberger B, Ganser A. High meningioma 1 (MN1) expression as a predictor for poor outcome in acute myeloid leukemia with normal cytogenetics. Blood. 2006; 108: 3898-3905]. Вместе с тем, было показано, что гиперэкспрессия гена MN1 является предиктором развития рецидива и коррелирует с другими показателями элиминации опухолевого клона, что косвенно указывает на возможность использования этого маркера с целью оценки ответа на противоопухолевую терапию [Carturan S et al. Variable but consistent pattern of Meningioma 1 gene (MN1) expression in different genetic subsets of acute myelogenous leukaemia and its potential use as a marker for minimal residual disease detection. Oncotarget. 2016 Sep 27].More recently, it was shown that overexpression of MN1, regardless of the involvement of this gene in the formation of a chimeric transcript, is an unfavorable prognostic factor in terms of disease-free survival [Heuser M, Beutel G, Krauter J,

, von Neuhoff N, Schlegelberger B, Ganser A. High meningioma 1 (MN1) expression as a predictor for poor outcome in acute myeloid leukemia with normal cytogenetics. Blood 2006; 108: 3898-3905]. However, it was shown that overexpression of the MN1 gene is a predictor of relapse and correlates with other indicators of elimination of the tumor clone, which indirectly indicates the possibility of using this marker to evaluate the response to antitumor therapy [Carturan S et al. Variable but consistent pattern of Meningioma 1 gene (MN1) expression in different genetic subsets of acute myelogenous leukaemia and its potential use as a marker for minimal residual disease detection. Oncotarget. 2016 Sep 27].

For the indicated tumor growth markers, the absolute number of copies is calculated using separate sets of calibrators for the analyzed and reference genes. Given the possibility of uneven degradation of plasmid DNA in calibrators, there is a possibility of distortion of the results of the study of relative expression. At the same time, if there are probabilistic deviations in the estimation of plasmid DNA copy number by spectrophotometry when creating calibrators based on sequential dilutions, the presence of the target and reference genes in the sequence can increase the accuracy of calculating the absolute values of relative gene expression.

The technical result of the claimed invention is the creation of plasmid DNA containing three sequences of calibrators, including copies of the target and reference genes, which makes it possible to mitigate the occurrence of possible errors and evaluate the effectiveness of the therapy in most cases of AML, including in patients without identified chromosomal aberrations.

The indicated technical result is ensured by the fact that the recombinant plasmid DNA pAL2-T-WBMAG used to create DNA calibrators for evaluating the effectiveness of therapy in patients with acute myeloid leukemia contains an insert of cDNA portions of the WT1, BAALC, MN1, ABL1 genes sequentially linked in the ligation reaction GUSB amplified in PCR-PB.

Since the combined frequency of detecting overexpression of the WT1, BAALC, MN1 genes in patients with AML is more than 90%, the creation of a unified system based on a comprehensive analysis allows us to evaluate the effectiveness of the therapy in almost all patients with AML. At the same time, a comprehensive assessment of several tumor markers seems appropriate also in connection with the change in the gene expression profile in the tumor population observed in some cases.

The WT1, BAALC, and MN1 genes act as markers of tumor progression, and the ABL1 and GUSB genes act as expression normalizers.

The invention is illustrated by figures 1-4.

In FIG. Figure 1 shows a physical map of the recombinant plasmid pAL2-T-WBMAG with the following notation: f1 origin - site of transcription initiation of single-stranded filamentous phage f1; T7 — early promoter of T7 phage for E. coli RNA polymerase; LacZ - β-galactosidase gene; AmpR - antibiotic resistance gene ampicillin; WT1 — sequence sequence of the cDNA of the WT1 gene; BAALC — sequence of the cDNA region of the BAALC gene; MN1 is the sequence of the cDNA region of the MN1 gene; ABL is the sequence of the cDNA region of the ABL gene; GUSB — GUSb cDNA region sequence; NotII, ClaI, EcoRI, HindIII - restriction endonuclease cleavage sites;

in FIG. 2 - levels of relative expression of the WT1 gene in healthy donors in bone marrow and peripheral blood;

in FIG. 3 - cumulative risk of relapse in the post-transplant period against the background of overexpression of the WT1, BAALC genes;

in FIG. 4 - comparison of expression levels of WT1, BAALC, MN1 genes with the level of donor chimerism and blast cells in a patient against a background of post-transplant relapse.

The recombinant plasmid DNA pAL2-T-WBMAG, which encodes the cDNA regions of the human genes WT1, BAALC, MN1, ABL1, GUSB, is obtained by the genetic engineering method: by inserting a DNA fragment consisting of nucleotide sequences of cDNA sequences of WT1 genes sequentially linked in a ligation reaction , MN1, BAALC, ABL1, GUSB, amplified using the clinical material of patients and healthy donors, into the vector pAL2-T.

The following genetic engineering constructs serve as the initial genetic material for plasmid construction:

- plasmid vector pAL2-T (Eurogen, cat. number TA002);

- conservative cDNA region of the WT1 gene amplified with cDNA obtained in the reverse transcription reaction from RNA isolated from the clinical material of patients with AML;

- a conservative cDNA region of the BAALC gene amplified with cDNA obtained in the reverse transcription reaction from RNA isolated from the clinical material of patients with AML;

- a conservative cDNA region of the MN1 gene amplified with cDNA obtained in the reverse transcription reaction from RNA isolated from the clinical material of patients with AML;

- a conserved region of the cDNA of the ABL1 gene amplified with cDNA obtained in the reverse transcription reaction from RNA isolated from the clinical material of healthy donors;

- a conservative cDNA region of the GUSb gene amplified with cDNA obtained in the reverse transcription reaction from RNA isolated from the clinical material of healthy donors;

- synthetic oligonucleotides

The resulting plasmid pAL2-T-WBMAG encoding sections of the cDNA sequences of the WT1, BAALC, MN1, ABL1, GUSB genes has a molecular weight of 2.61 MDa and a size of 4225 bp and contains the following elements:

- a 3009 bp region of pAL2-T plasmid, including the site of transcription initiation of a single-stranded filamentous phage f1, an early T7 phage promoter for E. coli RNA polymerase, triggering expression of the recombinant gene, AmpR ampicillin resistance gene for selection of plasmid-transformed cells E. coli and lactose operon, which allows the selection of transformed bacteria by the method of blue and white selection;

- ClaI / EcoRI cDNA fragment of the WT1 gene amplified with cDNA obtained in the reverse transcription reaction from RNA isolated from the clinical material of patients with AML;

- ClaI / EcoRI cDNA fragment of the BAALC gene amplified with cDNA obtained in the reverse transcription reaction from RNA isolated from the clinical material of patients with AML;

- ClaI / EcoRI cDNA fragment of the MN1 gene amplified with cDNA obtained in the reverse transcription reaction from RNA isolated from the clinical material of patients with AML;

- EcoRI / HindIII cDNA fragment of the ABL1 gene amplified with cDNA obtained in the reverse transcription reaction from RNA isolated from the clinical material of healthy donors;

- EcoRI / HindIII cDNA fragment of the GUSb gene amplified with cDNA obtained in the reverse transcription reaction from RNA isolated from the clinical material of healthy donors;

- a unique recognition site for NotI restriction enzyme for linearization of plasmid DNA, having the following coordinates - 4211.

The synthesis of plasmid DNA is as follows.

Plots of cDNA encoding transcript sequence analyzed by RT-PCR are amplified using synthetic oligonucleotides selected in such a way that the region amplified by RT-PCR is located inside the cloned fragment, and sites for specific recognition of restriction endonucleases are located at the 5'-ends of the oligonucleotides:

PCR is carried out in a volume of 25 μl using an HF Master Mix (Dialat) PCR reaction mixture containing high-precision Pfu polymerase. The concentration of primers in the reaction mixture is 300 mm (forward and reverse), the concentration of magnesium ions is 2.5 mm, cDNA is added in a volume of 5 μl.

The amplified sequences are subjected to electrophoretic separation in a 1.5% agarose gel, DNA fragments of the same size as the theoretical are cut out from the gel and purified using a Cleanup Standard gel double-stranded DNA fragment cleaning kit (Eurogen).

The resulting fragments are amplified in a PCR reaction using pairs of synthetic oligonucleotides selected in such a way that each primer at one end is complementary to the sequence of one gene, and the other end to the gene that will be located next to it in the genetic engineering construct.

Amplification of products for cloning is carried out by PCR. PCR is carried out in a volume of 25 μl using an HF Master Mix (Dialat) PCR reaction mixture containing high-precision Pfu polymerase. The reaction mixture contains primers at a concentration of 300 mm (forward and reverse), magnesium ions at a concentration of 2.5 mm. The gel-purified DNA fragment was added to the reaction mixture in an amount of 20-30 ng.

Amplification Protocol:

- primary denaturation - 2 minutes at 98 ° C

- amplification cycle (× 30)

- denaturation - 20 seconds at 98 ° C

- primer annealing - 30 seconds at 60 ° C

- elongation - 20 seconds at 72 ° C

- final elongation - 2 minutes at 72 ° C.

The amplified nucleotide sequences were subjected to electrophoretic separation in a 1.5% agarose gel, purified using a Cleanup Standard gel (Eurogen) double-stranded DNA fragment cleansing kit, and cross-linked into one 1180 bp fragment. using the NEBuilder® HiFi DNA Assembly (NEB) DNA ligation kit. For this, 100 ng of the desired DNA fragments and 50 ng of the pAL2-T vector, 10 μl of the NEB Master Mix mixture are mixed in one tube and the volume is adjusted with deionized water to a volume of 20 μl. The reaction mixture was incubated for 60 minutes at 50 ° C and chemically competent cells were transformed with it and plated on LB agaric ampicillin-containing medium. Colonies formed after 24 hours are tested for the presence of a genetically engineered insert using the PCR screening method with WT1_OL_F and ABL-GUSB_OL_R primers and Senger sequencing using M13 universal plasmid primers. Bacterial cells carrying the pAL2-T vector with the insertion of cDNA portions of the ABL1, GUSB, WT1, MN1, BAALC genes are inoculated in 0.5 L of LB nutrient medium containing ampicillin at a concentration of 100 μg / ml and cultured at 37 ° C on shaker for 8-12 hours. Plasmid DNA is then isolated from the mass of bacterial cells using the Maxiprep Purification Kit (Qiagen) plasmid DNA isolation kit. Isolated pAL2-T-WBMAG plasmid DNA was analyzed by restriction analysis at NotI and HindIII sites, inserts of the ABL1, GUSB, WT1, MN1, BAALC genes were sequenced and stored at -20 ° C.

For use as DNA catibrators, plasmid DNA pAL2-T-WBMAG is linearized at the NotI restriction enzyme recognition site, the DNA concentration is measured using a NanoDrop spectrophotometer, and the number of copies of plasmid DNA is calculated by the formula:

N = (C * 6.022 × 10 ²³ ) / (L * 650 * 1 × 10 ⁹ ),

where N is the number of copies of DNA in 1 μl of the initial solution, C is the concentration of plasmid DNA in the initial solution (ng / μl), L is the length of plasmid DNA (bp), and decimal dilutions of plasmid DNA containing 10 to 1,000,000 copies are prepared plasmid DNA in 5 microliters of buffer.

For amplification of the BAALC gene region, primers are used:

To amplify a portion of the MN1 gene, oligonucleotide sequences are used:

I

I

To amplify a portion of the WT1 gene, oligonucleotide sequences are used:

For amplification of the ABL1 gene region, oligonucleotide sequences are used:

To amplify a portion of the GUSB gene, oligonucleotide sequences are used:

Using the claimed recombinant plasmid DNA pAL2-T-WBMAG, the expression of the studied genes was analyzed from 108 patients aged 2 to 60 years (median - 27 years). The follow-up period averaged 6 months (1 to 47 months). Assessment of expression was carried out mainly in the post-transplant period (19 patients after related allogeneic hematopoietic stem cell transplantation (alloTGSC), 73 patients after unrelated alloTGSC and 16 patients after haploidentical alloTGSC). In addition, the material of healthy donors was examined (n = 10).

The relative expression of the WT1, BAALC, and MN1 genes in the analyzed samples was determined based on the equations of the calibration curves for the corresponding genes. For this, 5 successive decreasing decimal dilutions of plasmid DNA of known concentration containing amplifiable regions of the analyzed genes were included in each amplification. Amplification efficiency was determined as the angle of inclination of the calibration curve. For all pairs of primers, the amplification efficiency was from 90% to 100%, the correlation coefficient R ^∧ 2 was greater than or equal to 0.990.

To assess the reproducibility of the threshold cycle (Ct) values, all analyzed samples with each pair of primers were amplified in duplicate. The analysis included samples for which the difference in Ct values between repetitions was no more than 0.3 cycles.

The absolute amount of cDNA of each gene was determined based on the equation of the calibration curve using the iQ5 software from Bio-rad. The analysis included samples for which the ABL1 gene cDNA calculated from the calibration curve exceeded 10,000 copies per reaction. A relative WT1 gene expression level of more than 250 copies of the WT1 gene per 10,000 copies of the ABL gene was considered as marker overexpression. For the BAALC gene, hyperexpression was understood as the identification of more than 10 copies of this gene per 100 copies of the ABL gene, for the MN1 gene, the identification of more than 100 copies per 100 copies of the ABL gene.

When assessing the level of relative expression, we revealed the presence of significant differences in the level of WT1 gene expression in healthy donors in bone marrow and peripheral blood (Fig. 2), which coincides with the results of other researchers [K Inoue, Н Ogawa, Т Yamagami, Т Soma, Y Tani, T Tatekawa, Y Oji, H Tamaki, T Kyo, H Dohy, A Hiraoka, T Masaoka, T Kishimoto and H Sugiyama. Long-term follow-up of minimal residual disease in leukemia patients by monitoring WT1 (Wilms tumor gene) expression levels. Blood, 1996, 88, 2267-2278].

When analyzing the data of patients in the post-transplant period, the presence of a direct correlation between the expression level of the studied genes and detectable chimeric transcripts is noteworthy. For the WT1 gene, the correlation coefficient (Spearman's test) was 0.26 (p = 0.01), and for the BAALC gene, 0.27 (p = 0.0001). Comparable results were obtained when analyzing the correlation dependence of the change in the number of cells of the tumor population. When comparing the obtained expression data with the results of the study of donor chimerism (engraftment of donor cells after alloTGSC), an inverse correlation was observed, which is expressed in inverse correlation - the correlation coefficient for WT1 was -0.18 for the WT1 gene (p = 0.0006) and -0.21 for the BAALC gene ( p = 0.00001).

When assessing the cumulative risk of relapse after alloTGSC, data were obtained indicating a high probability of developing a relapse of the disease against the background of overexpression of the studied genes (Fig.3). We have shown that in the presence of overexpression of the WT1 and BAALC genes, there is a significant risk of relapse in patients in the post-transplant period - p = 0.009 and p = 0.027, respectively.

In FIG. 4 presents data indicative of the clinical significance of the evaluation of MN1 gene expression. With an increase in the expression of this gene, an increase in the expression of the WT1 and BAALC genes was also observed, which was accompanied by a decrease in the level of donor chimerism. These changes, in turn, reflected the quantitative values of the tumor clone cell population (the number of blast cells).

Thus, the invention improves the accuracy of the assessment of the relative expression of these genes due to the design of DNA calibrators that combine the sequences of all markers. A comprehensive analysis of the WT1, BAALC, and MN1 genes allows us to evaluate the effectiveness of the therapy in most cases of AML, including in patients without identified chromosomal aberrations.

Claims (2)

Recombinant plasmid DNA pAL2-T-WBMAG used to create DNA calibrators for evaluating the effectiveness of therapy in patients with acute myeloid leukemia, containing an insert of cDNA portions of the WT1, BAALC, MN1, ABL1, GUSB genes amplified in PCR- sequentially stitched in the ligation reaction RV, atgtctgaat tcgacctgca ggtcgaccat atgggagagc tcccaacgcg ttggatgcat 60 agcttgagta ttctatagtg tcacctaaat agcttggcgt aatcatggtc atagctgttt 120 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag 180 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg 240 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg 300 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc 360 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc 420 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg 480 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat 540 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag 600 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga 660 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg 720 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt 780 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac 840 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc 900 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt 960 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc 1020 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc 1080 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg 1140 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag 1200 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg 1260 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt 1320 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca 1380 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca 1440 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc 1500 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt 1560 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg 1620 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc 1680 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg 1740 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga 1800 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga 1860 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta 1920 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg 1980 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact 2040 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata 2100 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt 2160 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa 2220 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg atgcggtgtg aaataccgca 2280 cagatgcgta aggagaaaat accgcatcag gaaattgtaa gcgttaatat tttgttaaaa 2340 ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa 2400 atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac 2460 aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag 2520 ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt 2580 aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg 2640 gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca 2700 agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag 2760 ggcgcgtcca ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct 2820 cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa 2880 cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgaattg taatacgact 2940 cactataggg cgaattgggc ccgacgtcgc atgctcccgg ccgccatggc ggccgcggag 3000 acatttaaaa cgacggccag tgcca atatc gattacctgc ccagctgcct cgagagccag 3060 cccgctattc gcaatcaggg ttacagcacg gtcaccttcg acgggacgcc cagctacggt 3120 cacacgccct cgcaccatgc ggcgcagttc cccaaccact cattcaagca tgaggatccc 3180 atgggccagc agggctcgct gggtgagcag cagtactcgg tggaattcaa aaatcgatac 3240 tcgggcatgc tggaagatgg actgccctcc aatggtgtgc cccgatctac agccccaggt 3300 ggaataccca acccagagaa gaagacgaac tgtgagaccc agtgcccaaa tccccagagc 3360 ctcagctcag gccctctgac ccagaaacag aatggccttc agaccacaga ggctaaaaga 3420 gatgctaaga gaatgcctgc aaaagaagtc accattaatg taacagatag catccaacag 3480 atggacagaa ggaattctaa aatcgatacc attgacctgg actcgctgat ggcagagcac 3540 agcgctgcct ggtacatgcc cgctgacaag gccctggtgg acagcgcgga cgacgacaag 3600 acgttggcgc cctgggagaa ggccaaaccc cagaacccca acagcaaaga agcccacgac 3660 ctccctgcaa acaaggcctc agcatcccag cctggcagcc acttgcagtg cctgtctgtc 3720 cactgcgaat tctaaagaat tcaggaaaac cttctcgctg gacccagtga aaatgacccc 3780 aaccttttcg ttgcactgta tgattttgtg gccagtggag ataacactct aagcataact 3840 aaaggtgaaa agctccgggt cttaggctat aatcacaatg gggaatggtg tgaagcccaa 3900 accaaaaatg gccaaggctg ggtcccaagc aactacatca cgccagtcaa cagtctggag 3960 aaacactcct ggtaccatgg gcctgtgtcc cgcaatgccg ctgagtatct taaagctttt 4020 aagcttgcag aaacgattgc agggtttcac caggatccac ctctgatgtt cactgaagag 4080 taccagaaaa gtctgctaga gcagtaccat ctgggtctgg atcaaaaacg cagaaaatac 4140 gtggttggag agctcatttg gaattttgcc gatttcatga ctgaacagtc accgacgaga 4200 gtgct cgtaa tcatggtcat agctg 4225

Images