CN114164186B

CN114164186B - Oncolytic adenovirus, construction method and application thereof

Info

Publication number: CN114164186B
Application number: CN202111348993.8A
Authority: CN
Inventors: 李稀罕; 邹晓平
Original assignee: Nanjing Drum Tower Hospital
Current assignee: Nanjing Drum Tower Hospital
Priority date: 2021-11-15
Filing date: 2021-11-15
Publication date: 2023-09-12
Anticipated expiration: 2041-11-15
Also published as: CN114164186A

Abstract

The invention provides an oncolytic adenovirus, a construction method and application thereof, and the oncolytic adenovirus can kill tumor cells by combining PDT. The invention provides an oncolytic adenovirus, which is based on pAdEasy-1 adenovirus and is inserted with an E1A, CAT gene and a protein photosensitizer gene. The invention can decompose tumor cells by oncolytic adenovirus, and degrade hydrogen peroxide over-expressed in tumor tissue by virus-expressed catalase to generate oxygen, thereby providing oxygen source for virus-expressed protein photosensitizer, and can kill tumor cells in a combined way, so as to greatly improve the killing effect of tumor cells.

Description

Oncolytic adenovirus, construction method and application thereof

Technical Field

The invention belongs to the field of genetic engineering, and particularly relates to an oncolytic adenovirus, a construction method and application thereof.

Background

Photodynamic therapy (photodynamic therapy, PDT) of tumors is a new method for clinical treatment of tumors, which uses light source targeting to selectively destroy primary and recurrent tumors, avoid normal tissue damage, and improve the quality of life of patients; meanwhile, the kit can also be used as an auxiliary treatment means after tumor surgical excision to reduce the survival of residual tumor cells. PDT can act on various types of tumor cells and is thus widely used. PDT consists mainly of 3 important parts: light of a specific wavelength, a Photosensitizer (PS) and oxygen interact during treatment to produce cytotoxic reactive oxygen species (reactive oxygen species, ROS) that kill tumor cells through apoptosis or necrosis mechanisms.

Oxygen concentration at tissue sites in the course of photodynamic reactions determines the efficacy of photodynamic reactions, but the rapid growth of tumors results in insufficient blood supply to produce hypoxic cells, leading to general hypoxia in the tumor tissue, while local oxygen supply consumption in the PDT process aggravates tumor hypoxia, severely affecting the efficacy of PDT.

The photosensitizer is another key element of PDT, and includes chemical photosensitizer and protein photosensitizer, the chemical photosensitizer has porphyrin photosensitizer which has been practically used, its publication number is CN102174478A, its name is a photosensitizer binding protein/polypeptide and its application in photodynamic gene therapy, and it is disclosed that it can select a photosensitizer binding protein from human liver cDNA phage display library, and said photosensitizer binding protein can be combined with porphyrin photosensitizer in vitro, and can be transfected into various mammal cells by means of adenovirus or slow virus or other modes, and can produce photodynamic therapeutic effect after the artificial regulation and induction expression. However, chemical photosensitizers can lead to long-term skin photosensitivity and pigmentation due to their delayed action of phototoxicity, severely limiting the photodynamic effect of tumors. Protein photosensitizers are hydrolyzed by proteases and cleared in time, so that PDT side effects can be greatly reduced, and protein photosensitizers, such as Killerrad protein photosensitizers, are studied in the current photodynamic therapy of tumors.

Oncolytic adenovirus (oncogenic adenovirus) refers to a class of replication-selective oncolytic adenoviruses (replication-selective oncolytic virus) that have been identified as a hotspot for tumor-targeted therapies and clinical applications, either naturally or genetically engineered to have the function of selectively killing tumor cells. As an oncolytic adenovirus, the adenovirus has a higher application prospect for resisting tumors. However, unmodified adenoviruses suffer from poor ability to target tumor cells. Patent application publication number CN 111212656A, entitled oncolytic adenoviral vector and uses thereof, discloses viral replication restriction by insertion of microrna (miRNA) target sequences into viral genome and can be applied in photodynamic therapy of tumors, however the problem of hypoxia in photodynamic therapy is not solved.

In order to solve the above problems, we have sought an ideal technical solution.

Disclosure of Invention

The invention aims at overcoming the defects of the prior art, thereby providing an oncolytic adenovirus with good photodynamic treatment effect, a construction method thereof and application thereof in tumor treatment medicines.

The technical scheme of the invention is as follows: an oncolytic adenovirus, which is based on a pAdEasy-1 adenovirus, having inserted therein an E1A, CAT gene and a protein photosensitizer gene.

Noun interpretation:

E1A, adenovirus early region 1A, adenovirus early gene 1A

CAT, catalase, catalase

The protein photosensitizer gene can express a photosensitive protein, namely a protein photosensitizer, which is used in PDT, and the sequence of the protein photosensitizer gene cannot exceed 800bp in consideration of vector capacity, and the expressed photosensitive protein optical characteristics are subcellular localization and phototoxic cell ablation. The protein photosensitizer genes currently exist as follows: killered gene and SuperNova2 gene.

According to the technical scheme, when oncolytic adenovirus itself lyses cancer cells, hydrogen peroxide which is expressed in a large amount in cancer tissues is degraded by catalase expressed by the virus to generate oxygen, so that oxygen sources are provided for photosensitive proteins expressed by the virus, and pancreatic cancer cells are killed by combining photodynamic.

As a further improvement of the technical scheme, in order to improve the safety of oncolytic adenoviruses, and considering vector capacity, a miRNA target sequence is inserted at the 3-terminus of E1A. miRNA (microRNA) is a small, non-coding RNA of about 19-25 nucleotides in length that is endogenous to the organism and that is capable of negatively regulating gene expression at post-transcriptional levels by complementary mating with a target sequence on a target gene miRNA, resulting in degradation or translational inhibition of the miRNA. For pancreatic cancer tumor tissue, a miR145-5p target sequence, a miR96 target sequence, a miR143-3p target sequence or a Let-7d-5p target sequence can be inserted into E1A. For bile duct cancer tumor tissue, miR-122-5p target sequence, miR-148a-3p target sequence, miR-204-5p target sequence or miR-494-3p target sequence can be inserted into adenovirus. For gastric cancer tumor tissue, miR-139-5p target sequence, miR-133b target sequence, miR-365a-3p target sequence or miR-365b-3p target sequence can be inserted into E1A. For colorectal tumor tissue, a miR-551b target sequence, a miR-129 target sequence, a miR-138 target sequence or a miR-490-3P target sequence can be inserted in E1A. For liver cancer tumor tissue, miR-551b-5p target sequence, miR-6849-5p target sequence, miR-8060 target sequence or miR-650 target sequence can be inserted into E1A.

As a further improvement of the technical scheme, the miRNA target sequences are the combination of two or more miRNA target sequences, and the more miRNA target sequences are inserted, the better the targeting and the safety of oncolytic adenoviruses are.

As a further improvement of the technical scheme, the protein photosensitizer gene is a killered gene. Killered is the first phototoxic red fluorescent protein completely encoded by a gene, which is obtained by site-directed mutagenesis of two amino acid residues in jellyfish pigment protein, and has fluorescence excitation peaks and emission peaks of 585nm and 610nm, respectively, and a fluorescence quantum yield of 0.25, which represents the fluorescence intensity.

As a further improvement of the technical scheme, the CMV promoter and the polyA tail are respectively inserted into two ends of the E1A, CAT and protein photosensitizer genes.

As an implementation mode of the technical scheme, the E1A, CAT gene and the protein photosensitizer gene are sequentially inserted into pAdEasy-1 adenovirus, and the insertion sequence of the exogenous genes does not influence the integral replication of oncolytic adenovirus and the expression of each exogenous gene.

The construction method of the oncolytic adenovirus specifically comprises the following steps:

(1) Inserting an E1A, CAT gene and a protein photosensitizer gene into a shuttle plasmid pAdTrack-CMV polyclonal site, wherein a miRNA target sequence is inserted into the 3 end of the E1A; (2) Carrying out homologous recombination on the recombinant shuttle pelletization obtained in the step 1 and the skeleton plasmid pAdEasy-1 to obtain a recombinant adenovirus plasmid; (3) Recombinant adenovirus plasmids were packaged in 293A cells.

The invention also protects the application of the oncolytic adenovirus in preparing tumor medicaments, wherein tumors comprise pancreatic cancer, cholangiocarcinoma, liver cancer, colorectal cancer, gastric cancer, lung cancer, renal cancer, melanoma and the like.

The invention also protects a medicine comprising the oncolytic adenovirus, which is used for killing tumor tissues, and can be used before photodynamic therapy or combined application of photodynamic therapy, radiotherapy and chemotherapy and also can be used before tumor immunotherapy because the oncolytic adenovirus has the advantages of good targeting and high curative effect of killing tumor cells by combining PDT.

As a further improvement of the technical scheme, the oncolytic adenovirus is administered by in-situ intratumoral injection or intravenous injection.

Compared with the prior art, the invention has outstanding substantive characteristics and remarkable progress, and in particular, the invention utilizes a genetic engineering means to insert the oncolytic adenovirus into CAT genes and protein photosensitizer genes, and when the oncolytic adenovirus self lyses cancer cells, hydrogen peroxide which is expressed in a large amount in cancer tissues is degraded by catalase expressed by viruses to generate oxygen, thereby providing an oxygen source for the protein photosensitizer expressed by the viruses, and combining photodynamic to kill the cancer cells, thereby greatly improving the killing effect of the cancer cells. Furthermore, the oncolytic adenovirus is also inserted with a miRNA target sequence, so that the safety in adenovirus treatment is improved. The oncolytic adenovirus combined PDT treatment has the advantages of high curative effect, good targeting property and wide application range.

Drawings

FIG. 1 is a schematic diagram of a recombinant oncolytic adenovirus.

FIG. 2 is a morphology of primary oncolytic adenoviruses and control viruses in 293A cells.

FIG. 3 is a graph showing GFP signals from pancreatic cells and pancreatic cancer cells infected with oncolytic adenoviruses.

FIG. 4 is a graph showing the results of detection of the expression of foreign genes in pancreatic cells and pancreatic cancer cells.

FIG. 5 is a graph showing the detection of ROS levels in pancreatic cancer cells, aspc-1.

Fig. 6 is a bar graph of ROS level measurements corresponding to fig. 5.

FIG. 7 is a graph showing the proliferation potency of oncolytic adenoviruses against pancreatic cancer cells.

FIG. 8 is a graph showing the ability of oncolytic adenoviruses to promote apoptosis in pancreatic cancer cells.

Fig. 9 is a magnetic resonance assay of oncolytic adenovirus inhibiting the growth of nude mouse epidermoid tumor.

Detailed Description

The technical scheme of the invention is further described in detail through the following specific embodiments.

Example 1

Recombinant oncolytic adenovirus construction

Based on pAdEasy-1 adenovirus construction system, according to adenovirus replication essential gene E1A, catalase (CAT) gene and KillerRed gene in Genbank database, inserting into shuttle plasmid pAdTrack-CMV multiple cloning site in turn: (1) E1A, (2) CAT, (3) Killerrad, the CMV promoter and polyA tail were inserted at both ends of the foreign gene, respectively. In addition, to ensure the safety of oncolytic adenoviruses and pancreatic cancer specificity, miR145-5p, miR96, miR143-3p and Let-7d-5p target sequences which are remarkably low expressed in pancreatic cancer are also inserted at the 3 end of the E1A gene, and the four miRNA sequences are from a miRBase database. According to the total sequence design, the total sequence synthesis of the Jianjing Jinsri biological company, the total length of the gene fragment is 5917bp, and enzyme cutting sites are designed at two ends, and are respectively: 5'BgI II,3'Hind III. The detailed design is shown in fig. 1 below.

The synthesized insert and shuttle plasmid pAdTrack-CMV were digested with BgI II and HindIII, respectively, the digested products were purified after confirmation by 1.2% agarose electrophoresis, ligated overnight at 4℃with T4 DNA ligase, the ligation products were transformed into DH 5. Alpha. Competent cells, positive clones were selected, and sequencing after shaking confirmed that the inserts were correctly ligated to shuttle plasmid pAdTrack-CMV. The recombinant shuttle plasmid with correct connection is cultivated in an enlarged mode, and the extracted plasmid is preserved and named Vec-AdTrack-E1A-CAT-KR-miRT. Meanwhile, a recombinant shuttle plasmid without exogenous genes is constructed and named as Vec-AdTrack.

The Vec-AdTrack-E1A-CAT-KR-mirT and adenovirus backbone plasmid pAdEasy-1 are subjected to enzyme digestion linearization by using PmeI, then are respectively and electrically transformed into BJ5183 competent cells to construct homologous recombinant plasmids, the homologous recombinant plasmids are transformed into DH5 alpha competent cells after sequencing verification, positive clones are selected for sequencing verification again, and the recombinant plasmids are extracted for preservation and named as Vec-Ad-E1A-CAT-KR-mirT. Meanwhile, a control recombinant plasmid without exogenous gene is constructed and named as Vec-Ad.

The constructed Vec-Ad-E1A-CAT-KR-mirT plasmid is linearized by PacI, transfected into 293A cells with 70% confluency, green fluorescent protein and virus plaques appear under a fluorescent microscope after about 7 days, and oncolytic adenovirus is respectively obtained after three times of virus plaque purification. Extracting virus DNA by using QIAGEN DNA Blood Mini Kit, and then identifying by PCR to obtain primary oncolytic adenovirus, wherein the nucleotide sequence is SEQ ID NO. 1, named Adv-E1A-CAT-KR-miRT, and the seed is preserved at-80 ℃; the recombinant virus without exogenous gene is also constructed and named Adv, the seed is preserved at-80 ℃; amplifying and purifying the obtained two viruses for later use. The morphology of the primary oncolytic adenovirus in 293A cells is shown in FIG. 2.

Example 2

Cell level replication, expression and effects

Infection experiments with successfully constructed oncolytic adenoviruses Adv-E1A-CAT-KR-mirT were performed on HDPE from normal pancreatic cells and on three pancreatic cancer cells PANC-1, asPC-1 and BxPC-3 (four cells purchased from ATCC, USA). Based on the GFP signal (the virus carries the GFP gene itself) 4 days after infection, it was observed that Adv-E1A-CAT-KR-mirT replicated in all three pancreatic cancer cells, whereas the miRNA targeting design was less capable of infecting normal pancreatic cells, as shown in FIG. 3.

Total RNA and total protein in the four cells after infection are extracted and detected by using Real-time RT-PCR and Western blot methods. The results of the examination showed that all of these three exogenous genes were normally expressed in large amounts in pancreatic cancer cells, but the expression levels were very low in normal pancreatic cells as shown in FIG. 4.

Adv-E1A-CAT-KR-mirT and control viruses were each infected with pancreatic cancer cells, aspc-1, and light source at 585nm was irradiated daily for 20 minutes, followed by 3 days of irradiation, and ROS levels in the irradiated Aspc-1 cells were detected by Fluorometric Intracellular ROS Kit (Sigma). The results show that Adv-E1A-CAT-KR-mirT can generate high concentration of ROS after illumination, and that control viruses and non-illuminated oncolytic adenoviruses have low ROS content, as shown in FIGS. 5 and 6.

The effect of oncolytic adenoviruses and control viruses on the proliferation capacity of both pancreatic cancer cells MiaPaCa-2 and Aspc-1 at different multiplicity of infection was examined using CCK8 assay kit (Japan Co.). The test results show that the oncolytic adenovirus in the two pancreatic cancer cells has stronger cell proliferation inhibition capacity than the control virus after illumination, and the good pancreatic cancer proliferation inhibition capacity is shown as shown in figure 7.

The pro-apoptotic capacity of oncolytic adenoviruses was examined by flow cytometry, and the examination results are shown in fig. 8, in which oncolytic adenoviruses have a stronger pro-apoptotic capacity than control viruses after illumination in pancreatic cancer cells.

Example 3

Animal experiment

20 BALB/C-nu male nude mice are taken, 4-6 weeks old and the weight is 18-20 g. PANC-1 (low differentiation) was routinely subcultured in vitro. Cells in the logarithmic growth phase were digested with 0.125% pancreatin, washed 3 times with sterile PBS, and made into single cell suspensions. Nude mice were fixed and subcutaneously transplanted with a total injection of 200 μl (containing 2×10) ⁶ Individual cells). After operation, the nude mice are raised in SPF animal houses, and the weight, the appetite and the like of the nude mice are recorded every day. The oncolytic adenovirus was injected 2 times (3 days apart) at a total dose of 1 x 10 at 14 days post-tumor implantation ⁹ pfu, irradiated with 585nm laser for 20min daily from the start of oncolytic adenovirus injection, for 5 consecutive days. Tumor size was measured by small animal magnetic resonance after 2 weeks. The detection result is shown in fig. 8, and the oncolytic adenovirus has a remarkable tumor growth inhibition effect in combination with illumination.

Finally, it should be noted that the above-mentioned embodiments are only for illustrating the technical scheme of the present invention and are not limiting; while the invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that the present invention may be modified and equivalents substituted for elements thereof; without departing from the spirit of the invention, it is intended to cover the scope of the invention as claimed.

Sequence listing

<110> Nanjing drummer Hospital

<120> oncolytic adenovirus, its construction method and use

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catcatcaat aatatacctt attttggatt gaagccaata tgataatgag ggggtggagt 60

ttgtgacgtg gcgcggggcg tgggaacggg gcgggtgacg tagtagtgtg gcggaagtgt 120

gatgttgcaa gtgtggcgga acacatgtaa gcgacggatg tggcaaaagt gacgtttttg 180

gtgtgcgccg gtgtacacag gaagtgacaa ttttcgcgcg gttttaggcg gatgttgtag 240

taaatttggg cgtaaccgag taagatttgg ccattttcgc gggaaaactg aataagagga 300

agtgaaatct gaataatttt gtgttactca tagcgcgtaa tatttgtcta gggccgcggg 360

gactttgacc gtttacgtgg agactcgccc aggtgttttt ctcaggtgtt ttccgcgttc 420

cgggtcaaag ttggcgtttt attattatag tcagctgacg tgtagtgtat ttatacccgg 480

tgagttcctc aagaggccac tcttgagtgc cagcgagtag agttttctcc tccgagccgc 540

tccgacaccg ggactgaaaa tgagacatat tatctgccac ggaggtgtta ttaccgaaga 600

aatggccgcc agtcttttgg accagctgat cgaagaggta ctggctgata atcttccacc 660

tcctagccat tttgaaccac ctacccttca cgaactgtat gatttagacg tgacggcccc 720

cgaagatccc aacgaggagg cggtttcgca gatttttccc gactctgtaa tgttggcggt 780

gcaggaaggg attgacttac tcacttttcc gccggcgccc ggttctccgg agccgcctca 840

cctttcccgg cagcccgagc agccggagca gagagccttg ggtccggttt ctatgccaaa 900

ccttgtaccg gaggtgatcg atcttacctg ccacgaggct ggctttccac ccagtgacga 960

cgaggatgaa gagggtgagg agtttgtgtt agattatgtg gagcaccccg ggcacggttg 1020

caggtcttgt cattatcacc ggaggaatac gggggaccca gatattatgt gttcgctttg 1080

ctatatgagg acctgtggca tgtttgtcta cagtaagtga aaattatggg cagtgggtga 1140

tagagtggtg ggtttggtgt ggtaattttt tttttaattt ttacagtttt gtggtttaaa 1200

gaattttgta ttgtgatttt tttaaaaggt cctgtgtctg aacctgagcc tgagcccgag 1260

ccagaaccgg agcctgcaag acctacccgc cgtcctaaaa tggcgcctgc tatcctgaga 1320

cgcccgacat cacctgtgtc tagagaatgc aatagtagta cggatagctg tgactccggt 1380

ccttctaaca cacctcctga gatacacccg gtggtcccgc tgtgccccat taaaccagtt 1440

gccgtgagag ttggtgggcg tcgccaggct gtggaatgta tcgaggactt gcttaacgag 1500

cctgggcaac ctttggactt gagctgtaaa cgccccaggc cataaggtgt aaacctgtga 1560

ttgcgtgtgt ggttaacgcc tttgtttgct gaatgagttg atgtaagttt aataaagggt 1620

gagataatgt ttaacttgca tggcgtgtta aatggggcgg ggcttaaagg gtatataatg 1680

cgccgtgggc taatcttggt tacatctgac ctcatggagg cttgggagtg tttggaagat 1740

ttttctgctg tgcgtaactt gctggaacag agctctaaca gtacctcttg gttttggagg 1800

tttctgtggg gctcatccca ggcaaagtta gtctgcagaa ttaaggagga ttacaagtgg 1860

gaatttgaag agcttttgaa atcctgtggt gagctgtttg attctttgaa tctgggtcac 1920

caggcgcttt tccaagagaa ggtcatcaag actttggatt tttccacacc ggggcgcgct 1980

gcggctgctg ttgctttttt gagttttata aaggataagc accaactcgt ttgatggaag 2040

cattgtgagc tcatatttga caacgcgcat gcccccatgg gccggggtgc gtcagaatgt 2100

gatgggctcc agcattgatg gtcgccccgt cctgcccgca aactctacta ccttgaccta 2160

cgagaccgtg tctggaacgc cgttggagac tgcagcctcc gccgccgctt cagccgctgc 2220

agccaccgcc cgcgggattg tgactgactt tgctttcctg agcccgcttg caagcagtgc 2280

agcttcccgt tcatccgccc gcgatgacaa gttgacggct cttttggcac aattggattc 2340

tttgacccgg gaacttaatg tcgtttctca gcagctgttg gatctgcgcc agcaggtttc 2400

tgccctgaag gcttcctccc ctcccaatgc ggtttaaaac ataaataaaa aaccagactc 2460

tgtttggatt tggatcaagc aagtgtcttg ctgtctttat ttaggggttt tgcgcgcgcg 2520

gtaggcccgg gaccagcggt ctcggtcgtt gagggtcctg tgtatttttt ccaggacgtg 2580

gtaaaggtga ctctggatgt tcagatacat gggcataagc ccgtctctgg ggtggaggta 2640

gcaccactgc agagcttcat gctgcggggt ggtgttgtag atgatccagt cgtagcagga 2700

gcgctgggcg tggtgcctaa aaatgtcttt cagtagcaag ctgattgcca ggggcaggcc 2760

cttggtgtaa gtgtttacaa agcggttaag ctgggatggg tgcatacgtg gggatatgag 2820

atgcatcttg gactgtattt ttaggttggc tatgttccca gccatatccc tccggggatt 2880

catgttgtgc agaaccacca gcacagtgta tccggtgcac ttgggaaatt tgtcatgtag 2940

cttagaagga aatgcgtgga agaacttgga gacgcccttg tgacctccaa gattttccat 3000

gcattcgtcc ataatgatgg caatgggccc acgggcggcg gcctgggcga agatatttct 3060

gggatcacta acgtcatagt tgtgttccag gatgagatcg tcataggcca tttttacaaa 3120

gcgcgggcgg agggtgccag actgcggtat aatggttcca tccggcccag gggcgtagtt 3180

accctcacag atttgcattt cccacgcttt gagttcagat ggggggatca tgtctacctg 3240

cggggcgatg aagaaaacgg tttccggggt aggggagatc agctgggaag aaagcaggtt 3300

cctgagcagc tgcgacttac cgcagccggt gggcccgtaa atcacaccta ttaccgggtg 3360

caactggtag ttaagagagc tgcagctgcc gtcatccctg agcagggggg ccacttcgtt 3420

aagcatgtcc ctgactcgca tgttttccct gaccaaatcc gccagaaggc gctcgccgcc 3480

cagcgatagc agttcttgca aggaagcaaa gtttttcaac ggtttgagac cgtccgccgt 3540

aggcatgctt ttgagcgttt gaccaagcag ttccaggcgg tcccacagct cggtcacctg 3600

ctctacggca tctcgatcca gcatatctcc tcgtttcgcg ggttggggcg gctttcgctg 3660

tacggcagta gtcggtgctc gtccagacgg gccagggtca tgtctttcca cgggcgcagg 3720

gtcctcgtca gcgtagtctg ggtcacggtg aaggggtgcg ctccgggctg cgcgctggcc 3780

agggtgcgct tgaggctggt cctgctggtg ctgaagcgct gccggtcttc gccctgcgcg 3840

tcggccaggt agcatttgac catggtgtca tagtccagcc cctccgcggc gtggcccttg 3900

gcgcgcagct tgcccttgga ggaggcgccg cacgaggggc agtgcagact tttgagggcg 3960

tagagcttgg gcgcgagaaa taccgattcc ggggagtagg catccgcgcc gcaggccccg 4020

cagacggtct cgcattccac gagccaggtg agctctggcc gttcggggtc aaaaaccagg 4080

tttcccccat gctttttgat gcgtttctta cctctggttt ccatgagccg gtgtccacgc 4140

tcggtgacga aaaggctgtc cgtgtccccg tatacagact tgagaggcct gtcctcgagc 4200

ggtgttccgc ggtcctcctc gtatagaaac tcggaccact ctgagacaaa ggctcgcgtc 4260

caggccagca cgaaggaggc taagtgggag gggtagcggt cgttgtccac tagggggtcc 4320

actcgctcca gggtgtgaag acacatgtcg ccctcttcgg catcaaggaa ggtgattggt 4380

ttgtaggtgt aggccacgtg accgggtgtt cctgaagggg ggctataaaa gggggtgggg 4440

gcgcgttcgt cctcactctc ttccgcatcg ctgtctgcga gggccagctg ttggggtgag 4500

tactccctct gaaaagcggg catgacttct gcgctaagat tgtcagtttc caaaaacgag 4560

gaggatttga tattcacctg gcccgcggtg atgcctttga gggtggccgc atccatctgg 4620

tcagaaaaga caatcttttt gttgtcaagc ttggtggcaa acgacccgta gagggcgttg 4680

gacagcaact tggcgatgga gcgcagggtt tggtttttgt cgcgatcggc gcgctccttg 4740

gccgcgatgt ttagctgcac gtattcgcgc gcaacgcacc gccattcggg aaagacggtg 4800

gtgcgctcgt cgggcaccag gtgcacgcgc caaccgcggt tgtgcagggt gacaaggtca 4860

acgctggtgg ctacctctcc gcgtaggcgc tcgttggtcc agcagaggcg gccgcccttg 4920

cgcgagcaga atggcggtag ggggtctagc tgcgtctcgt ccggggggtc tgcgtccacg 4980

gtaaagaccc cgggcagcag gcgcgcgtcg aagtagtcta tcttgcatcc ttgcaagtct 5040

agcgcctgct gccatgcgcg ggcggcaagc gcgcgctcgt atgggttgag tgggggaccc 5100

catggcatgg ggtgggtgag cgcggaggcg tacatgccgc aaatgtcgta aacgtagagg 5160

ggctctctga gtattccaag atatgtaggg tagcatcttc caccgcggat gctggcgcgc 5220

acgtaatcgt atagttcgtg cgagggagcg aggaggtcgg gaccgaggtt gctacgggcg 5280

ggctgctctg ctcggaagac tatctgcctg aagatggcat gtgagttgga tgatatggtt 5340

ggacgctgga agacgttgaa gctggcgtct gtgagaccta ccgcgtcacg cacgaaggag 5400

gcgtaggagt cgcgcagctt gttgaccagc tcggcggtga cctgcacgtc tagggcgcag 5460

tagtccaggg tttccttgat gatgtcatac ttatcctgtc cctttttttt ccacagctcg 5520

cggttgagga caaactcttc gcggtctttc cagtactctt ggatcggaaa cccgtcggcc 5580

tccgaacggt aagagcctag catgtagaac tggttgacgg cctggtaggc gcagcatccc 5640

ttttctacgg gtagcgcgta tgcctgcgcg gccttccgga gcgaggtgtg ggtgagcgca 5700

aaggtgtccc tgaccatgac tttgaggtac tggtatttga agtcagtgtc gtcgcatccg 5760

ccctgctccc agagcaaaaa gtccgtgcgc tttttggaac gcggatttgg cagggcgaag 5820

gtgacatcgt tgaagagtat ctttcccgcg cgaggcataa agttgcgtgt gatgcggaag 5880

ggtcccggca cctcggaacg gttgttaatt acctgggcgg cgagcacgat ctcgtcaaag 5940

ccgttgatgt tgtggcccac aatgtaaagt tccaagaagc gcgggatgcc cttgatggaa 6000

ggcaattttt taagttcctc gtaggtgagc tcttcagggg agctgagccc gtgctctgaa 6060

agggcccagt ctgcaagatg agggttggaa gcgacgaatg agctccacag gtcacgggcc 6120

attagcattt gcaggtggtc gcgaaaggtc ctaaactggc gacctatggc cattttttct 6180

ggggtgatgc agtagaaggt aagcgggtct tgttcccagc ggtcccatcc aaggttcgcg 6240

gctaggtctc gcgcggcagt cactagaggc tcatctccgc cgaacttcat gaccagcatg 6300

aagggcacga gctgcttccc aaaggccccc atccaagtat aggtctctac atcgtaggtg 6360

acaaagagac gctcggtgcg aggatgcgag ccgatcggga agaactggat ctcccgccac 6420

caattggagg agtggctatt gatgtggtga aagtagaagt ccctgcgacg ggccgaacac 6480

tcgtgctggc ttttgtaaaa acgtgcgcag tactggcagc ggtgcacggg ctgtacatcc 6540

tgcacgaggt tgacctgacg accgcgcaca aggaagcaga gtgggaattt gagcccctcg 6600

cctggcgggt ttggctggtg gtcttctact tcggctgctt gtccttgacc gtctggctgc 6660

tcgaggggag ttacggtgga tcggaccacc acgccgcgcg agcccaaagt ccagatgtcc 6720

gcgcgcggcg gtcggagctt gatgacaaca tcgcgcagat gggagctgtc catggtctgg 6780

agctcccgcg gcgtcaggtc aggcgggagc tcctgcaggt ttacctcgca tagacgggtc 6840

agggcgcggg ctagatccag gtgataccta atttccaggg gctggttggt ggcggcgtcg 6900

atggcttgca agaggccgca tccccgcggc gcgactacgg taccgcgcgg cgggcggtgg 6960

gccgcggggg tgtccttgga tgatgcatct aaaagcggtg acgcgggcga gcccccggag 7020

gtaggggggg ctccggaccc gccgggagag ggggcagggg cacgtcggcg ccgcgcgcgg 7080

gcaggagctg gtgctgcgcg cgtaggttgc tggcgaacgc gacgacgcgg cggttgatct 7140

cctgaatctg gcgcctctgc gtgaagacga cgggcccggt gagcttgagc ctgaaagaga 7200

gttcgacaga atcaatttcg gtgtcgttga cggcggcctg gcgcaaaatc tcctgcacgt 7260

ctcctgagtt gtcttgatag gcgatctcgg ccatgaactg ctcgatctct tcctcctgga 7320

gatctccgcg tccggctcgc tccacggtgg cggcgaggtc gttggaaatg cgggccatga 7380

gctgcgagaa ggcgttgagg cctccctcgt tccagacgcg gctgtagacc acgccccctt 7440

cggcatcgcg ggcgcgcatg accacctgcg cgagattgag ctccacgtgc cgggcgaaga 7500

cggcgtagtt tcgcaggcgc tgaaagaggt agttgagggt ggtggcggtg tgttctgcca 7560

cgaagaagta cataacccag cgtcgcaacg tggattcgtt gatatccccc aaggcctcaa 7620

ggcgctccat ggcctcgtag aagtccacgg cgaagttgaa aaactgggag ttgcgcgccg 7680

acacggttaa ctcctcctcc agaagacgga tgagctcggc gacagtgtcg cgcacctcgc 7740

gctcaaaggc tacaggggcc tcttcttctt cttcaatctc ctcttccata agggcctccc 7800

cttcttcttc ttctggcggc ggtgggggag gggggacacg gcggcgacga cggcgcaccg 7860

ggaggcggtc gacaaagcgc tcgatcatct ccccgcggcg acggcgcatg gtctcggtga 7920

cggcgcggcc gttctcgcgg gggcgcagtt ggaagacgcc gcccgtcatg tcccggttat 7980

gggttggcgg ggggctgcca tgcggcaggg atacggcgct aacgatgcat ctcaacaatt 8040

gttgtgtagg tactccgccg ccgagggacc tgagcgagtc cgcatcgacc ggatcggaaa 8100

acctctcgag aaaggcgtct aaccagtcac agtcgcaagg taggctgagc accgtggcgg 8160

gcggcagcgg gcggcggtcg gggttgtttc tggcggaggt gctgctgatg atgtaattaa 8220

agtaggcggt cttgagacgg cggatggtcg acagaagcac catgtccttg ggtccggcct 8280

gctgaatgcg caggcggtcg gccatgcccc aggcttcgtt ttgacatcgg cgcaggtctt 8340

tgtagtagtc ttgcatgagc ctttctaccg gcacttcttc ttctccttcc tcttgtcctg 8400

catctcttgc atctatcgct gcggcggcgg cggagtttgg ccgtaggtgg cgccctcttc 8460

ctcccatgcg tgtgaccccg aagcccctca tcggctgaag cagggctagg tcggcgacaa 8520

cgcgctcggc taatatggcc tgctgcacct gcgtgagggt agactggaag tcatccatgt 8580

ccacaaagcg gtggtatgcg cccgtgttga tggtgtaagt gcagttggcc ataacggacc 8640

agttaacggt ctggtgaccc ggctgcgaga gctcggtgta cctgagacgc gagtaagccc 8700

tcgagtcaaa tacgtagtcg ttgcaagtcc gcaccaggta ctggtatccc accaaaaagt 8760

gcggcggcgg ctggcggtag aggggccagc gtagggtggc cggggctccg ggggcgagat 8820

cttccaacat aaggcgatga tatccgtaga tgtacctgga catccaggtg atgccggcgg 8880

cggtggtgga ggcgcgcgga aagtcgcgga cgcggttcca gatgttgcgc agcggcaaaa 8940

agtgctccat ggtcgggacg ctctggccgg tcaggcgcgc gcaatcgttg acgctctaga 9000

ccgtgcaaaa ggagagcctg taagcgggca ctcttccgtg gtctggtgga taaattcgca 9060

agggtatcat ggcggacgac cggggttcga gccccgtatc cggccgtccg ccgtgatcca 9120

tgcggttacc gcccgcgtgt cgaacccagg tgtgcgacgt cagacaacgg gggagtgctc 9180

cttttggctt ccttccaggc gcggcggctg ctgcgctagc ttttttggcc actggccgcg 9240

cgcagcgtaa gcggttaggc tggaaagcga aagcattaag tggctcgctc cctgtagccg 9300

gagggttatt ttccaagggt tgagtcgcgg gacccccggt tcgagtctcg gaccggccgg 9360

actgcggcga acgggggttt gcctccccgt catgcaagac cccgcttgca aattcctccg 9420

gaaacaggga cgagcccctt ttttgctttt cccagatgca tccggtgctg cggcagatgc 9480

gcccccctcc tcagcagcgg caagagcaag agcagcggca gacatgcagg gcaccctccc 9540

ctcctcctac cgcgtcagga ggggcgacat ccgcggttga cgcggcagca gatggtgatt 9600

acgaaccccc gcggcgccgg gcccggcact acctggactt ggaggagggc gagggcctgg 9660

cgcggctagg agcgccctct cctgagcggt acccaagggt gcagctgaag cgtgatacgc 9720

gtgaggcgta cgtgccgcgg cagaacctgt ttcgcgaccg cgagggagag gagcccgagg 9780

agatgcggga tcgaaagttc cacgcagggc gcgagctgcg gcatggcctg aatcgcgagc 9840

ggttgctgcg cgaggaggac tttgagcccg acgcgcgaac cgggattagt cccgcgcgcg 9900

cacacgtggc ggccgccgac ctggtaaccg catacgagca gacggtgaac caggagatta 9960

actttcaaaa aagctttaac aaccacgtgc gtacgcttgt ggcgcgcgag gaggtggcta 10020

taggactgat gcatctgtgg gactttgtaa gcgcgctgga gcaaaaccca aatagcaagc 10080

cgctcatggc gcagctgttc cttatagtgc agcacagcag ggacaacgag gcattcaggg 10140

atgcgctgct aaacatagta gagcccgagg gccgctggct gctcgatttg ataaacatcc 10200

tgcagagcat agtggtgcag gagcgcagct tgagcctggc tgacaaggtg gccgccatca 10260

actattccat gcttagcctg ggcaagtttt acgcccgcaa gatataccat accccttacg 10320

ttcccataga caaggaggta aagatcgagg ggttctacat gcgcatggcg ctgaaggtgc 10380

ttaccttgag cgacgacctg ggcgtttatc gcaacgagcg catccacaag gccgtgagcg 10440

tgagccggcg gcgcgagctc agcgaccgcg agctgatgca cagcctgcaa agggccctgg 10500

ctggcacggg cagcggcgat agagaggccg agtcctactt tgacgcgggc gctgacctgc 10560

gctgggcccc aagccgacgc gccctggagg cagctggggc cggacctggg ctggcggtgg 10620

cacccgcgcg cgctggcaac gtcggcggcg tggaggaata tgacgaggac gatgagtacg 10680

agccagagga cggcgagtac taagcggtga tgtttctgat cagatgatgc aagacgcaac 10740

ggacccggcg gtgcgggcgg cgctgcagag ccagccgtcc ggccttaact ccacggacga 10800

ctggcgccag gtcatggacc gcatcatgtc gctgactgcg cgcaatcctg acgcgttccg 10860

gcagcagccg caggccaacc ggctctccgc aattctggaa gcggtggtcc cggcgcgcgc 10920

aaaccccacg cacgagaagg tgctggcgat cgtaaacgcg ctggccgaaa acagggccat 10980

ccggcccgac gaggccggcc tggtctacga cgcgctgctt cagcgcgtgg ctcgttacaa 11040

cagcggcaac gtgcagacca acctggaccg gctggtgggg gatgtgcgcg aggccgtggc 11100

gcagcgtgag cgcgcgcagc agcagggcaa cctgggctcc atggttgcac taaacgcctt 11160

cctgagtaca cagcccgcca acgtgccgcg gggacaggag gactacacca actttgtgag 11220

cgcactgcgg ctaatggtga ctgagacacc gcaaagtgag gtgtaccagt ctgggccaga 11280

ctattttttc cagaccagta gacaaggcct gcagaccgta aacctgagcc aggctttcaa 11340

aaacttgcag gggctgtggg gggtgcgggc tcccacaggc gaccgcgcga ccgtgtctag 11400

cttgctgacg cccaactcgc gcctgttgct gctgctaata gcgcccttca cggacagtgg 11460

cagcgtgtcc cgggacacat acctaggtca cttgctgaca ctgtaccgcg aggccatagg 11520

tcaggcgcat gtggacgagc atactttcca ggagattaca agtgtcagcc gcgcgctggg 11580

gcaggaggac acgggcagcc tggaggcaac cctaaactac ctgctgacca accggcggca 11640

gaagatcccc tcgttgcaca gtttaaacag cgaggaggag cgcattttgc gctacgtgca 11700

gcagagcgtg agccttaacc tgatgcgcga cggggtaacg cccagcgtgg cgctggacat 11760

gaccgcgcgc aacatggaac cgggcatgta tgcctcaaac cggccgttta tcaaccgcct 11820

aatggactac ttgcatcgcg cggccgccgt gaaccccgag tatttcacca atgccatctt 11880

gaacccgcac tggctaccgc cccctggttt ctacaccggg ggattcgagg tgcccgaggg 11940

taacgatgga ttcctctggg acgacataga cgacagcgtg ttttccccgc aaccgcagac 12000

cctgctagag ttgcaacagc gcgagcaggc agaggcggcg ctgcgaaagg aaagcttccg 12060

caggccaagc agcttgtccg atctaggcgc tgcggccccg cggtcagatg ctagtagccc 12120

atttccaagc ttgatagggt ctcttaccag cactcgcacc acccgcccgc gcctgctggg 12180

cgaggaggag tacctaaaca actcgctgct gcagccgcag cgcgaaaaaa acctgcctcc 12240

ggcatttccc aacaacggga tagagagcct agtggacaag atgagtagat ggaagacgta 12300

cgcgcaggag cacagggacg tgccaggccc gcgcccgccc acccgtcgtc aaaggcacga 12360

ccgtcagcgg ggtctggtgt gggaggacga tgactcggca gacgacagca gcgtcctgga 12420

tttgggaggg agtggcaacc cgtttgcgca ccttcgcccc aggctgggga gaatgtttta 12480

aaaaaaaaaa agcatgatgc aaaataaaaa actcaccaag gccatggcac cgagcgttgg 12540

ttttcttgta ttccccttag tatgcggcgc gcggcgatgt atgaggaagg tcctcctccc 12600

tcctacgaga gtgtggtgag cgcggcgcca gtggcggcgg cgctgggttc tcccttcgat 12660

gctcccctgg acccgccgtt tgtgcctccg cggtacctgc ggcctaccgg ggggagaaac 12720

agcatccgtt actctgagtt ggcaccccta ttcgacacca cccgtgtgta cctggtggac 12780

aacaagtcaa cggatgtggc atccctgaac taccagaacg accacagcaa ctttctgacc 12840

acggtcattc aaaacaatga ctacagcccg ggggaggcaa gcacacagac catcaatctt 12900

gacgaccggt cgcactgggg cggcgacctg aaaaccatcc tgcataccaa catgccaaat 12960

gtgaacgagt tcatgtttac caataagttt aaggcgcggg tgatggtgtc gcgcttgcct 13020

actaaggaca atcaggtgga gctgaaatac gagtgggtgg agttcacgct gcccgagggc 13080

aactactccg agaccatgac catagacctt atgaacaacg cgatcgtgga gcactacttg 13140

aaagtgggca gacagaacgg ggttctggaa agcgacatcg gggtaaagtt tgacacccgc 13200

aacttcagac tggggtttga ccccgtcact ggtcttgtca tgcctggggt atatacaaac 13260

gaagccttcc atccagacat cattttgctg ccaggatgcg gggtggactt cacccacagc 13320

cgcctgagca acttgttggg catccgcaag cggcaaccct tccaggaggg ctttaggatc 13380

acctacgatg atctggaggg tggtaacatt cccgcactgt tggatgtgga cgcctaccag 13440

gcgagcttga aagatgacac cgaacagggc gggggtggcg caggcggcag caacagcagt 13500

ggcagcggcg cggaagagaa ctccaacgcg gcagccgcgg caatgcagcc ggtggaggac 13560

atgaacgatc atgccattcg cggcgacacc tttgccacac gggctgagga gaagcgcgct 13620

gaggccgaag cagcggccga agctgccgcc cccgctgcgc aacccgaggt cgagaagcct 13680

cagaagaaac cggtgatcaa acccctgaca gaggacagca agaaacgcag ttacaaccta 13740

ataagcaatg acagcacctt cacccagtac cgcagctggt accttgcata caactacggc 13800

gaccctcaga ccggaatccg ctcatggacc ctgctttgca ctcctgacgt aacctgcggc 13860

tcggagcagg tctactggtc gttgccagac atgatgcaag accccgtgac cttccgctcc 13920

acgcgccaga tcagcaactt tccggtggtg ggcgccgagc tgttgcccgt gcactccaag 13980

agcttctaca acgaccaggc cgtctactcc caactcatcc gccagtttac ctctctgacc 14040

cacgtgttca atcgctttcc cgagaaccag attttggcgc gcccgccagc ccccaccatc 14100

accaccgtca gtgaaaacgt tcctgctctc acagatcacg ggacgctacc gctgcgcaac 14160

agcatcggag gagtccagcg agtgaccatt actgacgcca gacgccgcac ctgcccctac 14220

gtttacaagg ccctgggcat agtctcgccg cgcgtcctat cgagccgcac tttttgagca 14280

agcatgtcca tccttatatc gcccagcaat aacacaggct ggggcctgcg cttcccaagc 14340

aagatgtttg gcggggccaa gaagcgctcc gaccaacacc cagtgcgcgt gcgcgggcac 14400

taccgcgcgc cctggggcgc gcacaaacgc ggccgcactg ggcgcaccac cgtcgatgac 14460

gccatcgacg cggtggtgga ggaggcgcgc aactacacgc ccacgccgcc accagtgtcc 14520

acagtggacg cggccattca gaccgtggtg cgcggagccc ggcgctatgc taaaatgaag 14580

agacggcgga ggcgcgtagc acgtcgccac cgccgccgac ccggcactgc cgcccaacgc 14640

gcggcggcgg ccctgcttaa ccgcgcacgt cgcaccggcc gacgggcggc catgcgggcc 14700

gctcgaaggc tggccgcggg tattgtcact gtgcccccca ggtccaggcg acgagcggcc 14760

gccgcagcag ccgcggccat tagtgctatg actcagggtc gcaggggcaa cgtgtattgg 14820

gtgcgcgact cggttagcgg cctgcgcgtg cccgtgcgca cccgcccccc gcgcaactag 14880

attgcaagaa aaaactactt agactcgtac tgttgtatgt atccagcggc ggcggcgcgc 14940

aacgaagcta tgtccaagcg caaaatcaaa gaagagatgc tccaggtcat cgcgccggag 15000

atctatggcc ccccgaagaa ggaagagcag gattacaagc cccgaaagct aaagcgggtc 15060

aaaaagaaaa agaaagatga tgatgatgaa cttgacgacg aggtggaact gctgcacgct 15120

accgcgccca ggcgacgggt acagtggaaa ggtcgacgcg taaaacgtgt tttgcgaccc 15180

ggcaccaccg tagtctttac gcccggtgag cgctccaccc gcacctacaa gcgcgtgtat 15240

gatgaggtgt acggcgacga ggacctgctt gagcaggcca acgagcgcct cggggagttt 15300

gcctacggaa agcggcataa ggacatgctg gcgttgccgc tggacgaggg caacccaaca 15360

cctagcctaa agcccgtaac actgcagcag gtgctgcccg cgcttgcacc gtccgaagaa 15420

aagcgcggcc taaagcgcga gtctggtgac ttggcaccca ccgtgcagct gatggtaccc 15480

aagcgccagc gactggaaga tgtcttggaa aaaatgaccg tggaacctgg gctggagccc 15540

gaggtccgcg tgcggccaat caagcaggtg gcgccgggac tgggcgtgca gaccgtggac 15600

gttcagatac ccactaccag tagcaccagt attgccaccg ccacagaggg catggagaca 15660

caaacgtccc cggttgcctc agcggtggcg gatgccgcgg tgcaggcggt cgctgcggcc 15720

gcgtccaaga cctctacgga ggtgcaaacg gacccgtgga tgtttcgcgt ttcagccccc 15780

cggcgcccgc gcggttcgag gaagtacggc gccgccagcg cgctactgcc cgaatatgcc 15840

ctacatcctt ccattgcgcc tacccccggc tatcgtggct acacctaccg ccccagaaga 15900

cgagcaacta cccgacgccg aaccaccact ggaacccgcc gccgccgtcg ccgtcgccag 15960

cccgtgctgg ccccgatttc cgtgcgcagg gtggctcgcg aaggaggcag gaccctggtg 16020

ctgccaacag cgcgctacca ccccagcatc gtttaaaagc cggtctttgt ggttcttgca 16080

gatatggccc tcacctgccg cctccgtttc ccggtgccgg gattccgagg aagaatgcac 16140

cgtaggaggg gcatggccgg ccacggcctg acgggcggca tgcgtcgtgc gcaccaccgg 16200

cggcggcgcg cgtcgcaccg tcgcatgcgc ggcggtatcc tgcccctcct tattccactg 16260

atcgccgcgg cgattggcgc cgtgcccgga attgcatccg tggccttgca ggcgcagaga 16320

cactgattaa aaacaagttg catgtggaaa aatcaaaata aaaagtctgg actctcacgc 16380

tcgcttggtc ctgtaactat tttgtagaat ggaagacatc aactttgcgt ctctggcccc 16440

gcgacacggc tcgcgcccgt tcatgggaaa ctggcaagat atcggcacca gcaatatgag 16500

cggtggcgcc ttcagctggg gctcgctgtg gagcggcatt aaaaatttcg gttccaccgt 16560

taagaactat ggcagcaagg cctggaacag cagcacaggc cagatgctga gggataagtt 16620

gaaagagcaa aatttccaac aaaaggtggt agatggcctg gcctctggca ttagcggggt 16680

ggtggacctg gccaaccagg cagtgcaaaa taagattaac agtaagcttg atccccgccc 16740

tcccgtagag gagcctccac cggccgtgga gacagtgtct ccagaggggc gtggcgaaaa 16800

gcgtccgcgc cccgcaggga agaaactctg gtgacgcaaa tagacgagcc tccctcgtac 16860

gaggaggcac taaagcaagg cctgcccacc acccgtccca tcgcgcccat ggctaccgga 16920

gtgctgggcc agcacacacc cgtaacgctg gacctgcctc cccccgccga cacccagcag 16980

aaacctgtgc tgccaggccc gaccgccgtt gttgtaaccc gtcctagccg cgcgtccctg 17040

cgccgcgccg ccagcggtcc gcgatcgttg cggcccgtag ccagtggcaa ctggcaaagc 17100

acactgaaca gcatcgtggg tctgggggtg caatccctga agcgccgacg atgcttctga 17160

atagctaacg tgtcgtatgt gtgtcatgta tgcgtccatg tcgccgccag aggagctgct 17220

gagccgccgc gcgcccgctt tccaagatgg ctaccccttc gatgatgccg cagtggtctt 17280

acatgcacat ctcgggccag gacgcctcgg agtacctgag ccccgggctg gtgcagtttg 17340

cccgcgccac cgagacgtac ttcagcctga ataacaagtt tagaaacccc acggtggcgc 17400

ctacgcacga cgtgaccaca gaccggtccc agcgtttgac gctgcggttc atccctgtgg 17460

accgtgagga tactgcgtac tcgtacaagg cgcggttcac cctagctgtg ggtgataacc 17520

gtgtgctgga catggcttcc acgtactttg acatccgcgg cgtgctggac aggggcccta 17580

cttttaagcc ctactctggc actgcctaca acgccctggc tcccaagggt gccccaaatc 17640

cttgcgaatg ggatgaagct gctactgctc ttgaaataaa cctagaagaa gaggacgatg 17700

acaacgaaga cgaagtagac gagcaagctg agcagcaaaa aactcacgta tttgggcagg 17760

cgccttattc tggtataaat attacaaagg agggtattca aataggtgtc gaaggtcaaa 17820

cacctaaata tgccgataaa acatttcaac ctgaacctca aataggagaa tctcagtggt 17880

acgaaactga aattaatcat gcagctggga gagtccttaa aaagactacc ccaatgaaac 17940

catgttacgg ttcatatgca aaacccacaa atgaaaatgg agggcaaggc attcttgtaa 18000

agcaacaaaa tggaaagcta gaaagtcaag tggaaatgca atttttctca actactgagg 18060

cgaccgcagg caatggtgat aacttgactc ctaaagtggt attgtacagt gaagatgtag 18120

atatagaaac cccagacact catatttctt acatgcccac tattaaggaa ggtaactcac 18180

gagaactaat gggccaacaa tctatgccca acaggcctaa ttacattgct tttagggaca 18240

attttattgg tctaatgtat tacaacagca cgggtaatat gggtgttctg gcgggccaag 18300

catcgcagtt gaatgctgtt gtagatttgc aagacagaaa cacagagctt tcataccagc 18360

ttttgcttga ttccattggt gatagaacca ggtacttttc tatgtggaat caggctgttg 18420

acagctatga tccagatgtt agaattattg aaaatcatgg aactgaagat gaacttccaa 18480

attactgctt tccactggga ggtgtgatta atacagagac tcttaccaag gtaaaaccta 18540

aaacaggtca ggaaaatgga tgggaaaaag atgctacaga attttcagat aaaaatgaaa 18600

taagagttgg aaataatttt gccatggaaa tcaatctaaa tgccaacctg tggagaaatt 18660

tcctgtactc caacatagcg ctgtatttgc ccgacaagct aaagtacagt ccttccaacg 18720

taaaaatttc tgataaccca aacacctacg actacatgaa caagcgagtg gtggctcccg 18780

ggttagtgga ctgctacatt aaccttggag cacgctggtc ccttgactat atggacaacg 18840

tcaacccatt taaccaccac cgcaatgctg gcctgcgcta ccgctcaatg ttgctgggca 18900

atggtcgcta tgtgcccttc cacatccagg tgcctcagaa gttctttgcc attaaaaacc 18960

tccttctcct gccgggctca tacacctacg agtggaactt caggaaggat gttaacatgg 19020

ttctgcagag ctccctagga aatgacctaa gggttgacgg agccagcatt aagtttgata 19080

gcatttgcct ttacgccacc ttcttcccca tggcccacaa caccgcctcc acgcttgagg 19140

ccatgcttag aaacgacacc aacgaccagt cctttaacga ctatctctcc gccgccaaca 19200

tgctctaccc tatacccgcc aacgctacca acgtgcccat atccatcccc tcccgcaact 19260

gggcggcttt ccgcggctgg gccttcacgc gccttaagac taaggaaacc ccatcactgg 19320

gctcgggcta cgacccttat tacacctact ctggctctat accctaccta gatggaacct 19380

tttacctcaa ccacaccttt aagaaggtgg ccattacctt tgactcttct gtcagctggc 19440

ctggcaatga ccgcctgctt acccccaacg agtttgaaat taagcgctca gttgacgggg 19500

agggttacaa cgttgcccag tgtaacatga ccaaagactg gttcctggta caaatgctag 19560

ctaactacaa cattggctac cagggcttct atatcccaga gagctacaag gaccgcatgt 19620

actccttctt tagaaacttc cagcccatga gccgtcaggt ggtggatgat actaaataca 19680

aggactacca acaggtgggc atcctacacc aacacaacaa ctctggattt gttggctacc 19740

ttgcccccac catgcgcgaa ggacaggcct accctgctaa cttcccctat ccgcttatag 19800

gcaagaccgc agttgacagc attacccaga aaaagtttct ttgcgatcgc accctttggc 19860

gcatcccatt ctccagtaac tttatgtcca tgggcgcact cacagacctg ggccaaaacc 19920

ttctctacgc caactccgcc cacgcgctag acatgacttt tgaggtggat cccatggacg 19980

agcccaccct tctttatgtt ttgtttgaag tctttgacgt ggtccgtgtg caccggccgc 20040

accgcggcgt catcgaaacc gtgtacctgc gcacgccctt ctcggccggc aacgccacaa 20100

cataaagaag caagcaacat caacaacagc tgccgccatg ggctccagtg agcaggaact 20160

gaaagccatt gtcaaagatc ttggttgtgg gccatatttt ttgggcacct atgacaagcg 20220

ctttccaggc tttgtttctc cacacaagct cgcctgcgcc atagtcaata cggccggtcg 20280

cgagactggg ggcgtacact ggatggcctt tgcctggaac ccgcactcaa aaacatgcta 20340

cctctttgag ccctttggct tttctgacca gcgactcaag caggtttacc agtttgagta 20400

cgagtcactc ctgcgccgta gcgccattgc ttcttccccc gaccgctgta taacgctgga 20460

aaagtccacc caaagcgtac aggggcccaa ctcggccgcc tgtggactat tctgctgcat 20520

gtttctccac gcctttgcca actggcccca aactcccatg gatcacaacc ccaccatgaa 20580

ccttattacc ggggtaccca actccatgct caacagtccc caggtacagc ccaccctgcg 20640

tcgcaaccag gaacagctct acagcttcct ggagcgccac tcgccctact tccgcagcca 20700

cagtgcgcag attaggagcg ccacttcttt ttgtcacttg aaaaacatgt aaaaataatg 20760

tactagagac actttcaata aaggcaaatg cttttatttg tacactctcg ggtgattatt 20820

tacccccacc cttgccgtct gcgccgttta aaaatcaaag gggttctgcc gcgcatcgct 20880

atgcgccact ggcagggaca cgttgcgata ctggtgttta gtgctccact taaactcagg 20940

cacaaccatc cgcggcagct cggtgaagtt ttcactccac aggctgcgca ccatcaccaa 21000

cgcgtttagc aggtcgggcg ccgatatctt gaagtcgcag ttggggcctc cgccctgcgc 21060

gcgcgagttg cgatacacag ggttgcagca ctggaacact atcagcgccg ggtggtgcac 21120

gctggccagc acgctcttgt cggagatcag atccgcgtcc aggtcctccg cgttgctcag 21180

ggcgaacgga gtcaactttg gtagctgcct tcccaaaaag ggcgcgtgcc caggctttga 21240

gttgcactcg caccgtagtg gcatcaaaag gtgaccgtgc ccggtctggg cgttaggata 21300

cagcgcctgc ataaaagcct tgatctgctt aaaagccacc tgagcctttg cgccttcaga 21360

gaagaacatg ccgcaagact tgccggaaaa ctgattggcc ggacaggccg cgtcgtgcac 21420

gcagcacctt gcgtcggtgt tggagatctg caccacattt cggccccacc ggttcttcac 21480

gatcttggcc ttgctagact gctccttcag cgcgcgctgc ccgttttcgc tcgtcacatc 21540

catttcaatc acgtgctcct tatttatcat aatgcttccg tgtagacact taagctcgcc 21600

ttcgatctca gcgcagcggt gcagccacaa cgcgcagccc gtgggctcgt gatgcttgta 21660

ggtcacctct gcaaacgact gcaggtacgc ctgcaggaat cgccccatca tcgtcacaaa 21720

ggtcttgttg ctggtgaagg tcagctgcaa cccgcggtgc tcctcgttca gccaggtctt 21780

gcatacggcc gccagagctt ccacttggtc aggcagtagt ttgaagttcg cctttagatc 21840

gttatccacg tggtacttgt ccatcagcgc gcgcgcagcc tccatgccct tctcccacgc 21900

agacacgatc ggcacactca gcgggttcat caccgtaatt tcactttccg cttcgctggg 21960

ctcttcctct tcctcttgcg tccgcatacc acgcgccact gggtcgtctt cattcagccg 22020

ccgcactgtg cgcttacctc ctttgccatg cttgattagc accggtgggt tgctgaaacc 22080

caccatttgt agcgccacat cttctctttc ttcctcgctg tccacgatta cctctggtga 22140

tggcgggcgc tcgggcttgg gagaagggcg cttctttttc ttcttgggcg caatggccaa 22200

atccgccgcc gaggtcgatg gccgcgggct gggtgtgcgc ggcaccagcg cgtcttgtga 22260

tgagtcttcc tcgtcctcgg actcgatacg ccgcctcatc cgcttttttg ggggcgcccg 22320

gggaggcggc ggcgacgggg acggggacga cacgtcctcc atggttgggg gacgtcgcgc 22380

cgcaccgcgt ccgcgctcgg gggtggtttc gcgctgctcc tcttcccgac tggccatttc 22440

cttctcctat aggcagaaaa agatcatgga gtcagtcgag aagaaggaca gcctaaccgc 22500

cccctctgag ttcgccacca ccgcctccac cgatgccgcc aacgcgccta ccaccttccc 22560

cgtcgaggca cccccgcttg aggaggagga agtgattatc gagcaggacc caggttttgt 22620

aagcgaagac gacgaggacc gctcagtacc aacagaggat aaaaagcaag accaggacaa 22680

cgcagaggca aacgaggaac aagtcgggcg gggggacgaa aggcatggcg actacctaga 22740

tgtgggagac gacgtgctgt tgaagcatct gcagcgccag tgcgccatta tctgcgacgc 22800

gttgcaagag cgcagcgatg tgcccctcgc catagcggat gtcagccttg cctacgaacg 22860

ccacctattc tcaccgcgcg taccccccaa acgccaagaa aacggcacat gcgagcccaa 22920

cccgcgcctc aacttctacc ccgtatttgc cgtgccagag gtgcttgcca cctatcacat 22980

ctttttccaa aactgcaaga tacccctatc ctgccgtgcc aaccgcagcc gagcggacaa 23040

gcagctggcc ttgcggcagg gcgctgtcat acctgatatc gcctcgctca acgaagtgcc 23100

aaaaatcttt gagggtcttg gacgcgacga gaagcgcgcg gcaaacgctc tgcaacagga 23160

aaacagcgaa aatgaaagtc actctggagt gttggtggaa ctcgagggtg acaacgcgcg 23220

cctagccgta ctaaaacgca gcatcgaggt cacccacttt gcctacccgg cacttaacct 23280

accccccaag gtcatgagca cagtcatgag tgagctgatc gtgcgccgtg cgcagcccct 23340

ggagagggat gcaaatttgc aagaacaaac agaggagggc ctacccgcag ttggcgacga 23400

gcagctagcg cgctggcttc aaacgcgcga gcctgccgac ttggaggagc gacgcaaact 23460

aatgatggcc gcagtgctcg ttaccgtgga gcttgagtgc atgcagcggt tctttgctga 23520

cccggagatg cagcgcaagc tagaggaaac attgcactac acctttcgac agggctacgt 23580

acgccaggcc tgcaagatct ccaacgtgga gctctgcaac ctggtctcct accttggaat 23640

tttgcacgaa aaccgccttg ggcaaaacgt gcttcattcc acgctcaagg gcgaggcgcg 23700

ccgcgactac gtccgcgact gcgtttactt atttctatgc tacacctggc agacggccat 23760

gggcgtttgg cagcagtgct tggaggagtg caacctcaag gagctgcaga aactgctaaa 23820

gcaaaacttg aaggacctat ggacggcctt caacgagcgc tccgtggccg cgcacctggc 23880

ggacatcatt ttccccgaac gcctgcttaa aaccctgcaa cagggtctgc cagacttcac 23940

cagtcaaagc atgttgcaga actttaggaa ctttatccta gagcgctcag gaatcttgcc 24000

cgccacctgc tgtgcacttc ctagcgactt tgtgcccatt aagtaccgcg aatgccctcc 24060

gccgctttgg ggccactgct accttctgca gctagccaac taccttgcct accactctga 24120

cataatggaa gacgtgagcg gtgacggtct actggagtgt cactgtcgct gcaacctatg 24180

caccccgcac cgctccctgg tttgcaattc gcagctgctt aacgaaagtc aaattatcgg 24240

tacctttgag ctgcagggtc cctcgcctga cgaaaagtcc gcggctccgg ggttgaaact 24300

cactccgggg ctgtggacgt cggcttacct tcgcaaattt gtacctgagg actaccacgc 24360

ccacgagatt aggttctacg aagaccaatc ccgcccgcca aatgcggagc ttaccgcctg 24420

cgtcattacc cagggccaca ttcttggcca attgcaagcc atcaacaaag cccgccaaga 24480

gtttctgcta cgaaagggac ggggggttta cttggacccc cagtccggcg aggagctcaa 24540

cccaatcccc ccgccgccgc agccctatca gcagcagccg cgggcccttg cttcccagga 24600

tggcacccaa aaagaagctg cagctgccgc cgccacccac ggacgaggag gaatactggg 24660

acagtcaggc agaggaggtt ttggacgagg aggaggagga catgatggaa gactgggaga 24720

gcctagacga ggaagcttcc gaggtcgaag aggtgtcaga cgaaacaccg tcaccctcgg 24780

tcgcattccc ctcgccggcg ccccagaaat cggcaaccgg ttccagcatg gctacaacct 24840

ccgctcctca ggcgccgccg gcactgcccg ttcgccgacc caaccgtaga tgggacacca 24900

ctggaaccag ggccggtaag tccaagcagc cgccgccgtt agcccaagag caacaacagc 24960

gccaaggcta ccgctcatgg cgcgggcaca agaacgccat agttgcttgc ttgcaagact 25020

gtgggggcaa catctccttc gcccgccgct ttcttctcta ccatcacggc gtggccttcc 25080

cccgtaacat cctgcattac taccgtcatc tctacagccc atactgcacc ggcggcagcg 25140

gcagcggcag caacagcagc ggccacacag aagcaaaggc gaccggatag caagactctg 25200

acaaagccca agaaatccac agcggcggca gcagcaggag gaggagcgct gcgtctggcg 25260

cccaacgaac ccgtatcgac ccgcgagctt agaaacagga tttttcccac tctgtatgct 25320

atatttcaac agagcagggg ccaagaacaa gagctgaaaa taaaaaacag gtctctgcga 25380

tccctcaccc gcagctgcct gtatcacaaa agcgaagatc agcttcggcg cacgctggaa 25440

gacgcggagg ctctcttcag taaatactgc gcgctgactc ttaaggacta gtttcgcgcc 25500

ctttctcaaa tttaagcgcg aaaactacgt catctccagc ggccacaccc ggcgccagca 25560

cctgtcgtca gcgccattat gagcaaggaa attcccacgc cctacatgtg gagttaccag 25620

ccacaaatgg gacttgcggc tggagctgcc caagactact caacccgaat aaactacatg 25680

agcgcgggac cccacatgat atcccgggtc aacggaatcc gcgcccaccg aaaccgaatt 25740

ctcttggaac aggcggctat taccaccaca cctcgtaata accttaatcc ccgtagttgg 25800

cccgctgccc tggtgtacca ggaaagtccc gctcccacca ctgtggtact tcccagagac 25860

gcccaggccg aagttcagat gactaactca ggggcgcagc ttgcgggcgg ctttcgtcac 25920

agggtgcggt cgcccgggca gggtataact cacctgacaa tcagagggcg aggtattcag 25980

ctcaacgacg agtcggtgag ctcctcgctt ggtctccgtc cggacgggac atttcagatc 26040

ggcggcgccg gccgtccttc attcacgcct cgtcaggcaa tcctaactct gcagacctcg 26100

tcctctgagc cgcgctctgg aggcattgga actctgcaat ttattgagga gtttgtgcca 26160

tcggtctact ttaacccctt ctcgggacct cccggccact atccggatca atttattcct 26220

aactttgacg cggtaaagga ctcggcggac ggctacgact gaatgttaag tggagaggca 26280

gagcaactgc gcctgaaaca cctggtccac tgtcgccgcc acaagtgctt tgcccgcgac 26340

tccggtgagt tttgctactt tgaattgccc gaggatcata tcgagggccc ggcgcacggc 26400

gtccggctta ccgcccaggg agagcttgcc cgtagcctga ttcgggagtt tacccagcgc 26460

cccctgctag ttgagcggga caggggaccc tgtgttctca ctgtgatttg caactgtcct 26520

aaccttggat tacatcaaga tctttgttgc catctctgtg ctgagtataa taaatacaga 26580

aattaaaata tactggggct cctatcgcca tcctgtaaac gccaccgtct tcacccgccc 26640

aagcaaacca aggcgaacct tacctggtac ttttaacatc tctccctctg tgatttacaa 26700

cagtttcaac ccagacggag tgagtctacg agagaacctc tccgagctca gctactccat 26760

cagaaaaaac accaccctcc ttacctgccg ggaacgtacg agtgcgtcac cggccgctgc 26820

accacaccta ccgcctgacc gtaaaccaga ctttttccgg acagacctca ataactctgt 26880

ttaccagaac aggaggtgag cttagaaaac ccttagggta ttaggccaaa ggcgcagcta 26940

ctgtggggtt tatgaacaat tcaagcaact ctacgggcta ttctaattca ggtttctcta 27000

gaatcggggt tggggttatt ctctgtcttg tgattctctt tattcttata ctaacgcttc 27060

tctgcctaag gctcgccgcc tgctgtgtgc acatttgcat ttattgtcag ctttttaaac 27120

gctggggtcg ccacccaaga tgattaggta cataatccta ggtttactca cccttgcgtc 27180

agcccacggt accacccaaa aggtggattt taaggagcca gcctgtaatg ttacattcgc 27240

agctgaagct aatgagtgca ccactcttat aaaatgcacc acagaacatg aaaagctgct 27300

tattcgccac aaaaacaaaa ttggcaagta tgctgtttat gctatttggc agccaggtga 27360

cactacagag tataatgtta cagttttcca gggtaaaagt cataaaactt ttatgtatac 27420

ttttccattt tatgaaatgt gcgacattac catgtacatg agcaaacagt ataagttgtg 27480

gcccccacaa aattgtgtgg aaaacactgg cactttctgc tgcactgcta tgctaattac 27540

agtgctcgct ttggtctgta ccctactcta tattaaatac aaaagcagac gcagctttat 27600

tgaggaaaag aaaatgcctt aatttactaa gttacaaagc taatgtcacc actaactgct 27660

ttactcgctg cttgcaaaac aaattcaaaa agttagcatt ataattagaa taggatttaa 27720

accccccggt catttcctgc tcaataccat tcccctgaac aattgactct atgtgggata 27780

tgctccagcg ctacaacctt gaagtcaggc ttcctggatg tcagcatctg actttggcca 27840

gcacctgtcc cgcggatttg ttccagtcca actacagcga cccaccctaa cagagatgac 27900

caacacaacc aacgcggccg ccgctaccgg acttacatct accacaaata caccccaagt 27960

ttctgccttt gtcaataact gggataactt gggcatgtgg tggttctcca tagcgcttat 28020

gtttgtatgc cttattatta tgtggctcat ctgctgccta aagcgcaaac gcgcccgacc 28080

acccatctat agtcccatca ttgtgctaca cccaaacaat gatggaatcc atagattgga 28140

cggactgaaa cacatgttct tttctcttac agtatgatta aatgagacat gattcctcga 28200

gtttttatat tactgaccct tgttgcgctt ttttgtgcgt gctccacatt ggctgcggtt 28260

tctcacatcg aagtagactg cattccagcc ttcacagtct atttgcttta cggatttgtc 28320

accctcacgc tcatctgcag cctcatcact gtggtcatcg cctttatcca gtgcattgac 28380

tgggtctgtg tgcgctttgc atatctcaga caccatcccc agtacaggga caggactata 28440

gctgagcttc ttagaattct ttaattatga aatttactgt gacttttctg ctgattattt 28500

gcaccctatc tgcgttttgt tccccgacct ccaagcctca aagacatata tcatgcagat 28560

tcactcgtat atggaatatt ccaagttgct acaatgaaaa aagcgatctt tccgaagcct 28620

ggttatatgc aatcatctct gttatggtgt tctgcagtac catcttagcc ctagctatat 28680

atccctacct tgacattggc tggaaacgaa tagatgccat gaaccaccca actttccccg 28740

cgcccgctat gcttccactg caacaagttg ttgccggcgg ctttgtccca gccaatcagc 28800

ctcgccccac ttctcccacc cccactgaaa tcagctactt taatctaaca ggaggagatg 28860

actgacaccc tagatctaga aatggacgga attattacag agcagcgcct gctagaaaga 28920

cgcagggcag cggccgagca acagcgcatg aatcaagagc tccaagacat ggttaacttg 28980

caccagtgca aaaggggtat cttttgtctg gtaaagcagg ccaaagtcac ctacgacagt 29040

aataccaccg gacaccgcct tagctacaag ttgccaacca agcgtcagaa attggtggtc 29100

atggtgggag aaaagcccat taccataact cagcactcgg tagaaaccga aggctgcatt 29160

cactcacctt gtcaaggacc tgaggatctc tgcaccctta ttaagaccct gtgcggtctc 29220

aaagatctta ttccctttaa ctaataaaaa aaaataataa agcatcactt acttaaaatc 29280

agttagcaaa tttctgtcca gtttattcag cagcacctcc ttgccctcct cccagctctg 29340

gtattgcagc ttcctcctgg ctgcaaactt tctccacaat ctaaatggaa tgtcagtttc 29400

ctcctgttcc tgtccatccg cacccactat cttcatgttg ttgcagatga agcgcgcaag 29460

accgtctgaa gataccttca accccgtgta tccatatgac acggaaaccg gtcctccaac 29520

tgtgcctttt cttactcctc cctttgtatc ccccaatggg tttcaagaga gtccccctgg 29580

ggtactctct ttgcgcctat ccgaacctct agttacctcc aatggcatgc ttgcgctcaa 29640

aatgggcaac ggcctctctc tggacgaggc cggcaacctt acctcccaaa atgtaaccac 29700

tgtgagccca cctctcaaaa aaaccaagtc aaacataaac ctggaaatat ctgcacccct 29760

cacagttacc tcagaagccc taactgtggc tgccgccgca cctctaatgg tcgcgggcaa 29820

cacactcacc atgcaatcac aggccccgct aaccgtgcac gactccaaac ttagcattgc 29880

cacccaagga cccctcacag tgtcagaagg aaagctagcc ctgcaaacat caggccccct 29940

caccaccacc gatagcagta cccttactat cactgcctca ccccctctaa ctactgccac 30000

tggtagcttg ggcattgact tgaaagagcc catttataca caaaatggaa aactaggact 30060

aaagtacggg gctcctttgc atgtaacaga cgacctaaac actttgaccg tagcaactgg 30120

tccaggtgtg actattaata atacttcctt gcaaactaaa gttactggag ccttgggttt 30180

tgattcacaa ggcaatatgc aacttaatgt agcaggagga ctaaggattg attctcaaaa 30240

cagacgcctt atacttgatg ttagttatcc gtttgatgct caaaaccaac taaatctaag 30300

actaggacag ggccctcttt ttataaactc agcccacaac ttggatatta actacaacaa 30360

aggcctttac ttgtttacag cttcaaacaa ttccaaaaag cttgaggtta acctaagcac 30420

tgccaagggg ttgatgtttg acgctacagc catagccatt aatgcaggag atgggcttga 30480

atttggttca cctaatgcac caaacacaaa tcccctcaaa acaaaaattg gccatggcct 30540

agaatttgat tcaaacaagg ctatggttcc taaactagga actggcctta gttttgacag 30600

cacaggtgcc attacagtag gaaacaaaaa taatgataag ctaactttgt ggaccacacc 30660

agctccatct cctaactgta gactaaatgc agagaaagat gctaaactca ctttggtctt 30720

aacaaaatgt ggcagtcaaa tacttgctac agtttcagtt ttggctgtta aaggcagttt 30780

ggctccaata tctggaacag ttcaaagtgc tcatcttatt ataagatttg acgaaaatgg 30840

agtgctacta aacaattcct tcctggaccc agaatattgg aactttagaa atggagatct 30900

tactgaaggc acagcctata caaacgctgt tggatttatg cctaacctat cagcttatcc 30960

aaaatctcac ggtaaaactg ccaaaagtaa cattgtcagt caagtttact taaacggaga 31020

caaaactaaa cctgtaacac taaccattac actaaacggt acacaggaaa caggagacac 31080

aactccaagt gcatactcta tgtcattttc atgggactgg tctggccaca actacattaa 31140

tgaaatattt gccacatcct cttacacttt ttcatacatt gcccaagaat aaagaatcgt 31200

ttgtgttatg tttcaacgtg tttatttttc aattgcagaa aatttcaagt catttttcat 31260

tcagtagtat agccccacca ccacatagct tatacagatc accgtacctt aatcaaactc 31320

acagaaccct agtattcaac ctgccacctc cctcccaaca cacagagtac acagtccttt 31380

ctccccggct ggccttaaaa agcatcatat catgggtaac agacatattc ttaggtgtta 31440

tattccacac ggtttcctgt cgagccaaac gctcatcagt gatattaata aactccccgg 31500

gcagctcact taagttcatg tcgctgtcca gctgctgagc cacaggctgc tgtccaactt 31560

gcggttgctt aacgggcggc gaaggagaag tccacgccta catgggggta gagtcataat 31620

cgtgcatcag gatagggcgg tggtgctgca gcagcgcgcg aataaactgc tgccgccgcc 31680

gctccgtcct gcaggaatac aacatggcag tggtctcctc agcgatgatt cgcaccgccc 31740

gcagcataag gcgccttgtc ctccgggcac agcagcgcac cctgatctca cttaaatcag 31800

cacagtaact gcagcacagc accacaatat tgttcaaaat cccacagtgc aaggcgctgt 31860

atccaaagct catggcgggg accacagaac ccacgtggcc atcataccac aagcgcaggt 31920

agattaagtg gcgacccctc ataaacacgc tggacataaa cattacctct tttggcatgt 31980

tgtaattcac cacctcccgg taccatataa acctctgatt aaacatggcg ccatccacca 32040

ccatcctaaa ccagctggcc aaaacctgcc cgccggctat acactgcagg gaaccgggac 32100

tggaacaatg acagtggaga gcccaggact cgtaaccatg gatcatcatg ctcgtcatga 32160

tatcaatgtt ggcacaacac aggcacacgt gcatacactt cctcaggatt acaagctcct 32220

cccgcgttag aaccatatcc cagggaacaa cccattcctg aatcagcgta aatcccacac 32280

tgcagggaag acctcgcacg taactcacgt tgtgcattgt caaagtgtta cattcgggca 32340

gcagcggatg atcctccagt atggtagcgc gggtttctgt ctcaaaagga ggtagacgat 32400

ccctactgta cggagtgcgc cgagacaacc gagatcgtgt tggtcgtagt gtcatgccaa 32460

atggaacgcc ggacgtagtc atatttcctg aagcaaaacc aggtgcgggc gtgacaaaca 32520

gatctgcgtc tccggtctcg ccgcttagat cgctctgtgt agtagttgta gtatatccac 32580

tctctcaaag catccaggcg ccccctggct tcgggttcta tgtaaactcc ttcatgcgcc 32640

gctgccctga taacatccac caccgcagaa taagccacac ccagccaacc tacacattcg 32700

ttctgcgagt cacacacggg aggagcggga agagctggaa gaaccatgtt ttttttttta 32760

ttccaaaaga ttatccaaaa cctcaaaatg aagatctatt aagtgaacgc gctcccctcc 32820

ggtggcgtgg tcaaactcta cagccaaaga acagataatg gcatttgtaa gatgttgcac 32880

aatggcttcc aaaaggcaaa cggccctcac gtccaagtgg acgtaaaggc taaacccttc 32940

agggtgaatc tcctctataa acattccagc accttcaacc atgcccaaat aattctcatc 33000

tcgccacctt ctcaatatat ctctaagcaa atcccgaata ttaagtccgg ccattgtaaa 33060

aatctgctcc agagcgccct ccaccttcag cctcaagcag cgaatcatga ttgcaaaaat 33120

tcaggttcct cacagacctg tataagattc aaaagcggaa cattaacaaa aataccgcga 33180

tcccgtaggt cccttcgcag ggccagctga acataatcgt gcaggtctgc acggaccagc 33240

gcggccactt ccccgccagg aaccatgaca aaagaaccca cactgattat gacacgcata 33300

ctcggagcta tgctaaccag cgtagccccg atgtaagctt gttgcatggg cggcgatata 33360

aaatgcaagg tgctgctcaa aaaatcaggc aaagcctcgc gcaaaaaaga aagcacatcg 33420

tagtcatgct catgcagata aaggcaggta agctccggaa ccaccacaga aaaagacacc 33480

atttttctct caaacatgtc tgcgggtttc tgcataaaca caaaataaaa taacaaaaaa 33540

acatttaaac attagaagcc tgtcttacaa caggaaaaac aacccttata agcataagac 33600

ggactacggc catgccggcg tgaccgtaaa aaaactggtc accgtgatta aaaagcacca 33660

ccgacagctc ctcggtcatg tccggagtca taatgtaaga ctcggtaaac acatcaggtt 33720

gattcacatc ggtcagtgct aaaaagcgac cgaaatagcc cgggggaata catacccgca 33780

ggcgtagaga caacattaca gcccccatag gaggtataac aaaattaata ggagagaaaa 33840

acacataaac acctgaaaaa ccctcctgcc taggcaaaat agcaccctcc cgctccagaa 33900

caacatacag cgcttccaca gcggcagcca taacagtcag ccttaccagt aaaaaagaaa 33960

acctattaaa aaaacaccac tcgacacggc accagctcaa tcagtcacag tgtaaaaaag 34020

ggccaagtgc agagcgagta tatataggac taaaaaatga cgtaacggtt aaagtccaca 34080

aaaaacaccc agaaaaccgc acgcgaacct acgcccagaa acgaaagcca aaaaacccac 34140

aacttcctca aatcgtcact tccgttttcc cacgttacgt aacttcccat tttaagaaaa 34200

ctacaattcc caacacatac aagttactcc gccctaaaac ctacgtcacc cgccccgttc 34260

ccacgccccg cgccacgtca caaactccac cccctcatta tcatattggc ttcaatccaa 34320

aataaggtat attattgatg atg 34343

Claims

1. An oncolytic adenovirus, characterized in that it is based on a pAdEasy-1 adenovirus, inserted with an E1A, CAT gene and a protein photosensitizer gene;

the protein photosensitizer gene is a KillerRed gene.

2. The oncolytic adenovirus of claim 1, wherein the 3-terminus of E1A is inserted with a miRNA target sequence.

3. The oncolytic adenovirus of claim 2, wherein the miRNA target sequence is a combination of two or more miRNA target sequences.

4. The oncolytic adenovirus of claim 1, wherein the E1A, CAT gene and the protein photosensitizer gene are flanked by a CMV promoter and a polyA tail, respectively.

5. The oncolytic adenovirus of claim 1, wherein the E1A, CAT gene and the protein photosensitizer gene are inserted sequentially into the pAdEasy-1 adenovirus.

6. The method for constructing an oncolytic adenovirus according to any one of claims 2-5, comprising: (1) Inserting an E1A, CAT gene and a protein photosensitizer gene into a shuttle plasmid pAdTrack-CMV polyclonal site, wherein a miRNA target sequence is inserted into the 3 end of the E1A; (2) Carrying out homologous recombination on the recombinant shuttle plasmid obtained in the step 1 and a skeleton plasmid pAdEasy-1 to obtain a recombinant adenovirus plasmid; (3) Recombinant adenovirus plasmids were packaged in 293A cells.

7. Use of an oncolytic adenovirus according to claims 2-5 in the manufacture of a oncolytic drug for photodynamic therapy.

8. A medicament comprising an oncolytic adenovirus according to any one of claims 2-5.

9. The medicament according to claim 8, characterized in that the oncolytic adenovirus is used before photodynamic therapy, or before combined photodynamic and chemoradiotherapy, or before tumour immunotherapy.