CN115074330B - 一种基于遗传密码子扩展技术的改造Vero细胞系 - Google Patents
一种基于遗传密码子扩展技术的改造Vero细胞系 Download PDFInfo
- Publication number
- CN115074330B CN115074330B CN202210412748.7A CN202210412748A CN115074330B CN 115074330 B CN115074330 B CN 115074330B CN 202210412748 A CN202210412748 A CN 202210412748A CN 115074330 B CN115074330 B CN 115074330B
- Authority
- CN
- China
- Prior art keywords
- cell line
- sequence
- modified
- vero cell
- codon
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 210000003501 vero cell Anatomy 0.000 title claims abstract description 38
- 238000005516 engineering process Methods 0.000 title claims abstract description 17
- 230000002068 genetic effect Effects 0.000 title claims abstract description 15
- 108020004705 Codon Proteins 0.000 claims abstract description 14
- 238000010276 construction Methods 0.000 claims abstract description 7
- 238000013519 translation Methods 0.000 claims abstract description 5
- 239000013612 plasmid Substances 0.000 claims description 18
- 108091033409 CRISPR Proteins 0.000 claims description 16
- 108700028939 Amino Acyl-tRNA Synthetases Proteins 0.000 claims description 14
- 238000003780 insertion Methods 0.000 claims description 11
- 230000037431 insertion Effects 0.000 claims description 11
- 101001000998 Homo sapiens Protein phosphatase 1 regulatory subunit 12C Proteins 0.000 claims description 10
- ZFOMKMMPBOQKMC-KXUCPTDWSA-N L-pyrrolysine Chemical compound C[C@@H]1CC=N[C@H]1C(=O)NCCCC[C@H]([NH3+])C([O-])=O ZFOMKMMPBOQKMC-KXUCPTDWSA-N 0.000 claims description 8
- 108091027544 Subgenomic mRNA Proteins 0.000 claims description 8
- 108020005038 Terminator Codon Proteins 0.000 claims description 7
- 238000010354 CRISPR gene editing Methods 0.000 claims description 6
- 102100035620 Protein phosphatase 1 regulatory subunit 12C Human genes 0.000 claims description 5
- 108020004566 Transfer RNA Proteins 0.000 claims description 3
- 108091093126 WHP Posttrascriptional Response Element Proteins 0.000 claims description 2
- 230000001124 posttranscriptional effect Effects 0.000 claims description 2
- 230000001105 regulatory effect Effects 0.000 claims description 2
- 241001127867 Memecylon malabaricum Species 0.000 claims 1
- 210000004027 cell Anatomy 0.000 abstract description 39
- 241000700605 Viruses Species 0.000 abstract description 15
- 239000001963 growth medium Substances 0.000 abstract description 9
- 229960005486 vaccine Drugs 0.000 abstract description 8
- 238000011160 research Methods 0.000 abstract description 2
- 108700010070 Codon Usage Proteins 0.000 abstract 1
- 230000003321 amplification Effects 0.000 abstract 1
- 238000003199 nucleic acid amplification method Methods 0.000 abstract 1
- 244000052769 pathogen Species 0.000 abstract 1
- 108090000623 proteins and genes Proteins 0.000 description 23
- 102000052866 Amino Acyl-tRNA Synthetases Human genes 0.000 description 12
- 102000004169 proteins and genes Human genes 0.000 description 12
- 239000000758 substrate Substances 0.000 description 11
- 150000001413 amino acids Chemical class 0.000 description 9
- 230000008685 targeting Effects 0.000 description 9
- 239000013598 vector Substances 0.000 description 9
- 108020004485 Nonsense Codon Proteins 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 8
- 238000012163 sequencing technique Methods 0.000 description 8
- 238000010586 diagram Methods 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 238000013461 design Methods 0.000 description 6
- 238000001890 transfection Methods 0.000 description 6
- 238000000034 method Methods 0.000 description 5
- 108040001032 pyrrolysyl-tRNA synthetase activity proteins Proteins 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 108020004414 DNA Proteins 0.000 description 4
- BAVYZALUXZFZLV-UHFFFAOYSA-N Methylamine Chemical compound NC BAVYZALUXZFZLV-UHFFFAOYSA-N 0.000 description 4
- 108091060545 Nonsense suppressor Proteins 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 230000035772 mutation Effects 0.000 description 4
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 description 4
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 239000012091 fetal bovine serum Substances 0.000 description 3
- 108091006047 fluorescent proteins Proteins 0.000 description 3
- 102000034287 fluorescent proteins Human genes 0.000 description 3
- 238000009472 formulation Methods 0.000 description 3
- 230000005847 immunogenicity Effects 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 description 3
- 230000003612 virological effect Effects 0.000 description 3
- IAKHMKGGTNLKSZ-INIZCTEOSA-N (S)-colchicine Chemical compound C1([C@@H](NC(C)=O)CC2)=CC(=O)C(OC)=CC=C1C1=C2C=C(OC)C(OC)=C1OC IAKHMKGGTNLKSZ-INIZCTEOSA-N 0.000 description 2
- 241000203069 Archaea Species 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 108700039887 Essential Genes Proteins 0.000 description 2
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 2
- 229930182816 L-glutamine Natural products 0.000 description 2
- 102000003960 Ligases Human genes 0.000 description 2
- 108090000364 Ligases Proteins 0.000 description 2
- 239000004472 Lysine Substances 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 241000359380 Methanosarcinales Species 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000013373 clone screening Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000001976 enzyme digestion Methods 0.000 description 2
- 239000003797 essential amino acid Substances 0.000 description 2
- 210000003527 eukaryotic cell Anatomy 0.000 description 2
- 238000005755 formation reaction Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 210000001236 prokaryotic cell Anatomy 0.000 description 2
- 229950010131 puromycin Drugs 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 229960004854 viral vaccine Drugs 0.000 description 2
- 101150067361 Aars1 gene Proteins 0.000 description 1
- 108700028369 Alleles Proteins 0.000 description 1
- 108020005098 Anticodon Proteins 0.000 description 1
- 238000010356 CRISPR-Cas9 genome editing Methods 0.000 description 1
- 238000010453 CRISPR/Cas method Methods 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 208000026350 Inborn Genetic disease Diseases 0.000 description 1
- 102000016397 Methyltransferase Human genes 0.000 description 1
- 108060004795 Methyltransferase Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 241000158504 Rhodococcus hoagii Species 0.000 description 1
- 241000192023 Sarcina Species 0.000 description 1
- 241000121220 Tricholoma matsutake Species 0.000 description 1
- 125000000266 alpha-aminoacyl group Chemical group 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000032823 cell division Effects 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 230000003021 clonogenic effect Effects 0.000 description 1
- 229960001338 colchicine Drugs 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000007877 drug screening Methods 0.000 description 1
- 108010048367 enhanced green fluorescent protein Proteins 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 208000016361 genetic disease Diseases 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 239000012212 insulator Substances 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 239000013028 medium composition Substances 0.000 description 1
- 230000031864 metaphase Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 230000006916 protein interaction Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 210000003705 ribosome Anatomy 0.000 description 1
- 238000007480 sanger sequencing Methods 0.000 description 1
- 229940054269 sodium pyruvate Drugs 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000014599 transmission of virus Effects 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000005727 virus proliferation Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0684—Cells of the urinary tract or kidneys
- C12N5/0686—Kidney cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/93—Ligases (6)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y601/00—Ligases forming carbon-oxygen bonds (6.1)
- C12Y601/01—Ligases forming aminoacyl-tRNA and related compounds (6.1.1)
- C12Y601/01026—Pyrrolysine-tRNAPyl ligase (6.1.1.26)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/32—Amino acids
- C12N2500/33—Amino acids other than alpha-amino carboxylic acids, e.g. beta-amino acids, taurine
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/60—Buffer, e.g. pH regulation, osmotic pressure
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2510/00—Genetically modified cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2830/00—Vector systems having a special element relevant for transcription
- C12N2830/36—Vector systems having a special element relevant for transcription being a transcription termination element
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Urology & Nephrology (AREA)
- Molecular Biology (AREA)
- Cell Biology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Medicinal Chemistry (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明涉及生物技术领域，具体公开了一种基于遗传密码子扩展技术的改造Vero细胞系，所述改造Vero细胞系的构建方法是基于基因密码子扩展技术，使用PylRS/tRNAPyl系统，定点插入一套能翻译终止密码子的序列，所述能翻译终止密码子序列如SEQ ID NO.1所示，该改造细胞系，在加入到特殊培养基后，限制性病毒仅能在改造细胞中增殖，但不在天然细胞中增殖，由此建立一个隔离在天然翻译系统之外的安全的病毒扩增和研究体系。本研究将通过：调整启动子，密码子偏好性，培养方案等工作，来建立以改造Vero细胞株为核心的正交系统。这个有效且安全的正交系统，不仅使我们在不接触野生病原的情况下在普通实验室研究病毒与细胞的相互关系，并且有可能大量培育这种限制性病毒来作为新型疫苗。
Description
技术领域
本发明涉及生物技术领域，具体涉及了一种基于遗传密码子扩展技术的改造Vero细胞系。
背景技术
病毒由于其体积微小，易传播性，一直危害人类健康而不易控制。由于安全性问题，对病毒的研究也只集中于蛋白片段和已有疫苗株的生物特性研究。如要进行野生病毒的感染性或免疫原性的研究，就必须在生物安全高等级实验室(P3或P4)中进行。随着人们对病毒基因组的认识，一些病毒上的突变被证明会影响病毒的增殖、复制、细胞适应性或免疫原性。这些特性对病毒传播机制及疫苗设计中增强疫苗的免疫原性、减小毒性均非常重要。对这些变异的反向修改和研究，我们也必须小心地评估其安全性，并在高等级生物安全实验室中进行。由于高等级生物安全实验运行和造价均成本较高，这从一定程度上限制了对病毒的研究。
基因密码子扩展技术(The genetic code expansion，GCE)技术是通过对现有编码蛋白的密码子表进行人为的扩展，引入新的编码规则，从而修饰或改变蛋白质的一种技术。其现在运用于多个领域，如遗传病的治疗，蛋白相互作用及生物医药领域。所有后生动物的蛋白质都是由20种经典氨基酸合成。这20种氨基酸的不同组合使蛋白质具有多种结构和功能，却同时限制了蛋白质特性的发展。尽管翻译后修饰机制在一定程度上解除了这种限制，但这依赖于极其强健且复杂的生物系统。为了能够直接释放氨基酸多样性的全部能力，科学家们在过去的几十年中，已经设计出多种方法将非经典氨基酸(noncanonicalamino acids,ncAA)直接掺入蛋白质中，从而大大扩展了蛋白质的特性。基因密码扩展技术便是其一，它允许在活细胞中将ncAA通过翻译系统特异的插入到任何蛋白质肽链上感兴趣的位点中。该技术通过工程改造的氨酰基-tRNA合成酶(aa-tRNA synthetase,aaRS)/tRNA对实现。改造后的aaRS可将同源的tRNA与特定的ncAA特异性氨酰化，专用于解码重新分配的密码子(通常为终止密码子，所匹配的aaRS/tRNA对称为无义抑制性aaRS/tRNA)。随后通过细胞核糖体识别tRNAncAA，允许ncAA在翻译过程中响应指定的密码子并且能够位点特异性地掺入合成肽中。
本研究组拟利用其中一种修改类型，“无义抑制性aaRS/tRNA系统”来建立限制性细胞系。当无义密码子被整合到病毒基因组中的必要基因时，这类具有提前终止密码子(premature termination codon,PTC)的病毒只能在有无义抑制性aaRS/tRNA对及对应ncAA存在的情况下进行复制。
作为本研究核心的无义抑制系统我们在这里详细进行介绍：几种无义抑制性aaRS/tRNA(氨酰合成酶/对应tRNA)系统是在古叠氮细菌中被发现，这种古细菌有着和现有生物不同的遗传密码子表。这些“无义抑制性aaRS/tRNA系统”被用于蛋白在真核或原核细胞中的条件限制性表达。其中，PylRS/tRNAPyl的应用最为广泛。PylRS指吡咯赖氨酸合成酶，tRNAPyl指吡咯赖氨酸tRNA。吡咯赖氨酸(Pyrrolysine，Pyl)作为第22种氨基酸，首次发现于巴氏甲烷八叠球菌(Methanosarcinales barkeri，M.barkeri)。通过识别框内琥珀密码子(UAG)掺入到甲胺甲基转移酶中，并在甲胺底物的分解代谢中起关键的催化残基作用。与Pyl相匹配的氨基酰-tRNA合成酶(pyrrolysyl-tRNA synthetase，PylRS)/tRNA(pyrrolysyl-tRNA，tRNAPyl)系统表达于M.barkeri及后发现的马氏甲烷八叠球菌(Methanosarcinales maze，M.mazei)基因组中(MbPylRS/tRNA；MmPylRS/tRNA)。当无义密码子(UAG)被整合到病毒基因组中的必要基因时，这类具有提前终止密码子(prematuretermination codon，PTC)的病毒只能在有无义抑制性PylRS/tRNAPyl对及对应吡咯赖氨酸存在的情况下进行复制。这类活的但复制缺陷疫苗就像一个介于灭活病毒疫苗和活病毒疫苗之间的中间体。灭活病毒疫苗是安全的，但并不总是具有抗原代表性，而活病毒疫苗则非常不安全。而该类疫苗保留了病毒高度的感染性及抗原特性。
相对于其他用于兼并ncAA的aaRS/tRNA对，PylRS/tRNAPyl系统具有明显的优势：①、首先，tRNAPyl已经是其同源PylRS的完美底物，并不与其他内源性aaRSs反应，因此不需要对tRNA定向改造。②、tRNAPyl的反密码子区域天然识别UAG密码子，从而避免了突变的需要。③、由于吡咯赖氨酸在结构上明显区别于其他内源氨基酸，因此，即便是突变的PylRS也不太可能会与其他内源氨基酸反应。④、tRNAPyl对应的终止密码子UAG在真核生物中使用率最低，可以在一定程度上降低细胞毒性。⑤、PylRS/tRNAPyl系统无论在是在原核或是真核细胞上都具有良好的正交性。正由于其广泛的正交性及功能多样性，PylRS/tRNAPyl系统已快速的实现遗传密码扩展技术从细菌到真核生物再到动物身上的应用。
虽然现有技术可以将正交系统PylRS/tRNAPyl以位于同一载体的方式导入Vero细胞中，获得稳定整合并表达tRNA合成酶和氨酰tRNA基因的细胞系，但是此细胞系并不能稳定遗传，在传代过程中转入细胞质中的质粒会丢失，这些细胞系就会失去其表达PylRS/tRNAPyl的功能，因此并不能持续表达目的基因。
发明内容
为解决背景技术中存在的问题，获得稳定传代并表达目的基因的限制性细胞系，本发明拟基于“基因密码子扩展技术(The genetic code expansion，GCE)”，将古细菌中一套能翻译终止子(UAG)的体系通过CRISPR/Cas9定点插入的方式，插入到现在用于疫苗生产的主流细胞Vero细胞中，使得人工设计的带有提前终止密码子的病毒只能在改造过的限制性Vero细胞中，加入到特殊培养基后限制性表达。由于设计病毒被引入复制缺陷，其在正常的细胞中无法进行复制从而保证安全性。
本发明所采取的技术方案为：
一种基于遗传密码子扩展技术的改造Vero细胞系，其特征在于，所述改造Vero细胞系的构建方法是基于基因密码子扩展技术，使用CRISPR/Cas9系统将携带PylRS/tRNAPyl系统的质粒插入到Vero细胞株中，定点插入一套能翻译终止密码子的序列，所述序列由2个隔离子、1个CAG启动子、1个马氏甲烷八叠球菌吡咯赖氨酸tRNA合成酶基因、1个WPRE转录后调控序列、1个ployA序列、4个hU6启动子-马氏甲烷八叠球菌吡咯赖氨酸tRNA基因序列组成，所述序列的碱基如SEQ ID NO.1所示。
SEQ ID NO.1所示序列定点插入的位置为PPP1R12C蛋白基因，所述PPP1R12C蛋白基因的碱基序列如SEQ ID NO.2所示。
SEQ ID NO.1所示序列定点插入的位置为PPP1R12C蛋白基因的第一个内含子内AAVS1位点，具体插入位置为NW_023666045.1，263811bp处。
本发明还提供了一种基于遗传密码子扩展技术的改造Vero细胞系的应用，使人工设计的带有提前终止密码子的病毒只能在改造的Vero细胞中，于特定培养基中限制性表达，所述特定培养基为MEM培养基，并添加有底物Nε-2-azidoethyloxycarbonyl-L-lysine，终浓度为1mM。
所述MEM培养基以质量百分比计由以下组分构成：86％ MEM、10％胎牛血清、1％非必需氨基酸、1％ L-谷氨酰胺、1％丙酮酸钠、1％HEPEs。
所述MEM培养基内还添加有嘌呤霉素，浓度为7μg/mL。
附图说明
图1Vero细胞电转效率实验a)电转效率表；b)电转效率细胞荧光图；
图2用于实验的Vero细胞核型分析图(其中染色体为58-60的细胞株为12株，占总株数的80％，符合实验研究的要求)；
图3改造Vero细胞中插入的序列图；
图4用于系统建立的目标质粒和插入位置示意图；
图5sgRNA的活性检测结果显示图；
图6打靶载体酶切鉴定结果显示图；
图7打靶载体图谱示意图；
图8阳性克隆PCR引物设计原则示意图(红色箭头验证引物的位置和方向。根据不同引物进行PCR可以判断目的基因的插入已否)；
图9基因敲入细胞电泳鉴定图(该图中在最后一图中1-F01和1-G08为敲入的纯合子，其他为杂合子，因为插入片段太大扩增时如为杂合子只会优先扩增短片段其和野生型一样，因此用第一和第二幅电泳图来判断是否基因插入，前两图所用引物的一端在插入基因上，如未插入则无条带，如前两图中的WT，注：第9和第12泳道为DNA Marker。1-8道为挑出的单克隆。10,11，13,14泳道为阳性对照。WT野生型，H2O阴性对照)；
图10突变GFP蛋白在改造细胞中的表达(a.突变GFP质粒转染改造细胞并加入NAEK底物，b.突变GFP质粒转染改造细胞未加入NAEK底物，c.正常GFP质粒转染改造细胞不加入NAEK底物)。
具体实施方式
本发明的具体实施方式仅限于进一步解释和说明本发明，并不对本发明的内容构成限制。下述实施例中所述的实验方法，如无特殊说明，均为常规方法；所述试剂与材料，如无特殊说明，均可从商业途径获得。
实施例1
构建正交系统的细胞一方：Vero细胞改造工作，用CRISPR-Cas9系统将携带PylRS/tRNAPyl系统插入到细胞株中。
一、对Vero细胞系的培养条件及转染条件进行摸索，并评估Vero细胞系克隆形成能力，对其进行核型分析。
1、按生物所生产用培养基配方配置培养基对Vero细胞进行培养，获得生长状况良好的Vero细胞，并进行保种。
2、用不同的脉冲电压，时间和次数来将荧光蛋白载体转入Vero细胞选取最佳转染效率，Vero细胞电转效率实验结果如图1。
3、对培养的Vero细胞用秋水仙素处理，让细胞的分裂停于中期。之后进行低渗、固定、制片处理并进行Giemsa染色观察细胞核型，核型分析结果如图2。
4、将细胞按1个/孔接种于96孔板，培养13天，观察克隆形成个数，计算得出此Vero细胞的克隆形成率为55％。
因此得出结论，此细胞可以进行细胞改造工作。
二、构建携带PylRS/tRNAPyl系统的sgRNA/Cas9质粒并活性检测后打靶载体共转染Vero细胞系，对转染后细胞的药杀及扩增；最后用PCR测序的方法筛选及鉴定阳性单克隆。
改造Vero细胞打靶策略：在PPP1R12C蛋白基因的AAVS1位点(此位点是位于PPP1R12C蛋白的第一个内含子内的一段特定序列，PPP1R12C不与任何疾病关联)插入2Xinsulator-CAGPr-Mma-PylRS-WPRE-pA-4X(hU6-Mma-PylT-tRNA)-loxP-puro△TK-loxP序列(如图3)。插入序列中包含了PylRS/tRNAPyl基因及其表达所需的相关结构。采用百奥赛图公司基于CRISPR/Cas9开发的EGE系统制备CL-KJY-001-AKIVero细胞系。
用于系统建立的目标质粒和插入位置示意图如图4。
1、靶序列的测序确认
不同细胞系，靶基因序列可能有所差异。为了保证所设计sgRNA/Cas9的效率，首先需要对Vero细胞系靶位点序列进行PCR扩增并测序验证，以保证sgRNA识别序列与Vero细胞系DNA序列完全一致。
PCR引物如下表1所示：
表1
对Vero细胞系DNA进行PCR及Sanger测序，结果证明：Vero细胞系靶序列与Genebank所给序列完全一致。
2、sgRNA/Cas9设计
基于sgRNA的设计原则，在靶位点区域共设计3条sgRNA，序列如下表2所示：
表2
| 5′Guide | Sequence(5′-3′) |
| Guide#1 | GCCCAAATTAAAGAAGTGAG AGG |
| Guide#2 | CACCTCTCACTTCTTTAATT TGG |
| Guide#3 | ACCTCTCACTTCTTTAATTT GGG |
3、sgRNA/Cas9的活性检测
采用百奥赛图研发的CRISPR/Cas9活性检测方法UCATM(Universal CRISPRActivity Assay，Biocytogen)方式进行sgRNA的活性检测(https://biocytogen.com/gene-editing/by-technologies/other-services/)。
检测结果如图5所示。从图中可以看出，sgRNA1的活性最高，因此选择CL-KJY-001-A-sgRNA1进行下一步实验。
4、打靶载体构建
4.1经过酶切鉴定和测序，确认打靶载体构建完成。如图6所示。
4.2打靶载体图谱。如图7所示。
5、阳性克隆筛选
CL-KJY-001-A-sgRNA1和打靶载体共转染Vero细胞系，经过药物筛选、混合克隆筛选、阳性克隆富集、PCR筛选，最终得到8个阳性克隆。PCR鉴定方法如下：
5.1鉴定引物设计
引物WPRE-F2和WPRE-R2设计在外源基因序列中，引物CL-KJY-001-A-L-GT-F和CL-KJY-001-A-R-GT-R设计在野生型基因序列中。
使用引物对CL-KJY-001-A-L-GT-F/WPRE-R2以及WPRE-F2/CL-KJY-001-A-R-GT-R这两对引物进行PCR时，能扩增出突变的产物。
CL-KJY-001-A-R-GT-F/CL-KJY-001-A-R-GT-R这对引物能扩增出野生型，突变和敲除等位基因的PCR产物，将PCR产物测序，根据测序结果和测序峰图可以判断具体基因型：纯合/杂合/野生型。
引物设计原则如图8所示：
引物信息如下表3和表4所示：
表3
表4
PCR条件(Enzyme:Kod-FX Touchdown)：
Enzyme:KOD-FX，具体参数如表5所示，
表5
5.2鉴定结果如图9所示。
5.3阳性克隆总结
表6
结论如表6所示：通过PCR产物测序，表明1-F01和1-G08为Vero细胞纯合敲进阳性克隆。
实施例2
转入细胞表达预实验：用荧光蛋白检测改造细胞系是否能表达含有UAG终止密码子的能力。
1、将pEGFP-C1质粒上EGFP区域的238位氨基酸进行点突变，将Tyr转变为终止密码子(TAC>TAG)，获得Mut质粒。同时，以pEGFP-C1作为对照质粒，称为WT质粒。
2、实验分3组：
(1)将Mut质粒转染改造细胞系加终浓度1mM的NAEK底物溶液；
(2)将Mut质粒转染改造细胞系不加NAEK底物溶液；
(3)将WT质粒转染改造细胞系不加NAEK底物溶液。
3、操作步骤(1)在六孔组织培养板中使用培养基培养改造细胞，培养环境为37℃，5％CO2，当细胞生长
至70-80％聚合度，进行质粒转染，同时根据实验要求添加NAEK底物溶液终浓度为1mM。(2)转染18h后，用显微镜观察转染是否成功，以及转染效率。
实验中改造细胞系使用上述5.3中的1-F01阳性克隆。
培养基成分配方如下表7所示：
表7
| 试剂 | 品牌 | 货号 | 配方 |
| MEM | Corning | 10-013-CV | 86％ |
| FBS(胎牛血清) | Excell Bio | FSP500 | 10％ |
| 非必需氨基酸 | Gibco | 11140-050 | 1％ |
| L-谷氨酰胺 | Gibco | 25030-081 | 1％ |
| 丙酮酸钠 | Gibco | 11360-070 | 1％ |
| HEPEs | Gibco | 15630-080 | 1％ |
| 嘌呤霉素 | 索莱宝 | P8230 | 7μg/mL |
NAEK(Nε-2-azidoethyloxycarbonyl-L-lysine)溶液配比：
原液:100mM NAEK粉末溶于0.2M NaOH和15％ DMSO中。
将新鲜的稀释液加入细胞中:原液：1M HEPES＝1:4进行稀释，现用现配。
4、实验结果
突变GFP蛋白在改造细胞中的表达结果如图10所示。从图中可以看出Mut质粒转染改造细胞并加入NAEK底物”可以表达荧光蛋白，证明改造Vero细胞系具有表达UAG终止密码子的能力。
序列表
<110> XX
<120> 一种基于遗传密码子扩展技术的改造Vero细胞系
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 5374
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
taaccgttta aacaattctg caggaatcta gttattaata gtaatcaatt acggggtcat 60
tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg 120
gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa 180
cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact 240
tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta 300
aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt 360
acatctacgt attagtcatc gctattacca tggtcgaggt gagccccacg ttctgcttca 420
ctctccccat ctcccccccc tccccacccc caattttgta tttatttatt ttttaattat 480
tttgtgcagc gatgggggcg gggggggggg gggggcgcgc gccaggcggg gcggggcggg 540
gcgaggggcg gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc 600
tccgaaagtt tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg 660
cgcggcgggc ggggagtcgc tgcgacgctg ccttcgcccc gtgccccgct ccgccgccgc 720
ctcgcgccgc ccgccccggc tctgactgac cgcgttactc ccacaggtga gcgggcggga 780
cggcccttct cctccgggct gtaattagcg cttggtttaa tgacggcttg tttcttttct 840
gtggctgcgt gaaagccttg aggggctccg ggagggccct ttgtgcgggg ggagcggctc 900
ggggggtgcg tgcgtgtgtg tgtgcgtggg gagcgccgcg tgcggctccg cgctgcccgg 960
cggctgtgag cgctgcgggc gcggcgcggg gctttgtgcg ctccgcagtg tgcgcgaggg 1020
gagcgcggcc gggggcggtg ccccgcggtg cggggggggc tgcgagggga acaaaggctg 1080
cgtgcggggt gtgtgcgtgg gggggtgagc agggggtgtg ggcgcgtcgg tcgggctgca 1140
accccccctg cacccccctc cccgagttgc tgagcacggc ccggcttcgg gtgcggggct 1200
ccgtacgggg cgtggcgcgg ggctcgccgt gccgggcggg gggtggcggc aggtgggggt 1260
gccgggcggg gcggggccgc ctcgggccgg ggagggctcg ggggaggggc gcggcggccc 1320
ccggagcgcc ggcggctgtc gaggcgcggc gagccgcagc cattgccttt tatggtaatc 1380
gtgcgagagg gcgcagggac ttcctttgtc ccaaatctgt gcggagccga aatctgggag 1440
gcgccgccgc accccctcta gcgggcgcgg ggcgaagcgg tgcggcgccg gcaggaagga 1500
aatgggcggg gagggccttc gtgcgtcgcc gcgccgccgt ccccttctcc ctctccagcc 1560
tcggggctgt ccgcgggggg acggctgcct tcggggggga cggggcaggg cggggttcgg 1620
cttctggcgt gtgaccggcg gctctagagc ctctgctaac catgttcatg ccttcttctt 1680
tttcctacag ctcctgggca acgtgctggt tattgtgctg tctcatcatt ttggcaaaga 1740
attgatttga taccgcattt aaatgccacc atggattaca aggacgacga tgacaagatg 1800
gataaaaaac cattagatgt tttaatatct gcgaccgggc tctggatgtc caggactggc 1860
acgctccaca aaatcaagca ccatgaggtc tcaagaagta aaatatacat tgaaatggcg 1920
tgtggagacc atcttgttgt gaataattcc aggagttgta gaacagccag agcattcaga 1980
catcataagt acagaaaaac ctgcaaacga tgtagggttt cggacgagga tatcaataat 2040
tttctcacaa gatcaaccga aagcaaaaac agtgtgaaag ttagggtagt ttctgctcca 2100
aaggtcaaaa aagctatgcc gaaatcagtt tcaagggctc cgaagcctct ggaaaattct 2160
gtttctgcaa aggcatcaac gaacacatcc agatctgtac cttcgcctgc aaaatcaact 2220
ccaaattcgt ctgttcccgc atcggctcct gctccttcac ttacaagaag ccagcttgat 2280
agggttgagg ctctcttaag tccagaggat aaaatttctc taaatatggc aaagcctttc 2340
agggaacttg agcctgaact tgtgacaaga agaaaaaacg attttcagcg gctctatacc 2400
aatgatagag aagactacct cggtaaactc gaacgtgata ttacgaaatt tttcgtagac 2460
cggggttttc tggagataaa gtctcctatc cttattccgg cggaatacgt ggagagaatg 2520
ggtattaata atgatactga actttcaaaa cagatcttcc gggtggataa aaatctctgc 2580
ttgaggccaa tgcttgcccc gactctttac aactatctgc gaaaactcga taggatttta 2640
ccaggcccaa taaaaatttt cgaagtcgga ccttgttacc ggaaagagtc tgacggcaaa 2700
gagcacctgg aagaatttac tatggtgaac ttctgtcaga tgggttcggg atgtactcgg 2760
gaaaatcttg aagctctcat caaagagttt ctggactatc tggaaatcga cttcgaaatc 2820
gtaggagatt cctgtatggt ctatggggat actcttgata taatgcacgg ggacctggag 2880
ctttcttcgg cagtcgtcgg gccagtttct cttgatagag aatggggtat tgacaaacca 2940
tggataggtg caggttttgg tcttgaacgc ttgctcaagg ttatgcacgg ctttaaaaac 3000
attaagaggg catcaaggtc cgaatcttac tataatggga tttcaaccaa tctgtaagcg 3060
atcgccaaat tcgatatcaa gcttatcgat aatcaacctc tggattacaa aatttgtgaa 3120
agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta 3180
atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa 3240
tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg 3300
tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc 3360
ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc 3420
cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg 3480
gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg ccacctggat tctgcgcggg 3540
acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg 3600
ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc 3660
ctttgggccg cctccccgca tcgataccgt cgacctcgac tgtgccttct agttgccagc 3720
catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg 3780
tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc 3840
tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg 3900
ctggggaact agtggcgcgc ctcgagacta gtaagcttgc atgcctgcag gtcgacgagg 3960
gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt agagagataa 4020
ttagaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt 4080
aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc 4140
ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacgaa 4200
acaccgttaa cggaaacctg atcatgtaga tcgaatggac tctaaatccg ttcagccggg 4260
ttagattccc ggggtttccg ccatttttct cgacgacgag ggcctatttc ccatgattcc 4320
ttcatatttg catatacgat acaaggctgt tagagagata attagaatta atttgactgt 4380
aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt 4440
tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta 4500
tttcgatttc ttggctttat atatcttgtg gaaaggacga aacaccggaa acctgatcat 4560
gtagatcgaa tggactctaa atccgttcag ccgggttaga ttcccggggt ttccgccatt 4620
tttctcgacc acgtggaggg cctatttccc atgattcctt catatttgca tatacgatac 4680
aaggctgtta gagagataat tagaattaat ttgactgtaa acacaaagat attagtacaa 4740
aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt 4800
aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat 4860
atcttgtgga aaggacgaaa caccagatcc ccggaaacct gatcatgtag atcgaatgga 4920
ctctaaatcc gttcagccgg gttagattcc cggggtttcc gccatttttg gtcgggcagg 4980
aagagggcct atttcccatg attccttcat atttgcatat acgatacaag gctgttagag 5040
agataattag aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta 5100
gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc 5160
atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag 5220
gacgaaacac cggaaacctg atcatgtaga tcgaatggac tctaaatccg ttcagccggg 5280
ttagattccc ggggtttccg ccatttttct cgactctaga ggatctgcgg ccgccaggtc 5340
tgaagaggag tttacgtcca gccaagctag cgtt 5374
<210> 2
<211> 27646
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
ccggcggccc ccggggcggg cgggcggcgg cggcggcagt tggggctcgg cgctcgctcg 60
ctcgctcgct gggcgggcgg gcggtgcgat gtccggagag gatggcccgg cggctggccc 120
gggggcggcg gcggcggctg cccgggagcg gcgacgggag cagctgcgac agtggggggc 180
gcgggcgggc gccgagcctg gccccggaga gcgccgcgcc cgcaccgtcc gcttcgagcg 240
cgccgccgag ttcctggcgg cctgtgcggg cggcgacctg gacgaagcgc gtctgatgct 300
gtgcgccgcc gaccctggcc ccggcgccga gctcgacccc gccgcgccgc cgcccgcccg 360
cgccgtgctg gactccacca acgccgacgg catcagcgcc ctgcaccagg tcagcgcccc 420
ctccggcgtc tcccggggcc aggcccaccc tccgcagcgc cgcctggggc atcctccttc 480
cccggttccg agccgccgtt gccagtctcg atccgcccgt cgttcctagc cctgggcttt 540
gccaccctat gctgacaccc cgtcccagcg cccccttacc agccctcttc gaccaccccg 600
cttccgaatt ggggctgctt caagtggccc cgggctttgc cactctgtgc ttaccaccct 660
gccccaggcc tccttaccag tccccttcga cctaccctct tccgcattgg agtcgcttta 720
actggccctg gctttgccag cctgtgctga cccctgcggt cccccttacc atccctccct 780
cgacttcccc tcttccgatg ttgagcctct ctagccggcc ctggactttg tctccctccc 840
tgccccgccc tctcctgaac ctgaggcagc tcccatagct cactctggtc tatctccctg 900
gccctggcca ttgtcacttt gcgctgcccc cctctcgccg ccgagtgccc ttgctgtgcc 960
gcctctactc tgccctctaa cgcggccgtc tctctccgga gtccggacca ctttgagctc 1020
tactggcttc tgcgccgcct ctggcccgct gtttcccctt tccaggcagg tcctgctttc 1080
actgacctgc attctctctc ctgaaccagt gctgctttct gcctgcagat tgtggcctgg 1140
gccacctcca cggctcggcc cagatccttc cccaccacct ccttcaggtt ccagcttcct 1200
ccaatccctc ttccccttgc tctgtgctgt gcgttgctgc ccaaggatgc tctttccaga 1260
gcacttcctt ctcggcgctg caccacgtgg tgtcctcgga gcggatcctg cccttgtctg 1320
ggtcctctcc gggcatctct gctccctacc ccaaccccca tgccatcttc actcgctggg 1380
ttcccttttc cttctccttc tggggcctgt gccatctctc gtttcttagg gtggtcttct 1440
ccgtgggatg tctgtccctt gcgtccccac ctccccttct tgtaggcctg catcatcacc 1500
gtttttctgg acaaccccaa agtccctgtc tccctggctt tagccacctc tccatcctct 1560
tgctttcttt gcctggacac cccctcctcc tgtggatttg ggtcacctct cacttcttta 1620
atttgggcag ctcccatacc ccctttacct ctcgctcttc cggcccctgt cacggcatct 1680
tgcaggggtc ccagagctcg gctagccctc ttcctccaag ctgggccccc atggccactt 1740
ccagacagga tcctgtgtcc cagagccggg accgccttct attcccaggg ccgattaata 1800
tggctctggt tctgggtacg tttatctgtc ccctccaccc ccccccagtg gagccactag 1860
ggacaggact ggtgacagaa aagccccatc cttaggcctc ctccctccta gtctccagat 1920
gttgggtctg acccccacct ccttttaagc agattcctta tctggtgaca cacccccatt 1980
tcccggagcc acctctctct ccccgccaga acccctaggg tttgctcact gatggagcca 2040
gagagggtcc tgggagggag agcttggcgg ggggtgggag ggaaggggga tgcgcgacct 2100
gctggttctc agtggccgcc ctgcgctgcc ctctcccgga gcctgagctg ctctggcgcg 2160
gccgtctggt gcatttcact gatccgggtg ctgctgcttc cttacacttc ccaagaggag 2220
aagcagtttg gaaaaacaaa atcagaataa gttggtcctg agttctaatt ttgggtcttc 2280
acctttctag ccccgaattt gtattgttcc tctgtgcatc agttttccct gtgagataag 2340
gccagtagcc ggccccgccc tggcagggct gtggcgagga gggcgtgtcc gtgtggaaaa 2400
ctccctttgt gagaatggtt cgtcctaggt gttcaccagg tcatggccgc ctctaccccc 2460
ttctctttct ccatccttct ttccttaaag agccccagtg ctgtctggga catactcctc 2520
cgcccggagc ggggtcccgg ggtccctctt ccctaaggcc ctgctctggg cctctgggtt 2580
tgagtccctg gcaagcccag gagaggcgct cagattccct gtccctcttc ctcgtcacca 2640
tcccatgccc ctggctctcc tgcactttcc ctacaggggt tcctggctct gctcttcaga 2700
ctgagccccc ttcccctgca tcccccagag gccccgggcc acctagttgg cctggacccg 2760
gcgagaggtc gccccagccc agtccaccag gctggctttc aggtggattc tcctcccact 2820
gtggggtgac tgcttggcaa actcactcct cggggtatcc caggaggcct ggagtattgg 2880
ggtgggctgg agttcaggga agaggggatt cccttctcag gttacgtggc cagcaagcaa 2940
gggagctggg tttgggtcag gtctgggtgt ggggtgacca gcttatgacg tttgcccata 3000
acagcctggt tttagcgctg aaaccctaag tcctatgaaa acaaggacgg ggtcactgtc 3060
tctgggtgac tcttgagtcc cggccagttt ctccacctgg ggctgtgttt ctcctgcatc 3120
cttcttgagg caggtcccca agcatcagcc ccctgctatg gcagtttccg agttcttagg 3180
gtaccccatg tgggtttatc aaccacttgg tgaggctggt acacttcccc cattcctgcc 3240
ctcccactgc cttagtggct aggggattgg ggactagagt aggaggggct aggagccagg 3300
attcttaggg ctgaacagag aagacctggg ggcctgggct tctgagtctg agggaggagg 3360
ggctgtggcc tgggctcctg ggtctgaggg aggaggggct gtggcctggg ctcctgggtc 3420
tgagggagga gggggctgga acctggaact cctggagtct gagggcaggc tggaactggg 3480
gactcctgga tctgagggag caggggctgg gcctgaactc cctgggtctg aggaggctgg 3540
tcctgggctc ctgggtttga ggaggagggg ctgtggcctg gactcctggg tctgaggaga 3600
gagggctggg gcctggactc ctgggtctga ggctgaggcc tggactcctg ggtgcgagga 3660
gggactgggg cctggactcc tgggtctgag gaggaggggc tgtggcctgg actctcagat 3720
cctccaggga gggagggctg ggggctggac tcctggggtc tcccagggag ggaggagggg 3780
ctgtggcctg ggctcctggg tctgagggag gagggggctg gaacctggac tcctgggtct 3840
gagggagcag gagctggagc ctggactcct ggatctgagg gagcaggggc tggagcctgg 3900
actcctgggt ctgagggagg aggagctggg gtcctgggct cctgggtttg agggaggagg 3960
ggctgtggcc tggactcctg ggtctgaggg aggaggggct gggggcctgg actcctgggt 4020
ctgagggagg agaggctggg ggcctggact cctgggtctg agggaggagg gactgggggc 4080
ctggactcct gggtctgagg gaggaggggc tgtggcctgg actcctgggt ctcagggagg 4140
aggggctggg ggcctggact cctgggtctc agggaggagg ggctgggggc ctggactcct 4200
gggtctcagg gaggaggggc tgggggcctg gactcctggg tctgaatggg gagaggctgg 4260
gggcctgggc ttctgcatct gaggatggaa aggctggggc ctggcctcct gggttgagtg 4320
ggggagggga tgggcctggc ctcctgggtt aaatggggag gggctgggcc tggacttcgg 4380
agtccctggt gcccaggcct caggcatctt tcacagggat gcctgtactg ggcaggtcct 4440
tgaaagggaa aggcccatcg ctctccttgc ccccctcccc cattgccatg acaactggat 4500
ggaaataaac gagtcgagtt catcccgttc ccagggcacg tgcggccccc ttcacagccg 4560
agtttccatg acctcatgct cttggccctc gtagctccct ccccccttct ccagatgggc 4620
agctttggag aggtgaggga cttagggggt aatttatcct gtggatctag gagtttagct 4680
tcactcctcc ctcggctcca gttcaggtcc cagggcccat ccagtgtcca caaggcctgg 4740
ggcacatttc tcccccaatc cccggacttt ggcttttgtc cccccaagtt ttggactcct 4800
aagggaagaa tgagaaatgc tggcccgtgt aagtccctgg ctgcagtgcc ccgtgcagag 4860
gaggcctcag tgaactggag tgtgacagcc tggggcccgg acacacaggt gtgcagctgt 4920
catctgggag tcctgcccag cccgctgagt gtgtccagca cggggcggcc ttcccccacc 4980
ctgcacagcc ctgggccgcg gcttcgctcc tgcacagtgt ctgccagggt ggtttctagg 5040
cttgaccctt ggaaggacct ggctgagtgt aaggcaggag gggctggggg ccaggactcc 5100
tggctctgaa ggaggagggg ctggaacctc ttccctagtc gcagccctgg aggcgccacc 5160
tgcgggtggt cggggatttt gctgtgtctg acaagtacca tgtggggttc gcacccggat 5220
gagaagcccc ctcccttccc cgctcacttc ctgtttgcag gtagccggga gtccttttgt 5280
ggtttccact gagcgccgaa ggcttggcgg acctgaccat tgggcaatcg ggggtatctt 5340
aaacagctgg cggcccgagg ctgttggggt cgttttcccc actgtcctag caccgtggcc 5400
ctggatctgt tttcgtggct gcctctggag tcctgccttg ctgggacacc gtggctggcg 5460
gaggtgcggc tgacggctgt ttcccacccc caggcctgca ttgatgagaa cctggaggtg 5520
gtgcgcttct tggtggagca aggcgccact gtgaaccagg cggacaacga gggctggacg 5580
cccctgcacg tggccgcctc ctgcggctac ctagacatcg ccaggtgagg caagggaggg 5640
ctgggcagga cctggtcggg gtgggggcct ggaccctcag ccctgaccaa cccccacccc 5700
caggtacctc ctgagccacg gggccaacat cgccgccgtc aacagtgacg gggacctgcc 5760
cctggacctg gccgagtcgg accccatgga ggggctgctg aaggcagaga tcgcccgccg 5820
aggtgggctc tggtgtgctg tgtgtggggt gggggacacc tcctcacctc tggtctccag 5880
gctgagagct gtagaggccg cctggacacc gaggctgacc ctgcttgcta ggattgtctt 5940
tctgtgtgcc tcagtttcct cacccgtgaa atggaggcat ggaggcctgc ctccaggatt 6000
gcttggagcc cagcagaggc acaggataag aactcaaacc cctaccaagt ggggttgtga 6060
tttcactcct tgagtagctg aggagattga aatgccaggg cggggtgggt gggtggaggg 6120
actcaccctg gagtgcgtta ggtaggattg ctggaaccct gccccaggtc tttctgggag 6180
gggatgcgtt tactcggggg ggagtagggg catgcagggg ttgccaggga ctggtctccc 6240
tgtcctctga gactcatggt accccaggag ccttcatatg gagtccagga gtccctgaaa 6300
ctgtgtctgg ccgggttcat ttttctagag aagggctgtt gcttctagca ggtgtgtgat 6360
ggccacacag gatccctggg tccccagcat ggcttctaaa gtcctgggct cacctccccg 6420
ctctgcctgt gactggcgac tctaggcatg tccccgcagc tcgcttccct gatttcctaa 6480
accaggacca ccggccccgc cgcctggggt gggtgtcggg gtctggctgg gtttggttgg 6540
aagggtgtga gcatggttcc ctgcgggttc cggggaaccg tgtggaagga aaggaagcct 6600
gacgaggaaa cgagataccg gcagccttcc agactgctga cgcggctgtg cttacagggc 6660
cgctggccag gctgaaaaag gataggattc cgcgggggtt cagggggcca caagagccac 6720
acaggaggag tagctagtga gacacgtggc ggcagagatc tgaagtttgg gagcccttct 6780
ttggttgaaa tggggagtgg gtcaaatcag ccgtagacct ggattctttc tcctccctcc 6840
ccccaacttt ttactttggg aaagcttgaa atttacagaa aagttggaac aatagcatgt 6900
gactacccat atgactttca ctgagtgtca ccagtgtgtg tgtggttttt tttagacagc 6960
tttttgagat gtaattcaca taccctacaa ttgtgcacac ttcagtgctt tttagtatat 7020
tcacagggtt gggcattcat gaccacattg attatagaac attttcatca tcacccagcg 7080
aaaccctgca accccttagc catcaccccc agccatgcag cccaccctca tgcagcgctc 7140
tgtcttctgt cgctgtagat ttgctgtttt gtatgcgtca cacagaggga atcatacagt 7200
atagaatcgt cagtgtttaa ctttttggta catttgtgtt ctctctctgt ccctcccttg 7260
cgatctcttc ccaggtacag acataaacac attttttcct atgcttcctc ctcctctttt 7320
ttccttccaa atctcccttt cttccttccc atccctgcct ttccattcct gagcgttttt 7380
tttttgaaat ggggtttcac tttttgccca ggctggagtg caatggcatg atggcatgat 7440
ttcggctcac tgcaacctct gtccctctgg gttcaagcga ttctcctgcc ttagcttctg 7500
ggtagctgag atgacaggcg tgtgccacca tgcccagcta attttgcatt tttagtagag 7560
atggagtttc accatgttga ccaggctggt ctggaactcc tgacctcagg cgatctgccg 7620
gcctcagcct tccaaagtgc tgggattaca ggcgtgagcc cggccatttt gagactgtgt 7680
tgcagacatt gtaaatcttc ccctgatgca cagcaggtat ctcctgccac aaggaaaacc 7740
tcctgcagaa ccacagtagg gatgcaacat gttaccccct gcgttgacct tgatgccaca 7800
ctgctacaca ctgttaactt cagtgctaca ctattacccc ctgcactaac cttgatgcca 7860
cacggttaca cactgtgtta acttcgatgc tacactatta cccactgcac taaccttgat 7920
gctacactct tgcctgcgtt tctccttgag attctttgta gcctctcccc ctctggttca 7980
gggcccagct agggatccag atctgggtga tttaggctcc ctctgtctgg gtcagtcttc 8040
ctcctccctc ggacctcagg gaggccagga aaggcctcca gggggtccgt gacctttccg 8100
acattgtatt ttcctgcaga aattgctcat aacttgcatc agcttctcag agggggccat 8160
gctttttccc tcccatgggg tgaggagaag gctggaaggg atacattggt gagggatatt 8220
ggcttgtggg atcagccggg aacgaaggcc actggtgcca ctggaaacac cctcagctgc 8280
gctgctcagg gtggggctct gctgccccaa atcacaaagc ctctgcctgt gacaccttgt 8340
cacacccagc tccacacagg agccttagag gggctgcagt gcgcagaacc tgggtcacag 8400
gctgcacccc agctctaagg agggcggagg cttttgcgcc ccctgcagga catgccgaca 8460
tgatgcagaa ttccccgagg tgctggcatg cagactggtg ctggccatag tgtggcccta 8520
gcagccactc acaggcaggg ctgggagagc agacagaccg gccacaggtg cggccatccc 8580
ctttgctttt ccctggctta cttgctgttt tcctctctct gggtctgggg ttcagtgaga 8640
gcacgtgatc tgctcagcca gtcaccctgt ggcctggagc tactgttccc ttcaatacca 8700
cgaaggcctc tgacccagtc gactcgggct gaatcgggca tgagtctttg caagaaccaa 8760
aacccattcc gcctggctca ggccagaaaa ggccattcga ggcctggagc agtggttcag 8820
acctgtaatc ccagcacttt gggaggccga gacaggagga tctcttgagc ccaggagttt 8880
gagaccagcc tcggaaacct agtgagaact catctctgta aaacaaaaca cattagccag 8940
gcgtgggaat gcacttgtgc tcccagctac tcagtaggct gagatgagaa gattgcttga 9000
acctgggagg tggaagatgc agtgaaccac tgccactcca gcctggggga caggcaagac 9060
cctgtctcaa ctaaaataaa acaaaaaagg cagattatgt acccatgttc atagcatcat 9120
tcacaatagc ccagaggtag gagcaaccta gtgcccatca tcaaatgaat ggaaaatcaa 9180
catgcggtcc ttccatgcaa tgagatatga ttcagtctta atcccagcac tttgggaagc 9240
tgaggtgggt ggatcacctg aggtcgggag ttcgagacca acctgaccaa catggagaaa 9300
ccctgtctct actaaaaata caaaattagc cgggctggtg gcacatccct gtaatctcag 9360
ctactcagga ggctgaggca ggagaatcgc ttgaacccgg gaggtggaga ttgcggtgag 9420
ccaagattgc accactgtgc tctagcctgg gtaacaagag gaaactctgt ctcaaaaaaa 9480
aaaacagaac aaaacacgca atgtagatga atcttgagga cgttatgcta agtgaagagt 9540
gaaactaagc cagtcacaga aggacagtta ctgtataatc cacatctgtg aggtattcag 9600
aatagtcaaa ataacagaca ggcagtagag tggctgtagc ccggagctgg gaggactggg 9660
gagaatagga ggtagtttag tgggtacact tttttttttt ttttaatgat ttctgtagtt 9720
ttttgtagag atggggtttc accacatgac ccccaggctg ctctggaact cttgagctca 9780
agtgatccac ccaccttggc ctcccaaagt gctgggatta caggtgttag ccattgtccc 9840
tgacctaggg tacacgattt tgattttgca agatgaaaaa attctggaga ttggttgcac 9900
aacaaccaat tgtgactact taatgcccct gaactggggt taagatggta aattttaggc 9960
cgggtgcggt ggctcacgcc tgtaatccca gcactttggg aggccgaggc aggcggatca 10020
cctgagctca ggagtttgag accagcctgg ccaacatggt gaaaccccat ctctactaaa 10080
aatacaaaag ttagccaggc atagtggtgt gcacctgtag tcccagctgc ttgggaggct 10140
gaggcaggaa aatcgcttga acctgggagg tggaggttgc agtgaactga gatcccgcca 10200
ctgcaacctc tgcctttcgg gttcaaatga atctcctgcc tcagccttcc aagaaactgg 10260
gattacaggc acccgccacc acgcccagct aatttttgtg tttttagtag agatggggtt 10320
tcactatgtt ggccagtttg gttttgaact cccgacctca ggtgatcctc ccgcctcggc 10380
ctcccaaagt gctgggatta caggcataag ccaccatgcc tggtcatata tatatatata 10440
tatttttaaa ggcaatctat cgccttatgt agctgaagtc catgagttac atggacttca 10500
ggcatagttt gatccagggg cacaagtgaa gtccagaacg tctctccgtc actctgctgc 10560
actccgtgct ggcttcactc ttaggcaggt tctctccata aggcactcag tgtgggtcct 10620
aactgcaggc ccgtatcgca gccaaaacac agcgcctctt tcccagtgcg tgatgctcac 10680
ggggccacct cggccagatg gcagctcctg agcccaccag tgttgcaaag gtgacaggaa 10740
gtcctggtga tgtgcccacc cgggagccag gctatgggcc cagtcacatt gaaaccatgt 10800
ggggcaaagt gtgggtgagg aaagtcaagg tgaggtcaca ggggaaggga gaatggattt 10860
tttaggcccg agcagcagca gctgtgctgc agggacacag cagcaccatg tcctacgcag 10920
aagggaccct ccctggccac tttgcacagg ggcatggaac tggcggcaat gagacatcat 10980
gtttttgaag catttgaagc cgactctctt ggaatcccaa catccgagtc agctggaaga 11040
gggggcgtta cacttggagg caggcagaac tgaccattgg ttcgctccaa gtgctgacaa 11100
gggcggacat gggagctgat ttctgcctgg tgggaaaggc gatgattaca gctactttgg 11160
gaaatggttg atcggcatcg attatagccg aaggccccat ctttgccttg ttctagcagt 11220
tccactcctg ggcaacctga gagaggcctc cccgcccatg gacaaaagtt catcgtcgta 11280
ttgtgtgcag tagtaagagg ttggagccca cactaaagac agaatggatc aatcactaaa 11340
ctaaatgagt gatctaagca ggggtcagca aactttctct ataaagggct agacagtgag 11400
tgtttgaggt gttgcaggac acaccgtccc tggcaccact gttccaccag tgctgtggag 11460
gggacactgc cagggatggt gtatgagtga atgggagtga ctgtgtcccg ttccactttt 11520
attgacaaag attgcctttc agaataggca aatccatgga gacagaaaga ttgctgggtg 11580
caaggggctg gggggaggga ggtctgagaa gtgacttttt ttttttttaa agacaggaac 11640
tcattctgcc tcccaggctg tagtgtagtg gagtgataat ggcttactgc agccttgacc 11700
tcctgggccc aagtgatcct cccacctcag cctcccaagt agctgagacc ataggtgtgc 11760
accaccacac ccagccaatt ttatttttct agagacaggg tctcctgtgt tgcccaggct 11820
gttctcagac tagcgggctc aagtgatctc ccatcttggc ctcccgaagt gctgggatta 11880
caggcgtgag ccatggtgcc tggctgggag tgacttcttg gatacagtat tcattaggga 11940
tcatgaaaaa gctcaggaat tggatagtgg tgatggttgc acaatattgg aggtatccta 12000
aatgccactg aattgtgcac tttaaaacag ggaagttgat aaattttacg ttgtttattt 12060
taccaccgcc accacaacaa aagataaaac acaacaggtg gtgagcaccc acaagctata 12120
gcttgctgag ccctggtcaa gggctctgtc tacagccaca gacagatctc agcaacatgt 12180
tgagcacaga aagcaaactg cagaggatgt gcagcagaca tgcagagggt gctgaccgtg 12240
gttgagtaaa aagccagtca ccctccctcc ccgtccctga attgagaccc atccggccat 12300
aataggattg aaccgttggt gactccttta cagaaatgac acagccaggc tcggtggctc 12360
atgcctgtaa tcccagcact ttgggaggcc aaggcaggag gatcccttga gcctgggaat 12420
tcgaggcttc agtgagccat aattgcacca ctgcactcca gcctggttga cagtgtgaga 12480
tcctgtctcc aaaaaaaaag ggggatcaga tgcagtggtt cacgcctgta atcccagcac 12540
tttgggaggc cgaggcgggc ggatcacgag gtcaggagtt cttcaagacc agcctgacca 12600
gcatggtgaa accccatctc tactaaaaat acaaaaataa attagccggg tgtggtggcg 12660
gcgcctatag tcccagctac tcaagacgct gaggcaggag aaccgcttga acccgggagg 12720
tggagcttac agtgagctga gatcgtgcca ctgcactcca gcctggggga cagagcaaga 12780
ctctgcctca aaaacaaaac aaaacaaaac aaaaaagaac tgcttgaggg caggagtttg 12840
cgaccagcct ggtcaaaatc aggagactcc tacctctctc acctctataa aaaataaaag 12900
taacggctgg gcgcagtggc tcaagcctgt aatcccagca ctttgggagg ccgagacggg 12960
cagatcacga ggtcaggaga tcgagaccat cctggctaac acggtgaaac cccgtctcta 13020
ctaaaaatac aaaaaaaaac tagccgggcg tggtggtgcc tgtagtccca gctactcggg 13080
aggctgaggc aggagaatgg cgtgaacccg ggaggtggag cttgcagtga gctgagatcc 13140
agccactgca ctccagtctg ggcgacagag cgagactcca tctcaaaaaa taaaaataaa 13200
aaataaaaaa aataaaaata actagccggg cgtagtggca tgtgcctgta gttctagcta 13260
cttgggaggc taaagtggga ggatgacttg aacccagcag atcgaggctg cagtgagctg 13320
tgattacgcc gctgcactcc accctgggtg acagagtgag gccctgactg tttaaaaaaa 13380
aaaccgtttg gccagacaca gtggctcacg ctgtgatccc agcacttcgg gaggccaagg 13440
tcggaggatt gcttgagccc aggagattga gaccagcctg ggcaagatat tgagactcca 13500
tctctgttta ttctttgaaa aaaaaaaaaa aaaaaaagac tgggtgcagg ggctcacacc 13560
tgtaatccta gcactttggg agactgaggc aggtggatca cctgaggtca ggagtttgag 13620
accagcctga ccaacatgat ggaaccctgt ttctgctaga aatgcaaaaa ttagctggac 13680
atggtggcag gcacctgtaa tcttagctac tagggaagct gaggcaggag aatcatttaa 13740
actcggtagg cagaggttgc agtgagaccc tgtctccaaa aaaaaaaaaa aaaagagttt 13800
atagcatgca aaagaataag tatcttcaga ggctgtcagt agatatgatt tcagcctcaa 13860
caagggtacc tgttttgatc caaaatcaac ttatatttaa ttttaggtca ttacttgcct 13920
tttacgtcta taatacagtg caacagttgg attacatgaa attgactggt tgggaagcat 13980
gcttttccca gattgctgta ggtggtcgca tgtttgcaaa cgggtggaac acctgggtag 14040
aaaaggagct gttgcactaa tggcacaagt tcttcattta gtggcttttt gcttggttta 14100
ttctcctcat gagaaaagtg gtaatggtga taaaatgtat gaagatgcta gattaagatt 14160
atattggggg gcggcacaac agctcatgcc tgtaatccca acactttggg agaccaaggc 14220
gggcagatca cctgaggtca ggagttcgag accagcctgc ccaatgtgtt gaaaccctgt 14280
ctcttctaaa aacaccaaaa ttagctcagc atggtgattt atgcctgtaa tccaagttac 14340
ttgggaggct gaggcaggag gatctctcga atctcggagg cggaagttgc agtgagattg 14400
cgccactgca ctccagcctg ggtgacagag taagactctg gctccaaaaa aaaaaaaaaa 14460
agttatattg ggagtgacag gaaaagttag atgtaattga gcactggatt tgctcagtaa 14520
acttcaggtt tatatatata tatgttatgt atataggaaa tatatgtata gtgtatatat 14580
ataggagatt gtgtgtgtgt gtgtgtgtgt gtgtatagat atatggtttt tttttttttt 14640
agagtcttgc tgtgtcgccc aggctggagt acagtggcgt aatctcggct tactgcaacc 14700
tctgactcct gggttcaggc gattctcctg cctcagcctc ccaagtagct gggactacag 14760
gcgcccacca ccacatccgg ctactgtttt tttttttttt tttttttttt ttgtaatttt 14820
agtagagatg gagtttcact atgttggcca ggctggtctt gaagtcctga gctcgtgatc 14880
cacctacctc ggcctcttaa agttgctggg attacaggca tgagccactg cagccagcca 14940
tggttttata ttaaaaataa taaaagtaag gtgactttct ggttgagatg gggaaaggcc 15000
tgtgatctcc attttccccg tgcttagcgc tgactcagag accctgagtg ggacccgtga 15060
ctgcatggag cacagttggg aaacagctgg gctagtctag gcctcccacg tctcagaggg 15120
gagcagcggt gaaagctctg ggcgagtccc agggccgaac cccagccagc ccagtgtata 15180
accctgggta ttccaggtcg tgggatgtga gtcctacctc gtgcctcttc tcctcctagg 15240
tgtggacgtg gaggcagcca agcgggcgga ggaggaattg ctacttcatg acacaaggtg 15300
ctggctgaat gggggcgcca tgccagaggc ccggcacccc cgcacaggcg cctctgccct 15360
gcacgtggct gctgccaagg gctacattga ggtgatgagg tgagtcgggc cggtgtgttc 15420
catcttgctg tcttatacct tcctgtggat cctgtccttt ccctttctct ttaaacgtat 15480
gtaccgaaga ctgtgtattt ccctgtcaat aacagcctta gctgtaccca caagttttga 15540
tgtgcggtct tttttaattt gtcattctgt gtaaaatatt tcccaatttt taatctttta 15600
ttatttattt attttaagac agggtcttgc tatatcgccc aggctggagt gcagtggtgc 15660
aatcgtggct cactgcagcc tcaaactccc caggctcagg tgatcctcct acctcagcct 15720
cctgaatagt ggggactaca ggcgcgcacc accatgcccc gctagttttt gtagagacag 15780
ggtttcacca tgttccacag gcttgtctca aactcctggg ctcaagtgat ctacctacct 15840
tagcctccca aagtgctggg attacaggtg tgagccactg tacttggcct aatttcaaaa 15900
tatacaggac ttctctagtc atcattttgt taccattttc ctggcttaat tgcattgtgg 15960
tcagagaatg ggttctatag gattctaact tttgaaatgt attgttttgc cttatatacc 16020
ccagtatatg taagttggaa aagaatggag tcgtcaagta cagtgttctc tgtaggttat 16080
taggtcagcc ttgtaatcat gctgttttat ttgtagtgct tctttatctg tagtgatatt 16140
tcccccgctg cttattctgt cagttactat gagaggtgca tttaaattct tgctatgatt 16200
atggactttg tttcttctta cagttctgtc agttacaatt tgtatatatt ttaagactgg 16260
attatttggt acatatatag tttatttttg tttttgagat ggagtctggc tctctgtcac 16320
ctaggttgga gtgcagtggt gtgatcatag ctcactgcaa cctccgcctc ttgggttcaa 16380
gcagttctct tgcctcagcc tccccagtat ctgtgactat gagagtaagc caccgcactt 16440
gaaaatactt tcgtattttc agtagagatg gagtttctct atgttgccta ggttggtctt 16500
gaactcctga cctcaggtga tctgcctgcc tcagccttcc aaagtgctgg ggttacaggc 16560
gtgagccact gcacccaacc aaggtgcatg tagagtttac attgctatgg tgcccattcc 16620
gtttctgtca tcctcacctc agtcttgggt cttattttgt tcctatttgg ctgcctttct 16680
cctttctatt aaagttgttg gttccagttc tcatccttag gctcctcatt cctttttcag 16740
ttccctcttc tctctttcat gacttacact ttattattta gtataaaaat gaagttaaca 16800
tgaattcctc cccctctctg ctcacctcta tgttgacagt ttttagtcag gttttctcag 16860
tgaggtacat aagatgactc ttaacagctc cagaccgctg tcccttctgc ttagcacctg 16920
ctgctgaagt agagctcctg gacctccgta tctggcccca gtcccagctc tgttgctgac 16980
tgggtccatc cctaagctga tcctttcgac aagggggact ggaaactgaa tggttgggac 17040
tggtgatgtg cccaggcctg caggccgtgt ccttttcctc ccatggcttc cacccctgaa 17100
tcctggaggc ggctgcatct cctccactgc acctgcagcc cagccagcag gaagagaggg 17160
gcaaaggagc aaacacccat tcctttccag agagcagtct gcaagtggca cgcaccactt 17220
aatgccaaca tcccgtttgc cagaccctag tcacatgacc gctcatcgct gcaagggaca 17280
gtaggagatt tgtctttagc tgggtggcct gtgtccggcc gggtgacctg tgtccggccg 17340
ggggcctgtg tgggtactgt tagtaaaggg agagatagag tccatattcg tgggcaacct 17400
cattcattct ctgccataga actgaagcac tttggactta gaaatattca ctgactataa 17460
tttcattact gcttctttgt aaggctgcat gtttacactc agatctattg actgcatttt 17520
ttattttatt tatttattta ttttgagacg gagtctcgct ctgtcaccca ggctggagtg 17580
cagtggcacg atctccactc actgcaatct ccatctcccg ggttcacgcc attctcctgc 17640
ctcagcctcc cgagtagctg ggactacagg cgcccgccac cacacccggc taattttttt 17700
gtatttttag tagagacagg gtttcaccat gttagccagg atggtctcga tctcctgacc 17760
ttgtgatccg cccgcctcgg cctcccaaag tgctgggata acaggtgtga gctaccgagc 17820
acggccattt atttttattt ttatttttga gatggagttt cactctgtca tgcatgctgg 17880
agtgcagtgg ctcaatctca gctcactgca acctctgccc cctgggttca agcacttctc 17940
ctgcctcaac cgcctgaata gctgtgatta caggtatgtg ccaccctgcc cggctaattt 18000
tttgtttgtg gtagagacag gttttgccaa gttggccaag atggtctcga actcctgacc 18060
tcaagtgatc ctcccgcctc agcctcccaa agtgctggaa ttacaggcat gagccactgc 18120
gcccagcctg actgcatttt aaaaagaatg taaccagcat tcgcatcaaa gatctgttgc 18180
aggctgggcg tggtggctca cgcctgtaat cccagcactt tgggaagttg atacaggtgg 18240
atcatgaggt caggagttca agaccagcct ggctaacaca gtgaaacccc gtctctctta 18300
aaaatacaaa aaattagctg ggcatggtgg cgggcgcctg tagtcccagc tacttggaag 18360
gctgaggcag gagaatggcg tgaacccagg aagcagaggt tgcagtgagt ggagatcatg 18420
ccactgcact ccagcctggg caacagtgtg agactctgtc tcaaaacaaa acaaaacaaa 18480
aacatttgtt gcagagtaga ggtatatgtt tatttgatgg tactgattcg atgcttattt 18540
tctgagcccc tgctgtgtgc caggcagtct gttagttgcc aaggaactgg gtaaacaaga 18600
gagatccggg cctatgtgcg gctcatacct cgtggggaga cagacatttt ggactgatgc 18660
tctttgctgc agcgctgtcc cgtgcatcac aggatgtggg gcagcctctc cggcctctac 18720
ctatgggacg tcgggagcat ccctggttgt gagaactaaa aatgtctctg gacgtggcca 18780
aatgtcccct ggggggcgca ttgtcccagt tgagaaccac tggcttaaga tgcaaatagg 18840
gtgatgggag aagacaagtg tgatgaggag gggctttggg gtcaggagac atgggggtga 18900
agggctggtg actgccaaaa aggggctcag cgtgagacgc tgcagggcac agaacactct 18960
ccgacgagag cagcaagtgg cgggtgggcg cttggggaaa tagcgggggg cccgcacagc 19020
tggggcaggg tgagctctgg gcaaagcgtg ggaaaggtga ggggcgaggt gggcggggcc 19080
agatagaggg acttgtgctt ataaggcttc ttcgtagaat atcccaggct gtccatctgt 19140
tcccactttg cgaagctctt tctcattgac acagaaactc attccagttc tccaaaaata 19200
gacacgtcct gccctgagct gagctcggag agtgcccttg gacgtggagc cctgcctttc 19260
cctcagattg aatggtttaa ggtgttattg ttttctggca gaaaactgtc ttgagtgagg 19320
gtttctcagc ccaggcgcta cccacatttt ggggcagatc cttctccttg tggggctgct 19380
gctgggcgct gctgggtgtg gagccgcctc cctggcctcc acccagccga tgccactagc 19440
ccctgcacca ggttatgaca acaaagatgt ctgcagacct gccgggtctc ccctggggga 19500
cagcattgcc ctggttggga gtcactgctt taagccagcc caggtcctag aacactactg 19560
ccctgagaca gccctttggg gtccaatcct tctgacgggc gtgggggctg gcacagggtc 19620
ctgcacctgc atcctccggg gctgcctcct tacaccccac tgctgtcacc ttggaatccc 19680
acagccaggg tgaccccctg ccggcaccag gagcaggttc cagaggccac ctgcagggcc 19740
agggtttaac gtgtagctgc tggtccaagg ggagggacaa ggcctggtgc agaggggcct 19800
cttgtggcca gggacgctgg gcagtggctc tttaccagcc tgtccagggg cgttttagtc 19860
tcgtggcctg ggacctcagt taccgcacca ctggctgaaa tcagcccccg gcccagggcc 19920
tcgccttggc ctctgaccct gagactccct ggcccccagg ttgctccttc aggctggcta 19980
cgacccagag ctccgggacg gggacggctg gactcccctg cacgcggcgg cacactgggg 20040
cgtggaggat gcctgccgcc tgctggccga gcatggcggg ggcatggact cgctgaccca 20100
cgcggtgagg gccagtgccc aggctggtgg ggggcagggc gcggggagga cccagcgcct 20160
ccttcattgt ctgctcctgc tcctccccca acccaggggc agcggccctg tgacctggct 20220
gatgaggaag tgctgagcct gttggaggaa ctggcccgga aacaggagga cgtgagtgtc 20280
agggctgagg ggcttgtccc agactcccca tgtcccctca actcctcaga ggagggtgca 20340
gagggagtcc ccccccgact tctgacaccc accaccccag caacctgctc tttcctgctt 20400
ggtgacccag ctccagcccc agcctcagga gagaccccca gccggggcag gatagagatg 20460
gggctgcaga gcttcctacc tgcctggtgg ggaggcagca gaaggatctg gacagggttg 20520
tctgacactc agacaaaata acaaaccagc tcagtgctcc tcatcacctc agggtggccc 20580
ttggcagcag ccatgtgcag gactgtccta gacttaggct gggctggtca cctgcaaggt 20640
gtcccccagc catccccggg gcagccttgc tgggaaaact tgcagtgaag gagtgaaggg 20700
cgtgagccct ggccagactg cctttgttgg aatcctggct ctgccatgga tctgccgagt 20760
cccagccagc cactttcctc tccaggcctc agtttcctca tctgtcaaat gggaatggta 20820
tggtgcgcac ctgtatgagg ggtcgtgcgt gacatgtgga ttgtgctgtc aggagctcgg 20880
gagtacgtgc cacgggacag cactagagcc atgccagcat tcagtgagct ctgtggggtg 20940
acaggaagct cccgaggcca ggcctagggc ttctggcgca tgggcagcgg agtggagaca 21000
gcttcctctt ttgaaaggtt ggcaggagca aggtggaggc tgacggatgt aggagaagaa 21060
ggtctcacac agtctggggg gcaagtgtgg gtgccctgag gtgcccaggg aggggagtgt 21120
aaggcagagc aaacccggga ggaccatgag tgccaggcga gaagcagggt gacttttgcc 21180
ctgagggtga tatggccaag gggggtttaa tgggtggggc aggatcagct taggtggccc 21240
agatatgagg ggagcccagc ctgggagcat ctggggacag tttctagaag acgtgagtac 21300
tacagcacta agagtttgct agaagggcag gaatgaaggg ccctcccagt actcctgggc 21360
accacatgca gaggcccgaa ggtgtgaaga cagtggtgaa ctgtgtgggg aaggtgtgaa 21420
gacagtggtg aactgtgggg aaggtgtgaa gacggtggca aactgtgtgg ggaaggtgtg 21480
aagatggtgg tgaactgtgt ggggaaggtg tgaagatggt gaactgtgtg ggaaaggtgt 21540
gaagatggcg gtgaactgtg tggggaagat gtgaagatgg cagtgaactg tgtgggaaag 21600
gtgtgaagac ggtgaactgt gtggggaagg tgtgaagaag gtgtggaaga cggtggtgaa 21660
ccgtgtgggg aaggtgcaag actatgtgaa ctgtgtgggg aaggtgtgaa gatggcagtg 21720
aactgtgtgg aaaggtgtga agacggtgaa ctgtgtgggg aaggtggaag acggtggtga 21780
atctggtgtg gggaaggggt gtgaagatgg tgggtgaact gggggtggag aaggtgtgaa 21840
gacggtgtga atcagtgggg aacgggtgaa gatggtggtg aactgtgtgg ggaaggtgtg 21900
aagacagtgg tgaactgtgt ggggaaggtg tgaagacggt ggtgaaactg tgtggggaag 21960
gtgtgaagac agtggtggaa ctgtatggga aggtgtgaag atggtggtga actgtgtggg 22020
aaggtgtgaa gacagtagtg tgaactgtaa aggtgtgaag acagtgaact gtgggaaggt 22080
gtgaagacgg tggtgaactg tgggaaggtg taagacagtg gaactgtggg gaaggtggaa 22140
gacggtggtg aactgtgggg aaggtgtgaa gacggggtgg tgaactgtgt gggaaaggtg 22200
tgaagatcgt ggtgaactgt gggaaggtgt ggaagacggt agaactgtgt gggaaaggtg 22260
tgaagacggt ggtgaactgt gtggagaagg tgtgaagaca gtgaactgtg tggggaaggt 22320
gtgaagatgg tgttgaactg tgtggggcag gtgtgaagat ggtgttgaac tgtgtggggc 22380
aggtgtgaag atggtgttga actttgtggg gcaggtgtga agacggtggt gaactgtgtg 22440
gggcaagtgt gaagacggtg ctgaactatg tggggaagga ggaggcgagt gccagcagca 22500
gaagccgggg cagctgaggg cccaccagtg ccctgggtgt gctgaggggt cgggatgagg 22560
tctgagcagg tcgcccctgg gagctagaag ggctcctctg gggatgagtc tggaggcttc 22620
tcatgggtgg gggccgtgag gacaagaggg cagtgcctgc acgtggcagc tgcaggcgcc 22680
tgctggacac agggcacctc ctctctctga ggtgtgccgc catctgtagg aaaaccagca 22740
agcgcttcca cgagccctcg gtgcccgcct gggcactgag ctcctgacag ctgttcatgg 22800
tggtattgct gtgggggacg tgtgctccac ccctcccctg cagcctcctt ccctccttgc 22860
agcttcggaa ccaaaaagaa gcttcccaga gccggggcca ggagccccaa gcgccctgta 22920
gcagcaaaca cagaaggtat agggagggag gagtcggggg ctggcggggt ggctggaggc 22980
cccgggcttg caccgcagca ccctctcgtt ccgcaacagg agctctgtgt gtcgtctgag 23040
cagccgagag aagatttccc tccaggacct gtccaaggag cgccggcctg gtggggctgg 23100
ggggccccct atccaggacg aggatgaggg ggaagaaggt cccgccggtg aggagcctga 23160
atctggactc ctgggtccaa gggaggaggg gtcgggttct ttgaatctag aggcagctgg 23220
cgtcttctgt gacctacccg gtattcccac atcacagagc cacgctctgc agaacccagg 23280
accctcaatg gcgtctcctc cccaccgcac cccagtccta agagccccgt ggtgagtgga 23340
tgccctgaga ggtcctgtga ggctgggagg gccgggaggt gggagttcat aaagcctgtg 23400
aacctctggc aggggcaggt agggtgaaga ctatagttcc cagagtcctc ctggacaacc 23460
ggcttccttt ttaggttgtg cttgccccag gggctgatgg gaattgtagt ttgttgcagt 23520
cctttggggg ctaacccttg aggtctgaga agaggaatcc tttctccctg agacctcctt 23580
cttctcccag ccgcctgaag aggccccctt ctccaggcgc tttggcctcc tgaagacagg 23640
gagttctggt gccctgggtc ccccggaaag gcggatggca gagggagccc ctggggctgg 23700
gctgcagcgc tcggcttcct cctcctggct ggaagggacc tccacccagg tgagtgaggg 23760
ttgggccggg tccgggccca ggcccctgcc cagatgaggg aactgcccct tcctcctcct 23820
ctccacacag gccaaggagc tccgtcttgc cagaattacc ccgaccccct cccggaagct 23880
gccggagccc tctgccctgt gagtccccaa ggcctgggcc cctcgggggt aggcggcaga 23940
tgtgggttcc aagcctgtct gtatgatcct gagaccatgt gccccccggg gtctgattcc 24000
tgctcccacc ccagagcctg tgctgtgttc ctcctggcgg ctggctccct ccctcaagcc 24060
tgtgttgaaa cgtcaacttc tccccatcat ccccccaccc cgaggcgcac ggaccacctg 24120
ctgccacctc tgagccgtgg gcacacgccc ctcccgctcc gctcccagtt ctcgcactgc 24180
tgcttctgtc cagcgtcctt tacccttctg accgcttggg tggagtgctg gtcttttcat 24240
ggctattgtc ggtgttgtgt atttctgctc agctctgact ctggtggccc agataatttg 24300
tttcttttgt acacggctgc atctccagtg ccggcacaca atgcacctgg gtaatgtttg 24360
ttgaatgaat gaattgatgc aggtctgagg tcaccaagcc tcctccttgc ttggagaact 24420
cctcgcctcc ctccaggatt ccggagcctg aatctccagc gaagccaaac gcccccacag 24480
cctccacagc gccctcagcg gactcccggg accgacggag gtgcagaggc tagggcagga 24540
agctgccgcc cccgcccccg ccccagcccc ggcccccagc cagcagagac tatgtcaagc 24600
cggagtcccg gggctctggg tgggttcctg agggagggct ggccccgggt ctgctgggag 24660
ttggagtccc ctcccatgga acttgcctcc cgcagagggt gtcagccacg gaatgggaca 24720
gggtccccag ggcccctccc tgaggttggg acccttccct caggtcctac cagatgcctg 24780
tgcgggatga ggagtcggaa tcccagagaa aagctcgctc ccgtctcatg cgccagtctc 24840
ggaggtccac acaggtgtgg ggtgggctgg gggcttgcac ccctgggtct gagggaggag 24900
gggctggggg gtggccaggg gcttgcaccc tgggtctgga gggagagggc caggggctgc 24960
accctgggcc tgagggagga ggggctgggg gcttgcaccc ctgggtctga gggaggaggg 25020
gctgggggct tgcacccctg ggtctgaggg aggaggggct ggggtctgga ctcttgggtc 25080
tgagggagga gggactgggg tctggactcc tgggtctgag ggaggagggg atggagtctg 25140
gactcctggg cctgagggag gagggactgg ggtctggact cctgggtctg agggaggagg 25200
gactggggtc tggactccta ggtctaaggg aggaggggat ggagtctgga ctcctgggcc 25260
tgaggaaaga ggagctgggc cctgacccca gcatcccctg actgtcccct ttccctgcca 25320
gggtgtgact ctgacagatc tgaaggaggc agagaagtct gcagggaagg ccccagagtc 25380
agagaagccg gcgcagagcc tggtgagagg ggtcagcgag ggcgtgtggg gtggtgcggg 25440
atgtcctggt cccctgactc accaacccct cccgacagga cccttcccgg aggccccgca 25500
tccctggagt ggagaactcc gacagccctg cccagagagg tgaggtctgc tctttggggg 25560
aggtcattcc cgggaatcat cgtctctccc agagtcttcc tgctttcctg ggggcttgac 25620
ccggggaccc gagcccccag aggggtctct ggaggtgcct agactgattc ctgcccctcc 25680
cccagcagag gcgcctgacg ggcagggtcc aggaccgcag gcggccaggg agccccgcaa 25740
ggtcggaaag gagtggaggg ggcctgcgga ggtgaggggg gctcaggtgg ggccgagctg 25800
gtgcagaggg tgggccgggg ggcgggcaaa gcccctccca tacccgctaa agcgcctccc 25860
atgcccattg aacccccctc tacctgccca cagggggagg aggcggagcc ggctgaccac 25920
agtccggagt ccaggtaggg gcggggtggg agaggtgggg cctgcgtgcg gcccgggcca 25980
gcccctgacg cgcgttccgc ccccgcccca gcaccctgga gggcggcccc tcagcccgca 26040
ggcagcggtc acagcgggac ctccacccag aagctgagcc agagtcggag gagcaggacg 26100
gaggctttag gaaggtgtga tcccacccct cctttggggc tccgcctgct aggtgcccac 26160
ctcctggctt ccccgccccc agcgtcgccc ctgccctcca cctgaccctc ctccgcctga 26220
ccttcctcca cccgaccccc agctgtatgc agagctgcgc agggagaacg agcggcttcg 26280
cgaggctctg accgagacca cgctgcggct ggcgcagctc aaggtggagc tggagcgggc 26340
cacgcaggta aaggcgccga ggccagggtc accagaactg gcagggccgc ctgggtgggg 26400
ctcagactgc tgggaggcag agcctggcct ggaggggccg atggggcttc gctccacctg 26460
agggcctggc cgagatctcg gaggcggggt ctgagcgaat gggggtgggg ctgtgggcct 26520
gctgggggcg gggcctggcc catgggggcg gggctggtgg gctgcctagt gggcgggcgg 26580
gtccccagca ggaatgtagg acccctcctc agagcagtgc tggcttcccc tgctgacccc 26640
tgtgctttct gcgtcctcac cagaggcaag agcgcttcgc tgagaggcca gcccttctgg 26700
agctggagag attcgtgagt agggacagtg gggacgggag ccagaggcgt tcccagcctc 26760
acatcactaa gtttggacca agagagccac attgcccttc ccagctggcc cctggccttc 26820
ccctgtcctc agcctcactc ggtttctgaa ctttccctgt tctttccagg agcgcagggc 26880
cctggaacgc aaggccgcag agctggagga agagctgaag gtggggtctg tgggcagagg 26940
gccgagggtg ggcgggggcg caacccacca gggctgccca gctgactctt tgccgtcccc 27000
caggccctgt ctgacctccg tgctgacaac cagcgcctca aggatgagaa tgcggcgttg 27060
atccgcgtta tcagcaaact ctccaagtga cctggaggga ctttcccgca cccgtattta 27120
tacagctgct tctgccacac gcaccccttc ccccgtgtga acacgagtga cagggctccg 27180
ggggaagtct cagagaagcc atagcccccc tgggacctcc gaacttggag gggagcagag 27240
tccccaggag ccaagggggc cacccgaggc caggatggag gtgggaggcc gagccaagta 27300
aggggacgtt gccagagggg accagggcca gagccgggac taggaaggaa ctgaggacca 27360
agaagtgcaa ggaccagtag ccaggaccag agtggccgcc tggaggtccc cctctcacgt 27420
gtgatgccac cccgtcaccc ccctcctgag cagggatcca agactgtgcc aagagtcctg 27480
ccggcctcag ccaggtgggc ctgtatatag ggtccatgtg caatagggag ggatgtcttc 27540
tattttttgc tgccccctcc acgcccactg tctggggcag ggggagaagg tattttcgag 27600
ataaagcaca ggcaccacaa ataaaagtcg tgaagttgcc actcaa 27646
Claims (1)
1.一种基于遗传密码子扩展技术的改造Vero细胞系，其特征在于，所述改造Vero细胞系的构建方法是基于基因密码子扩展技术，使用CRISPR/Cas9系统将携带PylRS/tRNAPyl系统的质粒插入到Vero细胞株中，定点插入一套能翻译终止密码子的序列，所述翻译终止密码子的序列由2个隔离子、1个CAG启动子、1个马氏甲烷八叠球菌吡咯赖氨酸tRNA合成酶基因、1个WPRE转录后调控序列、1个ployA序列、4个hU6启动子-马氏甲烷八叠球菌吡咯赖氨酸tRNA基因序列组成，所述翻译终止密码子的序列的碱基如SEQ ID NO.1所示；SEQ ID NO.1所示序列定点插入的位置为PPP1R12C蛋白基因的第一个内含子内AAVS1位点，具体插入位置为NW_023666045.1，263811bp处；
所述CRISPR/Cas9系统的sgRNA的序列为GCCCAAATTAAAGAAGTGAGAGG。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210412748.7A CN115074330B (zh) | 2022-04-19 | 2022-04-19 | 一种基于遗传密码子扩展技术的改造Vero细胞系 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210412748.7A CN115074330B (zh) | 2022-04-19 | 2022-04-19 | 一种基于遗传密码子扩展技术的改造Vero细胞系 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115074330A CN115074330A (zh) | 2022-09-20 |
| CN115074330B true CN115074330B (zh) | 2023-12-29 |
Family
ID=83246744
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210412748.7A Active CN115074330B (zh) | 2022-04-19 | 2022-04-19 | 一种基于遗传密码子扩展技术的改造Vero细胞系 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115074330B (zh) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107177593A (zh) * | 2016-03-10 | 2017-09-19 | 北京大学 | 利用优化的基因密码子扩展系统通读提前终止密码子疾病中的截短蛋白 |
| CN110835633A (zh) * | 2018-08-13 | 2020-02-25 | 北京大学 | 利用优化的基因密码子扩展系统制备ptc稳定细胞系及应用 |
| WO2021152086A1 (en) * | 2020-01-30 | 2021-08-05 | Bit Bio Limited | Safe harbor loci |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106929482A (zh) * | 2015-12-31 | 2017-07-07 | 北京大学 | 定点突变的流感病毒、其活疫苗及其制备方法和应用 |
-
2022
- 2022-04-19 CN CN202210412748.7A patent/CN115074330B/zh active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107177593A (zh) * | 2016-03-10 | 2017-09-19 | 北京大学 | 利用优化的基因密码子扩展系统通读提前终止密码子疾病中的截短蛋白 |
| CN110835633A (zh) * | 2018-08-13 | 2020-02-25 | 北京大学 | 利用优化的基因密码子扩展系统制备ptc稳定细胞系及应用 |
| WO2021152086A1 (en) * | 2020-01-30 | 2021-08-05 | Bit Bio Limited | Safe harbor loci |
Non-Patent Citations (1)
| Title |
|---|
| 马建岗.基因工程学原理.西安交通大学出版社,2018,(第4版),第289-290页. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115074330A (zh) | 2022-09-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| RU2760877C2 (ru) | Животные, отличные от человека, характеризующиеся экспансией гексануклеотидных повторов в локусе c9orf72 | |
| WO2020097372A1 (en) | Use of adeno-associated viral vectors to correct gene defects/ express proteins in hair cells and supporting cells in the inner ear | |
| CA3004665C (en) | Transposon system, kit comprising the same, and uses thereof | |
| EP3359676B1 (en) | Transposon system, kit comprising the same, and uses thereof | |
| CN110997925A (zh) | Cho细胞中的整合位点 | |
| KR20220013556A (ko) | 인간 만능 줄기 세포로부터 미세아교세포의 신속하고 결정적인 생성 | |
| CN111057721A (zh) | 人源化IL-4和/或IL-4Rα改造动物模型的制备方法及应用 | |
| KR20220097414A (ko) | X-연관 연소 망막층간분리 치료법을 위한 crispr 및 aav 전략 | |
| WO2019041344A1 (en) | METHODS AND COMPOSITIONS FOR THE TRANSFECTION OF SINGLE STRANDED DNA | |
| US20240018485A1 (en) | Method to reduce oncogenic potential of induced pluripotent stem cells from aged donors | |
| AU2018204332A1 (en) | Expression cassette | |
| AU2022200678A1 (en) | Nucleic acid molecules containing spacers and methods of use thereof | |
| CN115074330B (zh) | 一种基于遗传密码子扩展技术的改造Vero细胞系 | |
| WO2018126095A1 (en) | Transgenic animals and method for bioproduction | |
| KR20140015999A (ko) | 신규 ＭＡＲｓ 및 이를 이용하여 목적 단백질을 생산하는 방법 | |
| CN115175559A (zh) | 包含人源化pnpla3基因座的非人动物及其使用方法 | |
| US20020086427A1 (en) | Inducible eukaryotic expression system that regulates protein translation | |
| CN109295000B (zh) | 一种多能干细胞形成必需蛋白crept在诱导多能干细胞中的应用 | |
| JP5835772B2 (ja) | 骨芽細胞特異的発現を誘導するｄｎａ及びその塩基配列 | |
| CN115322993B (zh) | 一种用于猪基因组定点整合外源基因的安全位点及用其构建猪育种群方法 | |
| CN113846063B (zh) | 一种适用于同种异体移植的通用型人源干细胞以及构建方法 | |
| EA046478B1 (ru) | Быстрая и детерминистическая генерация микроглии из плюрипотентных стволовых клеток человека | |
| KR20230101839A (ko) | 세이프 하버 유전자좌위 | |
| JP2022090246A (ja) | 哺乳動物細胞用ノックインベクター | |
| CN113444722A (zh) | 单碱基编辑介导的剪接修复在制备治疗脊髓性肌萎缩症中的应用 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |