CN111593073A - 双报告基因骨架载体、四质粒假病毒包装系统、包装新冠肺炎假病毒 - Google Patents
双报告基因骨架载体、四质粒假病毒包装系统、包装新冠肺炎假病毒 Download PDFInfo
- Publication number
- CN111593073A CN111593073A CN202010191574.7A CN202010191574A CN111593073A CN 111593073 A CN111593073 A CN 111593073A CN 202010191574 A CN202010191574 A CN 202010191574A CN 111593073 A CN111593073 A CN 111593073A
- Authority
- CN
- China
- Prior art keywords
- gene
- reporter gene
- plasmid
- pseudovirus
- virus
- 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.)
- Granted
Links
- 239000013612 plasmid Substances 0.000 title claims abstract description 79
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 68
- 238000004806 packaging method and process Methods 0.000 title claims abstract description 61
- 241001112090 Pseudovirus Species 0.000 title claims abstract description 59
- 239000013598 vector Substances 0.000 title claims abstract description 34
- 206010035664 Pneumonia Diseases 0.000 title description 4
- 241001573881 Corolla Species 0.000 title description 2
- 241000700605 Viruses Species 0.000 claims abstract description 35
- 108700008625 Reporter Genes Proteins 0.000 claims abstract description 34
- 241000711573 Coronaviridae Species 0.000 claims abstract description 30
- 241001678559 COVID-19 virus Species 0.000 claims abstract description 20
- 101710091045 Envelope protein Proteins 0.000 claims abstract description 17
- 101710188315 Protein X Proteins 0.000 claims abstract description 17
- 108060001084 Luciferase Proteins 0.000 claims abstract description 15
- 239000005089 Luciferase Substances 0.000 claims abstract description 15
- 108091006047 fluorescent proteins Proteins 0.000 claims abstract description 12
- 102000034287 fluorescent proteins Human genes 0.000 claims abstract description 12
- 108700004030 rev Genes Proteins 0.000 claims abstract description 5
- 101150098213 rev gene Proteins 0.000 claims abstract description 5
- 101150047047 gag-pol gene Proteins 0.000 claims abstract description 3
- 239000005090 green fluorescent protein Substances 0.000 claims description 16
- 102100021696 Syncytin-1 Human genes 0.000 claims description 15
- 229940096437 Protein S Drugs 0.000 claims description 13
- 230000009977 dual effect Effects 0.000 claims description 13
- 108010043121 Green Fluorescent Proteins Proteins 0.000 claims description 12
- 102000004144 Green Fluorescent Proteins Human genes 0.000 claims description 12
- 101710198474 Spike protein Proteins 0.000 claims description 12
- 102000029301 Protein S Human genes 0.000 claims description 9
- 108090000331 Firefly luciferases Proteins 0.000 claims description 8
- 150000001413 amino acids Chemical class 0.000 claims description 8
- 238000011144 upstream manufacturing Methods 0.000 claims description 8
- 241000710831 Flavivirus Species 0.000 claims description 6
- 239000002773 nucleotide Substances 0.000 claims description 6
- 125000003729 nucleotide group Chemical group 0.000 claims description 6
- 102000004169 proteins and genes Human genes 0.000 claims description 5
- 241000711950 Filoviridae Species 0.000 claims description 4
- 238000013518 transcription Methods 0.000 claims description 4
- 230000035897 transcription Effects 0.000 claims description 4
- 241001430294 unidentified retrovirus Species 0.000 claims description 4
- 241000713666 Lentivirus Species 0.000 claims description 3
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical group C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 2
- 241000725619 Dengue virus Species 0.000 claims description 2
- 101100118093 Drosophila melanogaster eEF1alpha2 gene Proteins 0.000 claims description 2
- 241001115402 Ebolavirus Species 0.000 claims description 2
- 241000711549 Hepacivirus C Species 0.000 claims description 2
- 241000700721 Hepatitis B virus Species 0.000 claims description 2
- 241000701024 Human betaherpesvirus 5 Species 0.000 claims description 2
- 101710177509 Major immediate early protein Proteins 0.000 claims description 2
- 241000712079 Measles morbillivirus Species 0.000 claims description 2
- 241000713112 Orthobunyavirus Species 0.000 claims description 2
- 241000711798 Rabies lyssavirus Species 0.000 claims description 2
- 108010052090 Renilla Luciferases Proteins 0.000 claims description 2
- 241000725643 Respiratory syncytial virus Species 0.000 claims description 2
- 108091081024 Start codon Proteins 0.000 claims description 2
- 241000907316 Zika virus Species 0.000 claims description 2
- 108010027225 gag-pol Fusion Proteins Proteins 0.000 claims description 2
- 230000003494 hepatotrophic effect Effects 0.000 claims description 2
- 230000001553 hepatotropic effect Effects 0.000 claims description 2
- 230000003834 intracellular effect Effects 0.000 claims description 2
- 108010054624 red fluorescent protein Proteins 0.000 claims description 2
- 230000001105 regulatory effect Effects 0.000 claims description 2
- 102220097517 rs876659265 Human genes 0.000 claims description 2
- 241001529453 unidentified herpesvirus Species 0.000 claims description 2
- 241000712461 unidentified influenza virus Species 0.000 claims description 2
- 208000015181 infectious disease Diseases 0.000 abstract description 8
- 238000011160 research Methods 0.000 abstract description 5
- 238000012216 screening Methods 0.000 abstract description 3
- 229960005486 vaccine Drugs 0.000 abstract description 3
- 230000000840 anti-viral effect Effects 0.000 abstract description 2
- 238000011156 evaluation Methods 0.000 abstract 1
- 238000002360 preparation method Methods 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 29
- 108020004414 DNA Proteins 0.000 description 20
- 210000002966 serum Anatomy 0.000 description 10
- 230000003472 neutralizing effect Effects 0.000 description 9
- 238000001514 detection method Methods 0.000 description 8
- 238000001976 enzyme digestion Methods 0.000 description 8
- 238000000338 in vitro Methods 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 238000004113 cell culture Methods 0.000 description 5
- 238000010276 construction Methods 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 239000001963 growth medium Substances 0.000 description 5
- 101100203795 Severe acute respiratory syndrome coronavirus 2 S gene Proteins 0.000 description 4
- 230000003321 amplification Effects 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- 102100035765 Angiotensin-converting enzyme 2 Human genes 0.000 description 3
- 108090000975 Angiotensin-converting enzyme 2 Proteins 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 3
- 101710139375 Corneodesmosin Proteins 0.000 description 3
- 241000699666 Mus <mouse, genus> Species 0.000 description 3
- 239000012124 Opti-MEM Substances 0.000 description 3
- 125000003275 alpha amino acid group Chemical group 0.000 description 3
- 210000000170 cell membrane Anatomy 0.000 description 3
- 238000010367 cloning Methods 0.000 description 3
- 238000012217 deletion Methods 0.000 description 3
- 230000037430 deletion Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 108010048367 enhanced green fluorescent protein Proteins 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 230000003053 immunization Effects 0.000 description 3
- 238000002649 immunization Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 108020003175 receptors Proteins 0.000 description 3
- 102000005962 receptors Human genes 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 108020004705 Codon Proteins 0.000 description 2
- 102100031673 Corneodesmosin Human genes 0.000 description 2
- 208000001528 Coronaviridae Infections Diseases 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 102100021579 Enhancer of filamentation 1 Human genes 0.000 description 2
- 101000898310 Homo sapiens Enhancer of filamentation 1 Proteins 0.000 description 2
- 244000309467 Human Coronavirus Species 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 208000025370 Middle East respiratory syndrome Diseases 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 240000000220 Panda oleosa Species 0.000 description 2
- 235000016496 Panda oleosa Nutrition 0.000 description 2
- 208000035415 Reinfection Diseases 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
- 239000003085 diluting agent Substances 0.000 description 2
- 241001493065 dsRNA viruses Species 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 230000003278 mimic effect Effects 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 238000006386 neutralization reaction Methods 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000012264 purified product Substances 0.000 description 2
- 230000000241 respiratory effect Effects 0.000 description 2
- 210000002345 respiratory system Anatomy 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 241000282832 Camelidae Species 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 241000283153 Cetacea Species 0.000 description 1
- 241000288673 Chiroptera Species 0.000 description 1
- 206010011224 Cough Diseases 0.000 description 1
- 206010011732 Cyst Diseases 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 101710121417 Envelope glycoprotein Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 241001109669 Human coronavirus HKU1 Species 0.000 description 1
- 241000482741 Human coronavirus NL63 Species 0.000 description 1
- 241001428935 Human coronavirus OC43 Species 0.000 description 1
- 201000008197 Laryngitis Diseases 0.000 description 1
- 241000127282 Middle East respiratory syndrome-related coronavirus Species 0.000 description 1
- 241001292005 Nidovirales Species 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 101150010882 S gene Proteins 0.000 description 1
- 241000315672 SARS coronavirus Species 0.000 description 1
- 101100545229 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) ZDS2 gene Proteins 0.000 description 1
- 101100113084 Schizosaccharomyces pombe (strain 972 / ATCC 24843) mcs2 gene Proteins 0.000 description 1
- 101000629313 Severe acute respiratory syndrome coronavirus Spike glycoprotein Proteins 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 101100167209 Ustilago maydis (strain 521 / FGSC 9021) CHS8 gene Proteins 0.000 description 1
- 108010003533 Viral Envelope Proteins Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 239000000443 aerosol Substances 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 230000003281 allosteric effect Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 206010006451 bronchitis Diseases 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 208000031513 cyst Diseases 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000011841 epidemiological investigation Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000005099 host tropism Effects 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 201000009240 nasopharyngitis Diseases 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 238000012858 packaging process Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 229940023143 protein vaccine Drugs 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
Images
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
- 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
- C12N15/86—Viral vectors
-
- 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
- C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
-
- 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
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
-
- 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
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
- C12N2740/15041—Use of virus, viral particle or viral elements as a vector
- C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
-
- 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
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/20011—Coronaviridae
- C12N2770/20021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
-
- 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
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/20011—Coronaviridae
- C12N2770/20051—Methods of production or purification of viral material
- C12N2770/20052—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Virology (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
Abstract
双报告基因骨架载体，包括双报告基因骨架载体，其双报告基因选自荧光素酶和荧光蛋白，荧光蛋白报告基因优选mGFP基因，荧光素酶报告基因优选Fluc基因；双报告基因四质粒假病毒包装系统包括双报告基因骨架载体以及分别整合慢病毒gag‑pol基因、慢病毒rev基因以及含有目的病毒的包膜蛋白基因的3种真核表达包装质粒；包装新冠肺炎假病毒包括双报告基因四质粒假病毒包装系统、宿主细胞，所述宿主细胞优选自293T‑hACE2细胞。本发明对SARS‑CoV‑2进行具体实施，可快速包装出单周期感染且安全性更高的假病毒，可用于SARS‑Cov2等冠状病毒的研究，为抗病毒制剂和疫苗的评价提供了有力的筛选工具,具备广泛的应用价值。
Description
技术领域
本发明涉及基因工程和分子生物学领域，具体双报告基因骨架载体、四质 粒假病毒包装系统、包装新冠肺炎假病毒。
背景技术
冠状病毒(Coronavirus)在病毒学分类上属于巢状病毒目(order Nidovirals)、冠状病毒科(family Coronavirade)、冠状病毒属(genus Coronavirus)的成员，基因组为单股、正链的RNA，基因组全长在26～32kb之 间，是目前已知基因组最大的RNA病毒。冠状病毒在自然界的感染普非常广泛， 常见的哺乳类动物如犬、猫、鼠、猪、牛以及家禽类都易感。近几年来，又从 白鲸、骆驼，尤其是蝙蝠体内分离到多种类型的冠状病毒。人冠状病毒目前已 知有六种，分别是二十世纪60年代发现的人冠状病229E(HCoV-229E)和 HCoV-OC43，2003年出现的SARS，2004年在荷兰分离的HCoV-NL63，2005年在 香港鉴定的HCoV-HKU1以及2012年在中东地区出现的新型中东呼吸综合征 (Middle East respiratory syndromevirus，MERS)冠状病毒MERS-CoV。 SARS-CoV-2与SARS(severe acute respiratorysyndrome)的基因具有高度 的同源性。在2019-nCoV侵染宿主细胞过程中，病毒的刺突蛋白(spike protein,S protein)首先识别细胞膜上受体蛋白ACE2 (angiotensin-convertingenzyme 2)，进而介导并促使病毒包膜与细胞膜发 生融合，最终使病毒侵入宿主细胞。冠状病毒能够感染机体的呼吸道、消化道、 肝脏、肾脏以及神经系统，造成不同程度的病理性损伤，严重者甚至造成死亡。
自新型冠状病毒(SARS-CoV-2)肺炎疫情暴发以来，截至目前已引起全国 超过8万人被感染，3000多人死亡；而在全球范围内已导致超过200000人感 染，是一种传播性很强的病毒。SARS-CoV-2冠状病毒与传统冠状病毒的区别在 于对所有人都易感，既能感染上呼吸道，引起发热、咳嗽、喉炎等普通感冒症 状，又能感染下呼吸道，引起支气管炎、肺炎等急性呼吸道症状。SARS-CoV-2 与SARS冠状病毒的结构相似，是一种有包膜的单股正链RNA病毒，体现在病毒 表面的刺突蛋白S蛋白是病毒包膜上特异性的组织结构，在病毒的表面形成了 大量的刺突蛋白，在病毒入侵靶细胞以及病毒与细胞识别时发挥着重要作用， 同时SARS-CoV-2通过气溶胶传播，已有的研究表明其传播能力是SARS的十倍 以上。因此在非P3实验室操作SARS-CoV-2活病毒具有极大的生物安全隐患。
假病毒是指一种反转录病毒能够整合另外一种不同种类病毒的囊膜糖蛋 白，形成的具有外源性病毒的囊膜，而基因组保持着反转录病毒本身基因组特 性的病毒。假病毒由于核酸分子的缺陷性而只有一个细胞感染周期，因此具有 较高生物安全性，这对研究具有致病率高，传染性强、不易在体外培养的病毒 如HIV、SARS等提供了一种安全有效的研究方法。体外构建的假病毒还具有稳 定性强、宿主嗜性广等优点，因此被广泛应用于病毒宿主细胞的筛选、疫苗的 研发、中和抗原表位研究等方面。
目前有关SARS-CoV-2的基础研究较为薄弱，流行病学调查较少，而这些研 究普遍需要操作SARS-CoV-2活病毒，而操作SARS-CoV-2活病毒具有较强的生 物安全隐患，因此包装制备出生物安全性等级较低的SARS-CoV-2的假病毒具有 重要意义。
发明内容
双报告基因骨架载体，包括选自慢病毒遗传物质的双报告基因骨架载体， 所述双报告基因骨架载体的双报告基因选自荧光素酶和荧光蛋白，荧光素酶报 告基因的上游是调节转录的启动子,其中所述启动子优选自hEF1a启动子；所述 荧光蛋白报告基因的上游是增强型转录启动子E-IRES；所述双报告基因骨架载 体命名为pDLV19-mGFP-Fluc。
进一步的，所述双报告基因包括荧光蛋白报告基因和荧光素酶报告基因， 所述的荧光蛋白报告基因选自绿色荧光蛋白或红色荧光蛋白；所述荧光素酶报 告基因选自萤火虫荧光素酶或海肾荧光素酶。
进一步的，所述荧光蛋白报告基因选择修饰的绿色荧光蛋白mGFP基因，所 述绿色荧光蛋白mGFP基因的序列为SEQ ID NO：1；所述荧光素酶报告基因选 自密码子优化的萤火虫荧光素酶Fluc基因，所述萤火虫荧光素酶Fluc基因的 序列为SEQ ID NO：2。
进一步的，所述萤火虫荧光素酶Fluc基因的上游是hEF1a启动子，所述 hEF1a启动子基因序列为SEQ ID NO：3，所述绿色荧光蛋白mGFP基因的上游是 ECMV-IRES增强启动子，所述ECMV-IRES增强启动子的基因序列为SEQ ID NO： 4。
进一步的，所述绿色荧光蛋白mGFP基因位于荧光素酶Fluc基因的下游， 所述绿色荧光蛋白mGFP基因起始密码子序列之前添加了Kozak序列。
进一步的，所述pDLV19-mGFP-Fluc双报告基因骨架载体具有SEQ ID NO： 5的序列。
一种四质粒假病毒包装系统，由权利要求1～6中任一双报告基因骨架载体pDLV19-mGFP-Fluc、3种真核表达包装质粒组成；其中所述3种真核表达包装 质粒分别整合慢病毒gag-pol基因、慢病毒rev基因以及含有目的病毒的包膜 蛋白基因。
进一步的，所述3种真核表达包装质粒选自pCAGGS，质粒pCAGGS中CMV 启动子的序列优选替换为：
(3)EF1a启动子；
(4)包含有人巨细胞病毒主要即刻早期蛋白基因增强型启动子。
进一步的，所述含有慢病毒gag-pol的真核表达包装质粒具有SEQ ID NO： 6序列，所述包装质粒命名为psl4-gag-pol；所述含有慢病毒rev基因的真核 表达包装质粒具有SEQ ID NO：7序列，所述包装质粒命名为psl5-rev。
进一步的，所述pCAGGS真核表达包装质粒在宿主细胞水平表达目的病毒的 包膜蛋白，所述pCAGGS真核表达包装质粒细胞水平表达的包膜蛋白选自冠状病 毒、疱疹病毒、痘病毒、嗜肝病毒、丝状病毒、棒状病毒、流感病毒、副粘病 毒、黄病毒、弹状病毒、副粘病毒、黄病毒、包膜病毒、布尼亚病毒或逆转录 病毒的一种病毒或多种病毒。
进一步的，所述冠状病毒优选为SARS-CoV-2或SARS；所述嗜肝病毒优选 为乙肝病毒或丙肝病毒；所述丝状病毒优选为Ebola病毒；所述弹状病毒优选 为狂犬病毒；所述副粘病毒优选为麻疹病毒或呼吸道合胞病毒；所述黄病毒优 选为寨卡病毒或登革热病毒。
进一步的，所述真核表达包装质粒表达的包膜蛋白选自冠状病毒 SARS-CoV-2，所述双报告基因四质粒假病毒包装系统用于制备新冠假病毒时， 所述冠状病毒包膜蛋白选自：(1)刺突蛋白S全长氨基酸，即SEQ ID NO：8序 列、(2)刺突蛋白S的3’端缺失19个氨基酸，即SEQ ID NO：9序列、(3)刺 突蛋白S的3’端缺失28个氨基酸，即SEQ ID NO：10序列或(4)刺突蛋白S 的3’端缺失53个氨基酸且同时融合VSV包膜蛋白的胞内段，即SEQ ID NO：11序列；对应的所述pCAGGS真核表达包装质粒在宿主细胞水平表达的囊膜蛋 白的质粒命名分别为psl3-S、S19、S28、S53C。
一种包装新冠肺炎假病毒，其特征在于：包括权利要求7～12任一所述双 报告基因四质粒假病毒包装系统、宿主细胞，所述宿主细胞选自293、293T、 293sus、HEK293、HEK293T或HEK293FT细胞，所述宿主细胞表面稳定表达hACE2 蛋白。
需要补充说明内容如下：
本发明专利涉及到的假病毒优先考虑有包膜病毒的假病毒；
新冠假病毒的制备方法，其包括以下步骤：将权利要求1-6任一项的双报 告基因载体，和两个包装辅助质粒，以及包膜蛋白真核表达载体，合计4个质 粒共转染293T-hACE2，收集细胞培养上清，获得假病毒；其中，所述包膜表达 载体含有新冠病毒的包膜蛋白S基因及其改造体，所述的293T-hACE2是通过慢 病毒将hACE2整合到宿主细胞基因组中，所述的293T-hACE2宿主细胞膜表面稳 定表达hACE2蛋白，所述的hACE2是冠状病毒SARS-CoV-2进入哺乳动物细胞的 主要受体。
附图说明：
图1、hEF1α启动子启动Luc表达和IRES下的EGFP蛋白双报告检测体系(A)； 四个目的基因PCR扩增出条带(B)；
图2、建立四种包膜假病毒包装系统(图2A)，验证三种包膜变构体包装新冠 假病毒的效率(图2B)；
图3、启动子及报告基因优化的pDLV19-mGFP-Fluc质粒系统与传统未优化的 进行假病毒(VSVG囊膜)包装及体外复感染检测GFP的表达(A)和Luc(B) 的表达强度；
图4、四质粒体系包装出的新冠假病毒模拟SARS-CoV-2,应用于阳性血清的中 和抗体的检测；
如下具体实施方式将结合上述附图进一步说明本发明。
具体实施方式
下面结合具体实施例对本公开做进一步的详细说明，所述是对本发明的解 释而非限定，本公开主要是将真核表达SARS-CoV-2S基因或其修饰变构体作为 包膜包装质粒，通过构建的四质粒包装系统拯救出可模拟该囊膜抗原的假病毒， 并用于检测SARS-CoV-2S或截断体表达的蛋白在小鼠体内免疫后获得血清的中 和抗体生成情况。
本公开采用的试剂及耗材如下：Lipofectamine LTX(Invitrogen 15338100)，PBS(Hyclone SH30256.01)，DMEM高糖培养基(Gibco C11995500)，双抗(Gibco 15140-122)，胎牛血清(Gibco 10091-148)，
I Reduced Serum Medium(Gibco31985-070)，96孔细胞培养板(Corning 3599)，6孔细胞培养 板(Corning 3516)，6cm细胞培养板(Corning 430166)，T175细胞瓶(Corning 431080)。细胞系：
293T-hACE2贴壁细胞培养在37℃、含5％CO2的特定培养环境中(Thermo BB150细胞培养箱)，采用DMEM高糖完全培养基进行培养。
不同类型的包膜质粒构建方案：
分子构建以2019-nCoV的Spike蛋白(S蛋白)克隆作为重点(参考基因 组为GWHABKF00000000.genome.fasta)密码子优化后，分别将S全长序列 (1-3822bp)、S缺失C端19个氨基酸序列(1-3765bp)、S缺失C端28个氨基 酸序列(1-3738bp)、S缺失C端53个氨基酸序列(1-3663bp)克隆至pCAGGS 载体上。
实施例1 pCAGGS-SARS-CoV-2-S包膜质粒的构建
根据NCBI发布的SARS-CoV-2的S基因序列经过密码子优化设计后便于目 的基因在细胞内更高效的表达，所合成的目的基因序列由南京金斯瑞生物分别 合成至pCDNA3.1载体上，PCR扩增目的基因后，经过片段纯化试剂盒回收纯化 目的条带，将该片段和pVSV-GFP载体用限制性核酸内切酶(MCS1(Xhol)和 MCS2(Nhel))于37℃酶切3h，胶回收载体和目的片段后进行连接反应，再转至 感受态细胞，菌液PCR筛选阳性克隆及酶切和测序验证鉴定质粒构建情况(并 将构建的质粒命名为pCAGGS-SARS-CoV-2-S)，具体实施步骤如下：
引物合成及引物信息：引物由苏州金唯智生物生物科技有限公司合成，其 中构建扩增SARS-CoV-2-S基因所选PCR引物如表1所示：
表1 SARS-CoV-2-S基因扩增与检测引物
扩增SARS-CoV-2-S-△19-tail基因所选PCR引物及菌液PCR引物如表2所 示：
表2 SARS-CoV-2-S-△28-tail基因扩增
扩增SARS-CoV-2-S-△28-tail基因所选PCR引物及菌液PCR引物如表2所 示：
表3 SARS-CoV-2-S-△28-tail基因扩增
扩增SARS-CoV-2-S-△53-tail基因所选PCR引物及菌液PCR引物如表2所 示：
表4 SARS-CoV-2-S-△53-tail基因扩增
1)目的基因的获取：以带目的基因序列的pCDNA3.1质粒作为模板以表1-表4 引物进行PCR扩增；
2)参照AxyPrepTM PCR Cleanup kit说明书纯化上述酶切产物，用Nano-300 测定产物浓度；
3)将上述纯化产物以及载体进行双酶切(37℃酶切3h)；
4)用1％Agarose gel进行电泳，用相应的DNA maker作为对照，验证PCR产 物是否正确，割取凝胶条带，回收剩下的PCR产物，用Nano-300测定产物 浓度；
5)将上述纯化产物与载体进行连接(16℃连接过夜，连接比按1:5进行)；
6)参照E.coli DB3.1 Competent Cells(TaKaRa)说明书转化连接产物；
7)挑取LB(Kana)平板上的单克隆到事先加有200μL LB(Kana)培养基的无 菌1.5mL管中，37℃，250rpm，培养3h后，进行Colony PCR筛选阳性克 隆；
8)经琼脂糖凝胶电泳鉴定后，选取阳性克隆按1：500比例转接到15mL摇瓶中， 37℃、250rpm摇床培养14-16h；
9)按照TIANGEN无内毒素小提中量试剂盒说明书进行质粒提取；
10)将筛选的阳性质粒进行双酶切鉴定(Xho I和Nhe I在37℃酶切3h)；
11)酶切鉴定后，选取其中鉴定正确的质粒进行质粒测序；
12)将测序正确的质粒转染293T-hACE2后，收取细胞进行WB检测目的蛋白ACE2(新冠病毒的受体)的表达水平。
所构建的假病毒体系骨架质粒如图1A所示，含有hEF1α启动子启动Luc 表达和IRES下的EGFP蛋白双报告检测体系；根据实验结果可知，四个目的基 因分别进行PCR扩增后在相应位置处均出现特异性条带且条带分子大小均正 确，表明成功扩增出目的基因条带(图1B)，后续通过双酶切克隆分别如图1 所示的特定克隆区域中将启动子以及双报告基因整合在骨架质粒中，最终双报 告基因载体pDLV19-mGFP-FLuc构建成功。
实施例2 pDLV19-mGFP-FLuc假病毒包装体系的建立
为避免传统的活病毒在中和抗体检测实验中存在的生物安全隐患，本发明 公开了一种基于四质粒的双报告基因的假病毒包装系统，mGFP的增强性绿色荧 光有助于提高检测灵敏度，同时缩短了检测luc冷荧光活性的繁琐，节省操作 时间，本技术是以pDLV19-mGFP-FLuc骨架载体作为表达FLuc和mGFP的双报告 包装质粒作为核心，以psl4-gag-pol和psl5-rev作为辅助质粒，以构建的 psl3-S(包括其截短突变体)作为包膜包装质粒按一定比例进行体外宿主细胞 的瞬转，收集上清得到新冠假病毒，具体操作如下：
1)293T-hACE2细胞铺6孔板5个孔，1*106/孔，贴壁12-16h后转染；
2)pDLV19-mGFP-FLuc 0.67ug，pCAGGS-S/pCAGGS-S△19-tail/pCAGGS-S△ 28-tail/pCAGGS-S△53-tail/pCAGGS-VSVG(阳性包膜质粒)各0.33ug， psl4-gap-pol1.33ug，psl5-rev 0.67ug稀释于125ul opti-MEM并混匀， 6ul Lipofectamin 2000稀释于125ul opti-MEM并混匀；
3)将质粒稀释液与Lipofectamin 2000稀释液缓慢混匀，混匀后室温静置 20min；
4)将完全培养基更换成opti-MEM，将混合液加入到培养基中轻轻混匀，37℃， 5％CO2培养6h后更换成完全培养基；
5)细胞培养48h和72h后各收集一次病毒上清；
6)取100ul病毒上清感染预先铺有293T-hACE2细胞的96孔板中；
7)病毒感染72h后观察EGFP荧光表达情况和适用酶标仪读取Luc荧光强度；
8)根据假病毒包装情况确定所构建的四个包膜质粒的假病毒包装效率。
假病毒包装流程如图2A所示，四种包膜质粒中，表达全长S包膜质粒几乎 不能包装出假病毒(复感染有微弱荧光)，S△53-tail包膜质粒能包装出假病 毒的活性滴度最高，包装出的假病毒能够有效模拟假病毒体外感染293T-hACE2， 体外细胞水平结果如图2B所示，绿色荧光强度最高的代表假病毒的粒子数最 多。
实施例3 pDLV19-mGFP-Fluc假病毒包装体系对比实验
通过将pDLV19-mGFP-Fluc为骨架的报告质粒与pDLV19-GFP-luc为骨架的 报告质粒按实施例2进行假病毒包装及感染，观察GFP荧光表达和Luc荧光强 度，根据图3结果可知，以pDLV19-mGFP-Fluc为报告质粒的GFP(图3A)和 Luc(图3B)荧光表达强度较pDLV19-GFP-luc(非修饰载体包装的对照假病毒)， 侵染后表达的荧光强度更强，进一步提高了检测灵敏性，同时更便于肉眼的直 接对实验结果的定性分析，与对照相比，pDLV19-mGFP-Fluc的luc的阳性数值 比例更高，定量分析时组间差异更显著。
实施例4基于pDLV19-mGFP-FLuc假病毒体系测定蛋白疫苗免疫后小鼠血清中 特异性抗体的中和效果
为了验证包制备的SARS-CoV-S假病毒能否通过中和实验进行抗体中和活 性的检测，利用已知对SARS-Cov-2具有中和活性的小鼠血清进行假病毒中和实 验验证(所用假病毒为S△53-tail包膜质粒包装)，同时设置正常小鼠血清作 为阴性对照，具体操作步骤如下：
1)293T-hACE2细胞铺96孔板，1*104/孔，贴壁12-16h；
2)将血清进行梯度稀释后与等体积的假病毒混合(4000pfu)，设置复孔，同时 设置未与血清混合的假病毒对照和只加培养基的空白细胞对照；
3)于37摄氏度，5％CO2培养箱中放置1h；
4)将上一步中的血清-假病毒混合液接种至96孔板中，37摄氏度，5％CO2培养48h；
5)计算检测荧光素酶的活性(RLU)；
6)根据感染百分率(％)＝(检测血清RLU-空白细胞对照RLU)/(假病毒对照 RLU-空白细胞对照RLU)x100％；
根据结果可知(图4)，本公开的四质粒体系包装出的新冠假病毒可以充分 模拟SARS-CoV-2，用来检测疫苗免疫后的抗病毒抗体的中和效力，且本次实验 样本的IC50数值约为阳性血清样本稀释103倍时的中和抗体的浓度。表明本公 开的四质粒假病毒包装体系制备的新冠假病毒具备一次侵染宿主细胞的能力， 充分模拟了病毒的侵染的过程，是模拟新冠病毒侵染的有效工具。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细， 但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的 普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进， 这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求 为准。
序列表
<110> 苏州奥特铭医药科技有限公司
<120> 双报告基因骨架载体、四质粒假病毒包装系统、包装新冠肺炎假病毒
<130> 2020
<141> 2020-03-18
<160> 19
<170> SIPOSequenceListing 1.0
<210> 1
<211> 774
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 1
atggccacaa ccatgaaggg ggttaaggag gttatgaaga tttctctcga gatggactgc 60
acagtgaacg gcgacaagtt caagatcacc ggggatggta ctggagaacc atatgaggga 120
actcagaccc tgcacctgac agaaaaggag ggcaagcctc tgacctttag ctttgacgtc 180
ctcacacctg cctttcaata cggtaaccgc actttcacta aataccctgg caacatcccc 240
gacttcttta aacagacagt tagcggcggg gggtacacct gggagcgcaa gatgacttac 300
gaggacggag gcatctctaa cgtcaggagc gatatatctg tcaagggaga ttccttctat 360
tacaaaatcc acttcactgg agagttccct cctcatggac ccgtgatgca gcggaagacc 420
gtgaaatggg agcccagcac cgaagtcatg tatgtcgatg acaagagcga cggggtcctc 480
aagggcgacg tgaacatggc cctgctcctc aaggacggcc gccacctccg ggttgacttt 540
aacacttctt atatcccaaa gaagaaggtg gagaacatgc ctgactatca ttttattgac 600
caccgcattg aaatcctggg caatcctgaa gacaaacctg tcaaactgta cgagtgtgcc 660
gtcgcccgct actccctgct ccccgagaag aacaagtcca aagggaactc caagctggag 720
ggaaaaccca tccctaaccc actgctcggc ctgtactcca cacggaccgg gtga 774
<210> 2
<211> 1659
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 2
atggaagatg ccaagaacat caagaaaggc cctgccccct tctaccccct ggaagatggc 60
acagccggcg agcagctgca caaggccatg aagagatacg ccctggtgcc cggcaccatc 120
gccttcaccg acgcccacat cgaggtggac atcacctacg ccgagtattt cgagatgagc 180
gtgcggctgg ccgaggccat gaaacgctac ggcctgaaca ccaaccaccg gatcgtggtg 240
tgcagcgaga acagcctgca gttcttcatg cccgtgctgg gcgccctgtt catcggcgtg 300
gccgtggccc ctgccaacga catctacaac gagcgggagc tgctgaacag catgggcatc 360
agccagccca ccgtggtgtt cgtgagcaag aagggcctgc agaaaatcct gaacgtgcag 420
aagaagctgc ccatcatcca gaaaatcatc atcatggaca gcaagaccga ctaccagggc 480
ttccagagca tgtacacctt cgtgaccagc cacctgcccc ctggcttcaa cgagtacgac 540
ttcgtgcccg agagcttcga ccgggacaag accatcgccc tgatcatgaa cagcagcggc 600
agcaccggcc tgcctaaagg cgtggccctg cctcaccgga ccgcctgcgt gcggttcagc 660
cacgcccggg accccatctt cggcaaccag atcatccccg acaccgccat cctgagcgtg 720
gtgcccttcc accacggctt cggcatgttc accaccctgg gctacctgat ctgcggcttc 780
cgggtggtgc tgatgtaccg gttcgaggaa gagctgttcc tgcggagcct gcaggactac 840
aagatccaga gcgccctgct ggtgcccacc ctgttcagct ttttcgccaa gagcaccctg 900
atcgacaagt acgacctgag caacctgcac gagatcgcca gcggcggagc ccccctgtcc 960
aaagaagtgg gcgaagccgt cgccaagcgg ttccacctgc ccggcatccg gcagggctat 1020
ggcctgaccg agaccacaag cgccattctg atcacccccg agggcgacga caagcctggc 1080
gccgtgggca aggtggtgcc tttcttcgag gccaaggtgg tggacctgga caccggcaag 1140
accctgggcg tgaaccagcg gggcgagctg tgcgtgaggg gccccatgat catgagcggc 1200
tacgtgaaca accccgaggc caccaacgcc ctgattgaca aggacggctg gctgcacagc 1260
ggcgacatcg cctactggga cgaggacgag cacttcttca tcgtggaccg gctgaagagc 1320
ctgatcaagt acaagggcta ccaggtggcc ccagccgagc tggaaagcat cctgctgcag 1380
caccccaaca tcttcgatgc cggggtggcc ggactgcccg acgacgatgc cggcgagctg 1440
cctgccgccg tggtggtgct ggaacacggc aaaaccatga ccgagaaaga aatcgtggac 1500
tacgtggcca gccaggtgac caccgccaag aaactgagag gcggcgtggt gtttgtggac 1560
gaggtgccca agggcctgac aggcaagctg gacgcccgga agatccggga gatcctgatc 1620
aaggccaaga agggcggcaa gggcggcggc ggcagctaa 1659
<210> 3
<211> 1159
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 3
gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgaa 60
ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc 120
gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc 180
tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc cgtgtgtggt tcccgcgggc 240
ctggcctctt tacgggttat ggcccttgcg tgccttgaat tacttccacc tggctgcagt 300
acgtgattct tgatcccgag cttcgggttg gaagtgggtg ggagagttcg aggccttgcg 360
cttaaggagc cccttcgcct cgtgcttgag ttgaggcctg gcctgggcgc tggggccgcc 420
gcgtgcgaat ctggtggcac cttcgcgcct gtctcgctgc tttcgataag tctctagcca 480
tttaaaattt ttgatgacct gctgcgacgc tttttttctg gcaagatagt cttgtaaatg 540
cgggccaaga tcgatctgca cactggtatt tcggtttttg gggccgcggg cggcgacggg 600
gcccgtgcgt cccagcgcac atgttcggcg aggcggggcc tgcgagcgcg gccaccgaga 660
atcggacggg ggtagtctca agctggccgg cctgctctgg tgcctggcct cgcgccgccg 720
tgtatcgccc cgccctgggc ggcaaggctg gcccggtcgg caccagttgc gtgagcggaa 780
agatggccgc ttcccggccc tgctgcaggg agctcaaaat ggaggacgcg gcgctcggga 840
gagcgggcgg gtgagtcacc cacacaaagg aaaagggcct ttccgtcctc agccgtcgct 900
tcatgtgact ccacggagta ccgggcgccg tccaggcacc tcgattagtt ctcgatcgag 960
cttttggagt acgtcgtctt taggttgggg ggaggggttt tatgcgatgg agtttcccca 1020
cactgagtgg gtggagactg aagttaggcc agcttggcac ttgatgtaat tctccttgga 1080
atttgccctt tttgagtttg gatcttggtt cattctcaag cctcagacag tggttcaaag 1140
tttttttctt ccatttcag 1159
<210> 4
<211> 587
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 4
cccctctccc tccccccccc ctaacgttac tggccgaagc cgcttggaat aaggccggtg 60
tgcgtttgtc tatatgttat tttccaccat attgccgtct tttggcaatg tgagggcccg 120
gaaacctggc cctgtcttct tgacgagcat tcctaggggt ctttcccctc tcgccaaagg 180
aatgcaaggt ctgttgaatg tcgtgaagga agcagttcct ctggaagctt cttgaagaca 240
aacaacgtct gtagcgaccc tttgcaggca gcggaacccc ccacctggcg acaggtgcct 300
ctgcggccaa aagccacgtg tataagatac acctgcaaag gcggcacaac cccagtgcca 360
cgttgtgagt tggatagttg tggaaagagt caaatggctc tcctcaagcg tattcaacaa 420
ggggctgaag gatgcccaga aggtacccca ttgtatggga tctgatctgg ggcctcggta 480
cacatgcttt acatgtgttt agtcgaggtt aaaaaaacgt ctaggccccc cgaaccacgg 540
ggacgtggtt ttcctttgaa aaacacgatg ataatatggc cacaacc 587
<210> 5
<211> 10837
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 5
taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa 60
tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt 120
gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat 180
cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg 240
gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga 300
gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac 360
tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt 420
ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag 480
cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc 540
gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag 600
gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca 660
gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt 720
cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc 780
tttttacggt tcctggcctt ttgctggcct tgaagctgtc cctgatggtc gtcatctacc 840
tgcctggaca gcatggcctg caacgcgggc atcccgatgc cgccggaagc gagaagaatc 900
ataatgggga aggccatcca gcctcgcgtc gcgataaccc taattcgata gcatatgctt 960
cccgttgggt aacatatgct attgaattag ggttagtctg gatagtatat actactaccc 1020
gggaagcata tgctacccgt ttagggttca ccggtgatgc cggccacgat gcgtccggcg 1080
tagaggatct aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct 1140
tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta 1200
tgaccatgat tacgccaagc gcgcaattaa ccctcactaa agggaacaaa agctggagct 1260
gcaagcttaa tgtagtctta tgcaatactc ttgtagtctt gcaacatggt aacgatgagt 1320
tagcaacatg ccttacaagg agagaaaaag caccgtgcat gccgattggt ggaagtaagg 1380
tggtacgatc gtgccttatt aggaaggcaa cagacgggtc tgacatggat tggacgaacc 1440
actgaattgc cgcattgcag agatattgta tttaagtgcc tagctcgata cataaacggg 1500
tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg 1560
cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt 1620
gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt 1680
ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga 1740
ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa 1800
aaattttgac tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc 1860
gggggagaat tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat 1920
ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg 1980
gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc 2040
agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc 2100
atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa 2160
acaaaagtaa gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagat 2220
atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta 2280
ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga 2340
ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca 2400
atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat 2460
ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag 2520
cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg 2580
atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg 2640
agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa 2700
attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa 2760
aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac 2820
ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt 2880
ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca 2940
ttatcgtttc agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa 3000
gaagaaggtg gagagagaga cagagacaga tccattcgat tagtgaacgg atctcgacgg 3060
tatcggtttt aaaagaaaag gggggattgg ggggtacagt gcaggggaaa gaatagtaga 3120
cataatagca acagacatac aaactaaaga attacaaaaa caaattacaa aaattcaaaa 3180
ttttatcgat accgtcgacc tcgacggatc gggagatctc gaccgagctt tgcaaagatg 3240
gataaagttt taaacagaga ggaatctttg cagctaatgg accttctagg tcttgaaagg 3300
agtgggaatt ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga 3360
gaagttgggg ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa 3420
ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta 3480
tataagtgca gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca 3540
ggtaagtgcc gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt 3600
gccttgaatt acttccacct ggctgcagta cgtgattctt gatcccgagc ttcgggttgg 3660
aagtgggtgg gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt 3720
tgaggcctgg cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg 3780
tctcgctgct ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct 3840
ttttttctgg caagatagtc ttgtaaatgc gggccaagat cgatctgcac actggtattt 3900
cggtttttgg ggccgcgggc ggcgacgggg cccgtgcgtc ccagcgcaca tgttcggcga 3960
ggcggggcct gcgagcgcgg ccaccgagaa tcggacgggg gtagtctcaa gctggccggc 4020
ctgctctggt gcctggcctc gcgccgccgt gtatcgcccc gccctgggcg gcaaggctgg 4080
cccggtcggc accagttgcg tgagcggaaa gatggccgct tcccggccct gctgcaggga 4140
gctcaaaatg gaggacgcgg cgctcgggag agcgggcggg tgagtcaccc acacaaagga 4200
aaagggcctt tccgtcctca gccgtcgctt catgtgactc cacggagtac cgggcgccgt 4260
ccaggcacct cgattagttc tcgatcgagc ttttggagta cgtcgtcttt aggttggggg 4320
gaggggtttt atgcgatgga gtttccccac actgagtggg tggagactga agttaggcca 4380
gcttggcact tgatgtaatt ctccttggaa tttgcccttt ttgagtttgg atcttggttc 4440
attctcaagc ctcagacagt ggttcaaagt ttttttcttc catttcaggt gtcgtgagga 4500
attagcttga ccaagcttgg taccgagctc ggatccacta gtaacggccg ccagtgtgct 4560
ggaattatca caagtttgta caaaaaagca ggctacgcgt gccaccatgg aagatgccaa 4620
gaacatcaag aaaggccctg cccccttcta ccccctggaa gatggcacag ccggcgagca 4680
gctgcacaag gccatgaaga gatacgccct ggtgcccggc accatcgcct tcaccgacgc 4740
ccacatcgag gtggacatca cctacgccga gtatttcgag atgagcgtgc ggctggccga 4800
ggccatgaaa cgctacggcc tgaacaccaa ccaccggatc gtggtgtgca gcgagaacag 4860
cctgcagttc ttcatgcccg tgctgggcgc cctgttcatc ggcgtggccg tggcccctgc 4920
caacgacatc tacaacgagc gggagctgct gaacagcatg ggcatcagcc agcccaccgt 4980
ggtgttcgtg agcaagaagg gcctgcagaa aatcctgaac gtgcagaaga agctgcccat 5040
catccagaaa atcatcatca tggacagcaa gaccgactac cagggcttcc agagcatgta 5100
caccttcgtg accagccacc tgccccctgg cttcaacgag tacgacttcg tgcccgagag 5160
cttcgaccgg gacaagacca tcgccctgat catgaacagc agcggcagca ccggcctgcc 5220
taaaggcgtg gccctgcctc accggaccgc ctgcgtgcgg ttcagccacg cccgggaccc 5280
catcttcggc aaccagatca tccccgacac cgccatcctg agcgtggtgc ccttccacca 5340
cggcttcggc atgttcacca ccctgggcta cctgatctgc ggcttccggg tggtgctgat 5400
gtaccggttc gaggaagagc tgttcctgcg gagcctgcag gactacaaga tccagagcgc 5460
cctgctggtg cccaccctgt tcagcttttt cgccaagagc accctgatcg acaagtacga 5520
cctgagcaac ctgcacgaga tcgccagcgg cggagccccc ctgtccaaag aagtgggcga 5580
agccgtcgcc aagcggttcc acctgcccgg catccggcag ggctatggcc tgaccgagac 5640
cacaagcgcc attctgatca cccccgaggg cgacgacaag cctggcgccg tgggcaaggt 5700
ggtgcctttc ttcgaggcca aggtggtgga cctggacacc ggcaagaccc tgggcgtgaa 5760
ccagcggggc gagctgtgcg tgaggggccc catgatcatg agcggctacg tgaacaaccc 5820
cgaggccacc aacgccctga ttgacaagga cggctggctg cacagcggcg acatcgccta 5880
ctgggacgag gacgagcact tcttcatcgt ggaccggctg aagagcctga tcaagtacaa 5940
gggctaccag gtggccccag ccgagctgga aagcatcctg ctgcagcacc ccaacatctt 6000
cgatgccggg gtggccggac tgcccgacga cgatgccggc gagctgcctg ccgccgtggt 6060
ggtgctggaa cacggcaaaa ccatgaccga gaaagaaatc gtggactacg tggccagcca 6120
ggtgaccacc gccaagaaac tgagaggcgg cgtggtgttt gtggacgagg tgcccaaggg 6180
cctgacaggc aagctggacg cccggaagat ccgggagatc ctgatcaagg ccaagaaggg 6240
cggcaagggc ggcggcggca gcgactacaa ggacgacgat gacaagtaag tcgacaccca 6300
gctttcttgt acaaagtggt gataattctg cagatatcca tcacactggc ggccgctcga 6360
gttaattaag ctagcgcccc tctccctccc ccccccctaa cgttactggc cgaagccgct 6420
tggaataagg ccggtgtgcg tttgtctata tgttattttc caccatattg ccgtcttttg 6480
gcaatgtgag ggcccggaaa cctggccctg tcttcttgac gagcattcct aggggtcttt 6540
cccctctcgc caaaggaatg caaggtctgt tgaatgtcgt gaaggaagca gttcctctgg 6600
aagcttcttg aagacaaaca acgtctgtag cgaccctttg caggcagcgg aaccccccac 6660
ctggcgacag gtgcctctgc ggccaaaagc cacgtgtata agatacacct gcaaaggcgg 6720
cacaacccca gtgccacgtt gtgagttgga tagttgtgga aagagtcaaa tggctctcct 6780
caagcgtatt caacaagggg ctgaaggatg cccagaaggt accccattgt atgggatctg 6840
atctggggcc tcggtacaca tgctttacat gtgtttagtc gaggttaaaa aaacgtctag 6900
gccccccgaa ccacggggac gtggttttcc tttgaaaaac acgatgataa tatggccaca 6960
accatgaagg gggttaagga ggttatgaag atttctctcg agatggactg cacagtgaac 7020
ggcgacaagt tcaagatcac cggggatggt actggagaac catatgaggg aactcagacc 7080
ctgcacctga cagaaaagga gggcaagcct ctgaccttta gctttgacgt cctcacacct 7140
gcctttcaat acggtaaccg cactttcact aaataccctg gcaacatccc cgacttcttt 7200
aaacagacag ttagcggcgg ggggtacacc tgggagcgca agatgactta cgaggacgga 7260
ggcatctcta acgtcaggag cgatatatct gtcaagggag attccttcta ttacaaaatc 7320
cacttcactg gagagttccc tcctcatgga cccgtgatgc agcggaagac cgtgaaatgg 7380
gagcccagca ccgaagtcat gtatgtcgat gacaagagcg acggggtcct caagggcgac 7440
gtgaacatgg ccctgctcct caaggacggc cgccacctcc gggttgactt taacacttct 7500
tatatcccaa agaagaaggt ggagaacatg cctgactatc attttattga ccaccgcatt 7560
gaaatcctgg gcaatcctga agacaaacct gtcaaactgt acgagtgtgc cgtcgcccgc 7620
tactccctgc tccccgagaa gaacaagtcc aaagggaact ccaagctgga gggaaaaccc 7680
atccctaacc cactgctcgg cctgtactcc acacggaccg ggcaccacca ccatcaccac 7740
tgagtctagc ctagtgccat ttgttcagtg gttcgtaggg ctttccccca ctgtttggct 7800
ttcagttata tggatgatgt ggtattgggg gccaagtctg tacagcatct tgagtccctt 7860
tttaccgctg ttaccaattt tcttttgtct ttgggtatac atttaaaccc taacaaaaca 7920
aagagatggg gttactctct aaattttatg ggttatgtca ttggatgtta tgggtccttg 7980
ccacaagaac acatcataca aaaaatcaaa gaatgtttta gaaaacttcc tattaacagg 8040
cctattgatt ggaaagtatg tcaacgaatt gtgggtcttt tgggttttgc tgcccctttt 8100
acacaatgtg gttatcctgc gttgatgcct ttgtatgcat gtattcaatc taagcaggct 8160
ttcactttct cgccaactta caaggccttt ctgtgtaaac aatacctgaa cctttacccc 8220
gttgcccggc aacggccagg tctgtgccaa gtgtttgctg acgcaacccc cactggctgg 8280
ggcttggtca tgggccatca gcgcatgcgt ggaacctttt cggctcctct gccgatccat 8340
actgcggaac tcctagccgc ttgttttgct cgcagcaggt ctggagcaaa cattatcggg 8400
actgataact ctgttgtcct atcccgcaaa tatacatcgt ttccatggct gctaggctgt 8460
gctgccaact ggatcctgcg cgggacgtcc tttgtttacg tcccgtcggc gctgaatcct 8520
gcggacgacc cttctcgggg tcgcttggga ctctctcgtc cccttctccg tctgccgttc 8580
cgaccgacca cggggcgcac ctctctttac gcggactccc cgtctgtgcc ttctcatctg 8640
ccggaccgtg tgcacttcgc ttcacctctg cacgtcgcat ggagaccacc gtgaacgccc 8700
accaaatatt gcccaaggtc ttacataaga ggactcttgg actctcagca atgtcaacga 8760
ccgaccttga ggcatacttc aaagactgtt tgtttaaaga ctgggaggag ttgggggagg 8820
agattaggtt aaaggtcttt gtactaggag gctgtaggca taaattggtc tgcgcaccag 8880
caccatggcg caatcactag agcggggtac ctttaagacc aatgacttac aaggcagctg 8940
tagatcttag ccacttttta aaagaaaagg ggggactgga agggctaatt cactcccaac 9000
gaagacaaga tctgcttttt gcttgtactg ggtctctctg gttagaccag atctgagcct 9060
gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag 9120
tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac 9180
ccttttagtc agtgtggaaa atctctagca gtagtagttc atgtcatctt attattcagt 9240
atttataact tgcaaagaaa tgaatatcag agagtgagag gaacttgttt attgcagctt 9300
ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac 9360
tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggctctagc 9420
tatcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc 9480
gccccatggc tgactaattt tttttattta tgcagaggcc gaggccggat cccttgagtg 9540
gctttcatcc tggagcagac tttgcagtct gtggactgca acacaacatt gcctttatgt 9600
gtaactcttg gctgaagctc ttacaccaat gctgggggac atgtacctcc caggggccca 9660
ggaagactac gggaggctac accaacgtca atcagagggg cctgtgtagc taccgataag 9720
cggaccctca agagggcatt agcaatagtg tttataaggc ccccttgtta attcttgaag 9780
acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc 9840
ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt 9900
ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata 9960
atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt 10020
tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc 10080
tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat 10140
ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct 10200
atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca 10260
ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg 10320
catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa 10380
cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg 10440
ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga 10500
cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg 10560
cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt 10620
tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg 10680
agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc 10740
ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca 10800
gatcgctgag ataggtgcct cactgattaa gcattgg 10837
<210> 6
<211> 9121
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 6
gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 60
gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 120
ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 180
ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac 240
atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 300
cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 360
tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca 420
tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag 480
cgatgggggc gggggggggg ggggggcccc ccccaggcgg ggcggggcgg ggcgaggggc 540
ggggcggggc gaggcggaaa ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt 600
ttccttttat ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc gcgcggcggg 660
cgggagtcgt tgcgcgctgc cttccccccg tgccccgctc cgccgccgcc tcgcgccgcc 720
cgccccggct ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc 780
ctccgggctg taattagcgc ttggtttaat gacggcttgt ttcttttctg tggctgcgtg 840
aaagccttga ggggctccgg gagggccctt tgtgcggggg gagcggctcg gggggtgcgt 900
gcgtgtgtgt gtgcgtgggg agcgccgcgt gcggctccgc gctgcccggc ggctgtgagc 960
gctgcgggcg cggcgcgggg ctttgtgcgc tccgcagtgt gcgcgagggg agcgcggccg 1020
ggggcggtgc cccgcggtgc ggggggggct gcgaggggaa caaaggctgc gtgcggggtg 1080
tgtgcgtggg ggggtgagca gggggtgtgg gcgcgtcggt cgggctgcaa ccccccctgc 1140
acccccctcc ccgagttgct gagcacggcc cggcttcggg tgcggggctc cgtacggggc 1200
gtggcgcggg gctcgccgtg ccgggcgggg ggtggcggca ggtgggggtg ccgggcgggg 1260
cggggccgcc tcgggccggg gagggctcgg gggaaggggc gcggcggccc ccggagcgcc 1320
ggcggctgtc gaggcgcggc gagccgcagc cattgccttt tatggtaatc gtgcgagagg 1380
gcgcagggac ttcctttgtc ccaaatctgt gcggagccga aatctgggag gcgccgccgc 1440
accccctcta gcgggcgcgg ggcgaagcgg tgcggcgccg gcaggaagga aatgggcggg 1500
gagggccttc gtgcgtcgcc gcgccgccgt ccccttctcc ctctccagcc tcggggctgt 1560
ccgcgggggg acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt 1620
gtgaccggcg gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag 1680
ctcctgggca acgtgctggt tgttgtgctg tctcatcatt ttggcaaagc caccatgtac 1740
ccatacgatg ttccagatta cgctgaattc gagctcatcg atggtacccg ggcatgcctc 1800
gagatgggtg cgagagcgtc agtattaagc gggggagaat tagatcgatg ggaaaaaatt 1860
cggttaaggc cagggggaaa gaaaaaatat aaattaaaac atatagtatg ggcaagcagg 1920
gagctagaac gattcgcagt taatcctggc ctgttagaaa catcagaagg ctgtagacaa 1980
atactgggac agctacaacc atcccttcag acaggatcag aagaacttag atcattatat 2040
aatacagtag caaccctcta ttgtgtgcat caaaggatag agataaaaga caccaaggaa 2100
gctttagaca agatagagga agagcaaaac aaaagtaaga aaaaagcaca gcaagcagca 2160
gctgacacag gacacagcaa tcaggtcagc caaaattacc ctatagtgca gaacatccag 2220
gggcaaatgg tacatcaggc catatcacct agaactttaa atgcatgggt aaaagtagta 2280
gaagagaagg ctttcagccc agaagtgata cccatgtttt cagcattatc agaaggagcc 2340
accccacaag atttaaacac catgctaaac acagtggggg gacatcaagc agccatgcaa 2400
atgttaaaag agaccatcaa tgaggaagct gcagaatggg atagagtgca tccagtgcat 2460
gcagggccta ttgcaccagg ccagatgaga gaaccaaggg gaagtgacat agcaggaact 2520
actagtacta gtacccttca ggaacaaata ggatggatga cacataatcc acctatccca 2580
gtaggagaaa tctataaaag atggataatc ctgggattaa ataaaatagt aagaatgtat 2640
agccctacca gcattctgga cataagacaa ggaccaaagg aaccctttag agactatgta 2700
gaccgattct ataaaactct aagagccgag caagcttcac aagaggtaaa aaattggatg 2760
acagaaacct tgttggtcca aaatgcgaac ccagattgta agactatttt aaaagcattg 2820
ggaccaggag cgacactaga agaaatgatg acagcatgtc agggagtggg gggacccggc 2880
cataaagcaa gagttttggc tgaagcaatg agccaagtaa caaatccagc taccataatg 2940
atacagaaag gcaattttag gaaccaaaga aagactgtta agtgtttcaa ttgtggcaaa 3000
gaagggcaca tagccaaaaa ttgcagggcc cctaggaaaa agggctgttg gaaatgtgga 3060
aaggaaggac accaaatgaa agattgtact gagagacagg ctaatttttt agggaagatc 3120
tggccttccc acaagggaag gccagggaat tttcttcaga gcagaccaga gccaacagcc 3180
ccaccagaag agagcttcag gtttggggaa gagacaacaa ctccctctca gaagcaggag 3240
ccgatagaca aggaactgta tcctttagct tccctcagat cactctttgg cagcgacccc 3300
tcgtcacaat aaagataggg gggcaattaa aggaagctct attagataca ggagcagatg 3360
atacagtatt agaagaaatg aatttgccag gaagatggaa accaaaaatg atagggggaa 3420
ttggaggttt tatcaaagta ggacagtatg atcagatact catagaaatc tgcggacata 3480
aagctatagg tacagtatta gtaggaccta cacctgtcaa cataattgga agaaatctgt 3540
tgactcagat tggctgcact ttaaattttc ccattagtcc tattgagact gtaccagtaa 3600
aattaaagcc aggaatggat ggcccaaaag ttaaacaatg gccattgaca gaagaaaaaa 3660
taaaagcatt agtagaaatt tgtacagaaa tggaaaagga aggaaaaatt tcaaaaattg 3720
ggcctgaaaa tccatacaat actccagtat ttgccataaa gaaaaaagac agtactaaat 3780
ggagaaaatt agtagatttc agagaactta ataagagaac tcaagatttc tgggaagttc 3840
aattaggaat accacatcct gcagggttaa aacagaaaaa atcagtaaca gtactggatg 3900
tgggcgatgc atatttttca gttcccttag ataaagactt caggaagtat actgcattta 3960
ccatacctag tataaacaat gagacaccag ggattagata tcagtacaat gtgcttccac 4020
agggatggaa aggatcacca gcaatattcc agtgtagcat gacaaaaatc ttagagcctt 4080
ttagaaaaca aaatccagac atagtcatct atcaatacat ggatgatttg tatgtaggat 4140
ctgacttaga aatagggcag catagaacaa aaatagagga actgagacaa catctgttga 4200
ggtggggatt taccacacca gacaaaaaac atcagaaaga acctccattc ctttggatgg 4260
gttatgaact ccatcctgat aaatggacag tacagcctat agtgctgcca gaaaaggaca 4320
gctggactgt caatgacata cagaaattag tgggaaaatt gaattgggca agtcagattt 4380
atgcagggat taaagtaagg caattatgta aacttcttag gggaaccaaa gcactaacag 4440
aagtagtacc actaacagaa gaagcagagc tagaactggc agaaaacagg gagattctaa 4500
aagaaccggt acatggagtg tattatgacc catcaaaaga cttaatagca gaaatacaga 4560
agcaggggca aggccaatgg acatatcaaa tttatcaaga gccatttaaa aatctgaaaa 4620
caggaaaata tgcaagaatg aagggtgccc acactaatga tgtgaaacaa ttaacagagg 4680
cagtacaaaa aatagccaca gaaagcatag taatatgggg aaagactcct aaatttaaat 4740
tacccataca aaaggaaaca tgggaagcat ggtggacaga gtattggcaa gccacctgga 4800
ttcctgagtg ggagtttgtc aatacccctc ccttagtgaa gttatggtac cagttagaga 4860
aagaacccat aataggagca gaaactttct atgtagatgg ggcagccaat agggaaacta 4920
aattaggaaa agcaggatat gtaactgaca gaggaagaca aaaagttgtc cccctaacgg 4980
acacaacaaa tcagaagact gagttacaag caattcatct agctttgcag gattcgggat 5040
tagaagtaaa catagtgaca gactcacaat atgcattggg aatcattcaa gcacaaccag 5100
ataagagtga atcagagtta gtcagtcaaa taatagagca gttaataaaa aaggaaaaag 5160
tctacctggc atgggtacca gcacacaaag gaattggagg aaatgaacaa gtagatgggt 5220
tggtcagtgc tggaatcagg aaagtactat ttttagatgg aatagataag gcccaagaag 5280
aacatgagaa atatcacagt aattggagag caatggctag tgattttaac ctaccacctg 5340
tagtagcaaa agaaatagta gccagctgtg ataaatgtca gctaaaaggg gaagccatgc 5400
atggacaagt agactgtagc ccaggaatat ggcagctaga ttgtacacat ttagaaggaa 5460
aagttatctt ggtagcagtt catgtagcca gtggatatat agaagcagaa gtaattccag 5520
cagagacagg gcaagaaaca gcatacttcc tcttaaaatt agcaggaaga tggccagtaa 5580
aaacagtaca tacagacaat ggcagcaatt tcaccagtac tacagttaag gccgcctgtt 5640
ggtgggcggg gatcaagcag gaatttggca ttccctacaa tccccaaagt caaggagtaa 5700
tagaatctat gaataaagaa ttaaagaaaa ttataggaca ggtaagagat caggctgaac 5760
atcttaagac agcagtacaa atggcagtat tcatccacaa ttttaaaaga aaagggggga 5820
ttggggggta cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta 5880
aagaattaca aaaacaaatt acaaaaattc aaaattttcg ggtttattac agggacagca 5940
gagatccagt ttggaaagga ccagcaaagc tcctctggaa aggtgaaggg gcagtagtaa 6000
tacaagataa tagtgacata aaagtagtgc caagaagaaa agcaaagatc atcagggatt 6060
atggaaaaca gatggcaggt gatgattgtg tggcaagtag acaggatgag gattaagcta 6120
gcagatctta attaattaag gatctttttc cctctgccaa aaattatggg gacatcatga 6180
agccccttga gcatctgact tctggctaat aaaggaaatt tattttcatt gcaatagtgt 6240
gttggaattt tttgtgtctc tcactcggaa ggacatatgg gagggcaaat catttaaaac 6300
atcagaatga gtatttggtt tagagtttgg caacatatgc ccatatgctg gctgccatga 6360
acaaaggttg gctataaaga ggtcatcagt atatgaaaca gccccctgct gtccattcct 6420
tattccatag aaaagccttg acttgaggtt agattttttt tatattttgt tttgtgttat 6480
ttttttcttt aacatcccta aaattttcct tacatgtttt actagccaga tttttcctcc 6540
tctcctgact actcccagtc atagctgtcc ctcttctctt atggagatcc ctcgacctgc 6600
agcccaagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc 6660
acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga 6720
gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg 6780
tcgtgccagc ggatccgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc 6840
ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt 6900
tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag 6960
gaggcttttt tggaggccta ggcttttgca aaaagctaac ttgtttattg cagcttataa 7020
tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca 7080
ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgga tccgctgcat 7140
taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc 7200
tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca 7260
aaggcggtaa tacggttatc cacagaatca gggataacgc aggaaagaac atgtgagcaa 7320
aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc 7380
tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga 7440
caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc 7500
cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 7560
ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct 7620
gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg 7680
agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta 7740
gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct 7800
acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa 7860
gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt 7920
gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta 7980
cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat 8040
caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa 8100
gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct 8160
cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta 8220
cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct 8280
caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg 8340
gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa 8400
gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt 8460
cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta 8520
catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca 8580
gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta 8640
ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct 8700
gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg 8760
cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac 8820
tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact 8880
gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa 8940
atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt 9000
ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat 9060
gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg 9120
g 9121
<210> 7
<211> 5159
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 7
gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 60
gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 120
ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 180
ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac 240
atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 300
cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 360
tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca 420
tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag 480
cgatgggggc gggggggggg ggggggcccc ccccaggcgg ggcggggcgg ggcgaggggc 540
ggggcggggc gaggcggaaa ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt 600
ttccttttat ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc gcgcggcggg 660
cgggagtcgt tgcgcgctgc cttccccccg tgccccgctc cgccgccgcc tcgcgccgcc 720
cgccccggct ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc 780
ctccgggctg taattagcgc ttggtttaat gacggcttgt ttcttttctg tggctgcgtg 840
aaagccttga ggggctccgg gagggccctt tgtgcggggg gagcggctcg gggggtgcgt 900
gcgtgtgtgt gtgcgtgggg agcgccgcgt gcggctccgc gctgcccggc ggctgtgagc 960
gctgcgggcg cggcgcgggg ctttgtgcgc tccgcagtgt gcgcgagggg agcgcggccg 1020
ggggcggtgc cccgcggtgc ggggggggct gcgaggggaa caaaggctgc gtgcggggtg 1080
tgtgcgtggg ggggtgagca gggggtgtgg gcgcgtcggt cgggctgcaa ccccccctgc 1140
acccccctcc ccgagttgct gagcacggcc cggcttcggg tgcggggctc cgtacggggc 1200
gtggcgcggg gctcgccgtg ccgggcgggg ggtggcggca ggtgggggtg ccgggcgggg 1260
cggggccgcc tcgggccggg gagggctcgg gggaaggggc gcggcggccc ccggagcgcc 1320
ggcggctgtc gaggcgcggc gagccgcagc cattgccttt tatggtaatc gtgcgagagg 1380
gcgcagggac ttcctttgtc ccaaatctgt gcggagccga aatctgggag gcgccgccgc 1440
accccctcta gcgggcgcgg ggcgaagcgg tgcggcgccg gcaggaagga aatgggcggg 1500
gagggccttc gtgcgtcgcc gcgccgccgt ccccttctcc ctctccagcc tcggggctgt 1560
ccgcgggggg acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt 1620
gtgaccggcg gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag 1680
ctcctgggca acgtgctggt tgttgtgctg tctcatcatt ttggcaaagc caccatgtac 1740
ccatacgatg ttccagatta cgctgaattc gagctcatcg atggtacccg ggcatgcctc 1800
gagatggcag gaagaagcgg agacagcgac gaagacctcc tcaaggcagt cagactcatc 1860
aagtttctct atcaaagcaa cccacctccc aatcccgagg ggacccgaca ggcccgaagg 1920
aatagaagaa gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc 1980
cttagcactt atctgggacg atctgcggag cctgtgcctc ttcagctacc accgcttgag 2040
agacttactc ttgattgtaa cgaggattgt ggaacttctg ggacgcaggg ggtgggaagc 2100
cctcaaatat tggtggaatc tcctacaata ttggagtcag gagctaaaga ataggctagc 2160
agatcttaat taattaagga tctttttccc tctgccaaaa attatgggga catcatgaag 2220
ccccttgagc atctgacttc tggctaataa aggaaattta ttttcattgc aatagtgtgt 2280
tggaattttt tgtgtctctc actcggaagg acatatggga gggcaaatca tttaaaacat 2340
cagaatgagt atttggttta gagtttggca acatatgccc atatgctggc tgccatgaac 2400
aaaggttggc tataaagagg tcatcagtat atgaaacagc cccctgctgt ccattcctta 2460
ttccatagaa aagccttgac ttgaggttag atttttttta tattttgttt tgtgttattt 2520
ttttctttaa catccctaaa attttcctta catgttttac tagccagatt tttcctcctc 2580
tcctgactac tcccagtcat agctgtccct cttctcttat ggagatccct cgacctgcag 2640
cccaagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac 2700
aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt 2760
gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc 2820
gtgccagcgg atccgcatct caattagtca gcaaccatag tcccgcccct aactccgccc 2880
atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt 2940
tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga 3000
ggcttttttg gaggcctagg cttttgcaaa aagctaactt gtttattgca gcttataatg 3060
gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt 3120
ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatc cgctgcatta 3180
atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc 3240
gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa 3300
ggcggtaata cggttatcca cagaatcagg gataacgcag gaaagaacat gtgagcaaaa 3360
ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 3420
cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 3480
ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 3540
accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 3600
catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 3660
gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 3720
tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 3780
agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 3840
actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 3900
gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc 3960
aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg 4020
gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca 4080
aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt 4140
atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca 4200
gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg 4260
atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca 4320
ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt 4380
cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt 4440
agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca 4500
cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca 4560
tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga 4620
agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact 4680
gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga 4740
gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg 4800
ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc 4860
tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga 4920
tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat 4980
gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt 5040
caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt 5100
atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgg 5159
<210> 8
<211> 3822
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 8
atgttcgtgt ttctggtgct gctgcctctg gtgagctccc agtgcgtgaa cctgaccaca 60
cggacacagc tgccccctgc ctacaccaac agcttcacaa ggggcgtgta ctaccccgac 120
aaggtgttta gatctagcgt gctgcactcc acacaggatc tgtttctgcc tttcttttct 180
aacgtgacct ggttccacgc tatccacgtg tccggcacca acggaacaaa gaggttcgac 240
aacccagtgc tgccctttaa cgatggcgtg tacttcgcct ccaccgagaa gtctaacatc 300
atcagaggct ggatctttgg aaccacactg gacagcaaga cacagtccct gctgatcgtg 360
aacaacgcca ccaacgtggt catcaaggtg tgcgagttcc agttttgtaa cgatccattc 420
ctgggcgtgt actaccacaa gaacaacaag tcttggatgg agagcgagtt tcgcgtgtac 480
tcctctgcca acaactgtac atttgagtac gtgtcccagc ccttcctgat ggacctggag 540
ggcaagcagg gaaacttcaa gaacctgcgg gagttcgtgt ttaagaacat cgatggctac 600
tttaagatct actccaagca caccccaatc aacctggtgc gcgacctgcc acagggcttc 660
tctgccctgg agccactggt ggatctgccc atcggaatca acatcaccag gtttcagaca 720
ctgctggccc tgcacagaag ctacctgaca ccaggcgaca gctcctctgg atggaccgct 780
ggagctgctg cctactacgt gggctacctg cagccccgga ccttcctgct gaagtacaac 840
gagaacggaa ccatcacaga cgctgtggat tgcgccctgg accccctgtc tgagaccaag 900
tgtacactga agagctttac cgtggagaag ggcatctacc agacaagcaa cttccgggtg 960
cagcctaccg agtccatcgt gcgctttccc aacatcacaa acctgtgccc ttttggagag 1020
gtgttcaacg ctacccgctt cgcctccgtg tacgcttgga accggaagcg catctccaac 1080
tgcgtggccg actactctgt gctgtacaac agcgccagct tcagcacctt caagtgctac 1140
ggcgtgagcc caacaaagct gaacgacctg tgctttacca acgtgtacgc tgattccttc 1200
gtgatcaggg gagacgaggt gcgccagatc gctcccggcc agacaggaaa gatcgctgac 1260
tacaactaca agctgcctga cgatttcacc ggctgcgtga tcgcctggaa ctctaacaac 1320
ctggatagca aagtgggcgg aaactacaac tacctgtaca ggctgtttag aaagtctaac 1380
ctgaagccat tcgagcggga catctccaca gagatctacc aggctggctc taccccatgc 1440
aacggagtgg agggcttcaa ctgttacttc cctctgcaga gctacggatt ccagccaaca 1500
aacggcgtgg gataccagcc ctaccgcgtg gtggtgctgt cttttgagct gctgcacgct 1560
cctgctacag tgtgcggacc aaagaagagc accaacctgg tgaagaacaa gtgcgtgaac 1620
ttcaacttta acggactgac cggcacagga gtgctgaccg agtctaacaa gaagttcctg 1680
ccttttcagc agttcggccg ggacatcgcc gataccacag acgctgtgcg cgaccctcag 1740
accctggaga tcctggatat cacaccatgc tccttcggcg gagtgtctgt gatcacacca 1800
ggaaccaaca caagcaacca ggtggccgtg ctgtaccagg acgtgaactg taccgaggtg 1860
cccgtggcta tccacgccga tcagctgacc cctacatgga gggtgtactc taccggcagc 1920
aacgtgttcc agacaagagc cggctgtctg atcggagctg agcacgtgaa caacagctac 1980
gagtgcgaca tccctatcgg cgccggaatc tgtgcttcct accagaccca gacaaactcc 2040
ccaaggagag ccaggtctgt ggctagccag tccatcatcg cctacaccat gagcctgggc 2100
gccgagaact ccgtggctta ctccaacaac tctatcgcta tccctaccaa cttcacaatc 2160
tccgtgacca cagagatcct gccagtgagc atgaccaaga catccgtgga ctgcacaatg 2220
tacatctgtg gagattccac cgagtgctct aacctgctgc tgcagtacgg ctctttctgt 2280
acccagctga acagagccct gacaggaatc gctgtggagc aggacaagaa cacacaggag 2340
gtgttcgccc aggtgaagca gatctacaag accccaccca tcaaggactt tggcggattc 2400
aactttagcc agatcctgcc cgatcctagc aagccatcca agaggtcttt tatcgaggac 2460
ctgctgttca acaaggtgac cctggctgat gccggcttca tcaagcagta cggcgattgc 2520
ctgggagaca tcgctgccag agacctgatc tgtgcccaga agtttaacgg actgaccgtg 2580
ctgcctccac tgctgacaga tgagatgatc gctcagtaca catctgctct gctggccggc 2640
accatcacaa gcggatggac cttcggcgct ggagctgccc tgcagatccc ctttgccatg 2700
cagatggctt acagattcaa cggcatcgga gtgacccaga acgtgctgta cgagaaccag 2760
aagctgatcg ccaaccagtt taactccgct atcggcaaga tccaggactc tctgagctcc 2820
acagctagcg ccctgggaaa gctgcaggat gtggtgaacc agaacgctca ggccctgaac 2880
accctggtga agcagctgtc tagcaacttc ggcgccatct cctctgtgct gaacgatatc 2940
ctgagcaggc tggacaaggt ggaggctgag gtgcagatcg acaggctgat cacaggaaga 3000
ctgcagtccc tgcagaccta cgtgacacag cagctgatca gggctgctga gatcagggct 3060
tctgccaacc tggctgccac caagatgagc gagtgcgtgc tgggccagtc caagagagtg 3120
gacttttgtg gcaagggata ccacctgatg agcttcccac agtccgcccc tcacggagtg 3180
gtgtttctgc acgtgaccta cgtgccagct caggagaaga acttcaccac agctcccgcc 3240
atctgccacg atggcaaggc ccactttcct cgggagggcg tgttcgtgag caacggaacc 3300
cactggtttg tgacacagcg caacttctac gagccacaga tcatcaccac agacaacaca 3360
ttcgtgtccg gcaactgtga cgtggtcatc ggaatcgtga acaacaccgt gtacgatcct 3420
ctgcagccag agctggactc ttttaaggag gagctggata agtacttcaa gaaccacacc 3480
agccctgacg tggatctggg cgacatctct ggaatcaacg ccagcgtggt gaacatccag 3540
aaggagatcg accggctgaa cgaggtggct aagaacctga acgagtccct gatcgatctg 3600
caggagctgg gcaagtacga gcagtacatc aagtggccct ggtacatctg gctgggcttc 3660
atcgccggac tgatcgctat cgtgatggtg accatcatgc tgtgctgtat gacaagctgc 3720
tgttcctgcc tgaagggctg ctgttcttgt ggaagctgct gtaagtttga cgaggacgat 3780
agcgagcctg tgctgaaggg cgtgaagctg cactacacct aa 3822
<210> 9
<211> 3765
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 9
atgttcgtgt ttctggtgct gctgcctctg gtgagctccc agtgcgtgaa cctgaccaca 60
cggacacagc tgccccctgc ctacaccaac agcttcacaa ggggcgtgta ctaccccgac 120
aaggtgttta gatctagcgt gctgcactcc acacaggatc tgtttctgcc tttcttttct 180
aacgtgacct ggttccacgc tatccacgtg tccggcacca acggaacaaa gaggttcgac 240
aacccagtgc tgccctttaa cgatggcgtg tacttcgcct ccaccgagaa gtctaacatc 300
atcagaggct ggatctttgg aaccacactg gacagcaaga cacagtccct gctgatcgtg 360
aacaacgcca ccaacgtggt catcaaggtg tgcgagttcc agttttgtaa cgatccattc 420
ctgggcgtgt actaccacaa gaacaacaag tcttggatgg agagcgagtt tcgcgtgtac 480
tcctctgcca acaactgtac atttgagtac gtgtcccagc ccttcctgat ggacctggag 540
ggcaagcagg gaaacttcaa gaacctgcgg gagttcgtgt ttaagaacat cgatggctac 600
tttaagatct actccaagca caccccaatc aacctggtgc gcgacctgcc acagggcttc 660
tctgccctgg agccactggt ggatctgccc atcggaatca acatcaccag gtttcagaca 720
ctgctggccc tgcacagaag ctacctgaca ccaggcgaca gctcctctgg atggaccgct 780
ggagctgctg cctactacgt gggctacctg cagccccgga ccttcctgct gaagtacaac 840
gagaacggaa ccatcacaga cgctgtggat tgcgccctgg accccctgtc tgagaccaag 900
tgtacactga agagctttac cgtggagaag ggcatctacc agacaagcaa cttccgggtg 960
cagcctaccg agtccatcgt gcgctttccc aacatcacaa acctgtgccc ttttggagag 1020
gtgttcaacg ctacccgctt cgcctccgtg tacgcttgga accggaagcg catctccaac 1080
tgcgtggccg actactctgt gctgtacaac agcgccagct tcagcacctt caagtgctac 1140
ggcgtgagcc caacaaagct gaacgacctg tgctttacca acgtgtacgc tgattccttc 1200
gtgatcaggg gagacgaggt gcgccagatc gctcccggcc agacaggaaa gatcgctgac 1260
tacaactaca agctgcctga cgatttcacc ggctgcgtga tcgcctggaa ctctaacaac 1320
ctggatagca aagtgggcgg aaactacaac tacctgtaca ggctgtttag aaagtctaac 1380
ctgaagccat tcgagcggga catctccaca gagatctacc aggctggctc taccccatgc 1440
aacggagtgg agggcttcaa ctgttacttc cctctgcaga gctacggatt ccagccaaca 1500
aacggcgtgg gataccagcc ctaccgcgtg gtggtgctgt cttttgagct gctgcacgct 1560
cctgctacag tgtgcggacc aaagaagagc accaacctgg tgaagaacaa gtgcgtgaac 1620
ttcaacttta acggactgac cggcacagga gtgctgaccg agtctaacaa gaagttcctg 1680
ccttttcagc agttcggccg ggacatcgcc gataccacag acgctgtgcg cgaccctcag 1740
accctggaga tcctggatat cacaccatgc tccttcggcg gagtgtctgt gatcacacca 1800
ggaaccaaca caagcaacca ggtggccgtg ctgtaccagg acgtgaactg taccgaggtg 1860
cccgtggcta tccacgccga tcagctgacc cctacatgga gggtgtactc taccggcagc 1920
aacgtgttcc agacaagagc cggctgtctg atcggagctg agcacgtgaa caacagctac 1980
gagtgcgaca tccctatcgg cgccggaatc tgtgcttcct accagaccca gacaaactcc 2040
ccaaggagag ccaggtctgt ggctagccag tccatcatcg cctacaccat gagcctgggc 2100
gccgagaact ccgtggctta ctccaacaac tctatcgcta tccctaccaa cttcacaatc 2160
tccgtgacca cagagatcct gccagtgagc atgaccaaga catccgtgga ctgcacaatg 2220
tacatctgtg gagattccac cgagtgctct aacctgctgc tgcagtacgg ctctttctgt 2280
acccagctga acagagccct gacaggaatc gctgtggagc aggacaagaa cacacaggag 2340
gtgttcgccc aggtgaagca gatctacaag accccaccca tcaaggactt tggcggattc 2400
aactttagcc agatcctgcc cgatcctagc aagccatcca agaggtcttt tatcgaggac 2460
ctgctgttca acaaggtgac cctggctgat gccggcttca tcaagcagta cggcgattgc 2520
ctgggagaca tcgctgccag agacctgatc tgtgcccaga agtttaacgg actgaccgtg 2580
ctgcctccac tgctgacaga tgagatgatc gctcagtaca catctgctct gctggccggc 2640
accatcacaa gcggatggac cttcggcgct ggagctgccc tgcagatccc ctttgccatg 2700
cagatggctt acagattcaa cggcatcgga gtgacccaga acgtgctgta cgagaaccag 2760
aagctgatcg ccaaccagtt taactccgct atcggcaaga tccaggactc tctgagctcc 2820
acagctagcg ccctgggaaa gctgcaggat gtggtgaacc agaacgctca ggccctgaac 2880
accctggtga agcagctgtc tagcaacttc ggcgccatct cctctgtgct gaacgatatc 2940
ctgagcaggc tggacaaggt ggaggctgag gtgcagatcg acaggctgat cacaggaaga 3000
ctgcagtccc tgcagaccta cgtgacacag cagctgatca gggctgctga gatcagggct 3060
tctgccaacc tggctgccac caagatgagc gagtgcgtgc tgggccagtc caagagagtg 3120
gacttttgtg gcaagggata ccacctgatg agcttcccac agtccgcccc tcacggagtg 3180
gtgtttctgc acgtgaccta cgtgccagct caggagaaga acttcaccac agctcccgcc 3240
atctgccacg atggcaaggc ccactttcct cgggagggcg tgttcgtgag caacggaacc 3300
cactggtttg tgacacagcg caacttctac gagccacaga tcatcaccac agacaacaca 3360
ttcgtgtccg gcaactgtga cgtggtcatc ggaatcgtga acaacaccgt gtacgatcct 3420
ctgcagccag agctggactc ttttaaggag gagctggata agtacttcaa gaaccacacc 3480
agccctgacg tggatctggg cgacatctct ggaatcaacg ccagcgtggt gaacatccag 3540
aaggagatcg accggctgaa cgaggtggct aagaacctga acgagtccct gatcgatctg 3600
caggagctgg gcaagtacga gcagtacatc aagtggccct ggtacatctg gctgggcttc 3660
atcgccggac tgatcgctat cgtgatggtg accatcatgc tgtgctgtat gacaagctgc 3720
tgttcctgcc tgaagggctg ctgttcttgt ggaagctgct gttaa 3765
<210> 10
<211> 3738
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 10
atgttcgtgt ttctggtgct gctgcctctg gtgagctccc agtgcgtgaa cctgaccaca 60
cggacacagc tgccccctgc ctacaccaac agcttcacaa ggggcgtgta ctaccccgac 120
aaggtgttta gatctagcgt gctgcactcc acacaggatc tgtttctgcc tttcttttct 180
aacgtgacct ggttccacgc tatccacgtg tccggcacca acggaacaaa gaggttcgac 240
aacccagtgc tgccctttaa cgatggcgtg tacttcgcct ccaccgagaa gtctaacatc 300
atcagaggct ggatctttgg aaccacactg gacagcaaga cacagtccct gctgatcgtg 360
aacaacgcca ccaacgtggt catcaaggtg tgcgagttcc agttttgtaa cgatccattc 420
ctgggcgtgt actaccacaa gaacaacaag tcttggatgg agagcgagtt tcgcgtgtac 480
tcctctgcca acaactgtac atttgagtac gtgtcccagc ccttcctgat ggacctggag 540
ggcaagcagg gaaacttcaa gaacctgcgg gagttcgtgt ttaagaacat cgatggctac 600
tttaagatct actccaagca caccccaatc aacctggtgc gcgacctgcc acagggcttc 660
tctgccctgg agccactggt ggatctgccc atcggaatca acatcaccag gtttcagaca 720
ctgctggccc tgcacagaag ctacctgaca ccaggcgaca gctcctctgg atggaccgct 780
ggagctgctg cctactacgt gggctacctg cagccccgga ccttcctgct gaagtacaac 840
gagaacggaa ccatcacaga cgctgtggat tgcgccctgg accccctgtc tgagaccaag 900
tgtacactga agagctttac cgtggagaag ggcatctacc agacaagcaa cttccgggtg 960
cagcctaccg agtccatcgt gcgctttccc aacatcacaa acctgtgccc ttttggagag 1020
gtgttcaacg ctacccgctt cgcctccgtg tacgcttgga accggaagcg catctccaac 1080
tgcgtggccg actactctgt gctgtacaac agcgccagct tcagcacctt caagtgctac 1140
ggcgtgagcc caacaaagct gaacgacctg tgctttacca acgtgtacgc tgattccttc 1200
gtgatcaggg gagacgaggt gcgccagatc gctcccggcc agacaggaaa gatcgctgac 1260
tacaactaca agctgcctga cgatttcacc ggctgcgtga tcgcctggaa ctctaacaac 1320
ctggatagca aagtgggcgg aaactacaac tacctgtaca ggctgtttag aaagtctaac 1380
ctgaagccat tcgagcggga catctccaca gagatctacc aggctggctc taccccatgc 1440
aacggagtgg agggcttcaa ctgttacttc cctctgcaga gctacggatt ccagccaaca 1500
aacggcgtgg gataccagcc ctaccgcgtg gtggtgctgt cttttgagct gctgcacgct 1560
cctgctacag tgtgcggacc aaagaagagc accaacctgg tgaagaacaa gtgcgtgaac 1620
ttcaacttta acggactgac cggcacagga gtgctgaccg agtctaacaa gaagttcctg 1680
ccttttcagc agttcggccg ggacatcgcc gataccacag acgctgtgcg cgaccctcag 1740
accctggaga tcctggatat cacaccatgc tccttcggcg gagtgtctgt gatcacacca 1800
ggaaccaaca caagcaacca ggtggccgtg ctgtaccagg acgtgaactg taccgaggtg 1860
cccgtggcta tccacgccga tcagctgacc cctacatgga gggtgtactc taccggcagc 1920
aacgtgttcc agacaagagc cggctgtctg atcggagctg agcacgtgaa caacagctac 1980
gagtgcgaca tccctatcgg cgccggaatc tgtgcttcct accagaccca gacaaactcc 2040
ccaaggagag ccaggtctgt ggctagccag tccatcatcg cctacaccat gagcctgggc 2100
gccgagaact ccgtggctta ctccaacaac tctatcgcta tccctaccaa cttcacaatc 2160
tccgtgacca cagagatcct gccagtgagc atgaccaaga catccgtgga ctgcacaatg 2220
tacatctgtg gagattccac cgagtgctct aacctgctgc tgcagtacgg ctctttctgt 2280
acccagctga acagagccct gacaggaatc gctgtggagc aggacaagaa cacacaggag 2340
gtgttcgccc aggtgaagca gatctacaag accccaccca tcaaggactt tggcggattc 2400
aactttagcc agatcctgcc cgatcctagc aagccatcca agaggtcttt tatcgaggac 2460
ctgctgttca acaaggtgac cctggctgat gccggcttca tcaagcagta cggcgattgc 2520
ctgggagaca tcgctgccag agacctgatc tgtgcccaga agtttaacgg actgaccgtg 2580
ctgcctccac tgctgacaga tgagatgatc gctcagtaca catctgctct gctggccggc 2640
accatcacaa gcggatggac cttcggcgct ggagctgccc tgcagatccc ctttgccatg 2700
cagatggctt acagattcaa cggcatcgga gtgacccaga acgtgctgta cgagaaccag 2760
aagctgatcg ccaaccagtt taactccgct atcggcaaga tccaggactc tctgagctcc 2820
acagctagcg ccctgggaaa gctgcaggat gtggtgaacc agaacgctca ggccctgaac 2880
accctggtga agcagctgtc tagcaacttc ggcgccatct cctctgtgct gaacgatatc 2940
ctgagcaggc tggacaaggt ggaggctgag gtgcagatcg acaggctgat cacaggaaga 3000
ctgcagtccc tgcagaccta cgtgacacag cagctgatca gggctgctga gatcagggct 3060
tctgccaacc tggctgccac caagatgagc gagtgcgtgc tgggccagtc caagagagtg 3120
gacttttgtg gcaagggata ccacctgatg agcttcccac agtccgcccc tcacggagtg 3180
gtgtttctgc acgtgaccta cgtgccagct caggagaaga acttcaccac agctcccgcc 3240
atctgccacg atggcaaggc ccactttcct cgggagggcg tgttcgtgag caacggaacc 3300
cactggtttg tgacacagcg caacttctac gagccacaga tcatcaccac agacaacaca 3360
ttcgtgtccg gcaactgtga cgtggtcatc ggaatcgtga acaacaccgt gtacgatcct 3420
ctgcagccag agctggactc ttttaaggag gagctggata agtacttcaa gaaccacacc 3480
agccctgacg tggatctggg cgacatctct ggaatcaacg ccagcgtggt gaacatccag 3540
aaggagatcg accggctgaa cgaggtggct aagaacctga acgagtccct gatcgatctg 3600
caggagctgg gcaagtacga gcagtacatc aagtggccct ggtacatctg gctgggcttc 3660
atcgccggac tgatcgctat cgtgatggtg accatcatgc tgtgctgtat gacaagctgc 3720
tgttcctgcc tgaagtaa 3738
<210> 11
<211> 3663
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 11
atgttcgtgt ttctggtgct gctgcctctg gtgagctccc agtgcgtgaa cctgaccaca 60
cggacacagc tgccccctgc ctacaccaac agcttcacaa ggggcgtgta ctaccccgac 120
aaggtgttta gatctagcgt gctgcactcc acacaggatc tgtttctgcc tttcttttct 180
aacgtgacct ggttccacgc tatccacgtg tccggcacca acggaacaaa gaggttcgac 240
aacccagtgc tgccctttaa cgatggcgtg tacttcgcct ccaccgagaa gtctaacatc 300
atcagaggct ggatctttgg aaccacactg gacagcaaga cacagtccct gctgatcgtg 360
aacaacgcca ccaacgtggt catcaaggtg tgcgagttcc agttttgtaa cgatccattc 420
ctgggcgtgt actaccacaa gaacaacaag tcttggatgg agagcgagtt tcgcgtgtac 480
tcctctgcca acaactgtac atttgagtac gtgtcccagc ccttcctgat ggacctggag 540
ggcaagcagg gaaacttcaa gaacctgcgg gagttcgtgt ttaagaacat cgatggctac 600
tttaagatct actccaagca caccccaatc aacctggtgc gcgacctgcc acagggcttc 660
tctgccctgg agccactggt ggatctgccc atcggaatca acatcaccag gtttcagaca 720
ctgctggccc tgcacagaag ctacctgaca ccaggcgaca gctcctctgg atggaccgct 780
ggagctgctg cctactacgt gggctacctg cagccccgga ccttcctgct gaagtacaac 840
gagaacggaa ccatcacaga cgctgtggat tgcgccctgg accccctgtc tgagaccaag 900
tgtacactga agagctttac cgtggagaag ggcatctacc agacaagcaa cttccgggtg 960
cagcctaccg agtccatcgt gcgctttccc aacatcacaa acctgtgccc ttttggagag 1020
gtgttcaacg ctacccgctt cgcctccgtg tacgcttgga accggaagcg catctccaac 1080
tgcgtggccg actactctgt gctgtacaac agcgccagct tcagcacctt caagtgctac 1140
ggcgtgagcc caacaaagct gaacgacctg tgctttacca acgtgtacgc tgattccttc 1200
gtgatcaggg gagacgaggt gcgccagatc gctcccggcc agacaggaaa gatcgctgac 1260
tacaactaca agctgcctga cgatttcacc ggctgcgtga tcgcctggaa ctctaacaac 1320
ctggatagca aagtgggcgg aaactacaac tacctgtaca ggctgtttag aaagtctaac 1380
ctgaagccat tcgagcggga catctccaca gagatctacc aggctggctc taccccatgc 1440
aacggagtgg agggcttcaa ctgttacttc cctctgcaga gctacggatt ccagccaaca 1500
aacggcgtgg gataccagcc ctaccgcgtg gtggtgctgt cttttgagct gctgcacgct 1560
cctgctacag tgtgcggacc aaagaagagc accaacctgg tgaagaacaa gtgcgtgaac 1620
ttcaacttta acggactgac cggcacagga gtgctgaccg agtctaacaa gaagttcctg 1680
ccttttcagc agttcggccg ggacatcgcc gataccacag acgctgtgcg cgaccctcag 1740
accctggaga tcctggatat cacaccatgc tccttcggcg gagtgtctgt gatcacacca 1800
ggaaccaaca caagcaacca ggtggccgtg ctgtaccagg acgtgaactg taccgaggtg 1860
cccgtggcta tccacgccga tcagctgacc cctacatgga gggtgtactc taccggcagc 1920
aacgtgttcc agacaagagc cggctgtctg atcggagctg agcacgtgaa caacagctac 1980
gagtgcgaca tccctatcgg cgccggaatc tgtgcttcct accagaccca gacaaactcc 2040
ccaaggagag ccaggtctgt ggctagccag tccatcatcg cctacaccat gagcctgggc 2100
gccgagaact ccgtggctta ctccaacaac tctatcgcta tccctaccaa cttcacaatc 2160
tccgtgacca cagagatcct gccagtgagc atgaccaaga catccgtgga ctgcacaatg 2220
tacatctgtg gagattccac cgagtgctct aacctgctgc tgcagtacgg ctctttctgt 2280
acccagctga acagagccct gacaggaatc gctgtggagc aggacaagaa cacacaggag 2340
gtgttcgccc aggtgaagca gatctacaag accccaccca tcaaggactt tggcggattc 2400
aactttagcc agatcctgcc cgatcctagc aagccatcca agaggtcttt tatcgaggac 2460
ctgctgttca acaaggtgac cctggctgat gccggcttca tcaagcagta cggcgattgc 2520
ctgggagaca tcgctgccag agacctgatc tgtgcccaga agtttaacgg actgaccgtg 2580
ctgcctccac tgctgacaga tgagatgatc gctcagtaca catctgctct gctggccggc 2640
accatcacaa gcggatggac cttcggcgct ggagctgccc tgcagatccc ctttgccatg 2700
cagatggctt acagattcaa cggcatcgga gtgacccaga acgtgctgta cgagaaccag 2760
aagctgatcg ccaaccagtt taactccgct atcggcaaga tccaggactc tctgagctcc 2820
acagctagcg ccctgggaaa gctgcaggat gtggtgaacc agaacgctca ggccctgaac 2880
accctggtga agcagctgtc tagcaacttc ggcgccatct cctctgtgct gaacgatatc 2940
ctgagcaggc tggacaaggt ggaggctgag gtgcagatcg acaggctgat cacaggaaga 3000
ctgcagtccc tgcagaccta cgtgacacag cagctgatca gggctgctga gatcagggct 3060
tctgccaacc tggctgccac caagatgagc gagtgcgtgc tgggccagtc caagagagtg 3120
gacttttgtg gcaagggata ccacctgatg agcttcccac agtccgcccc tcacggagtg 3180
gtgtttctgc acgtgaccta cgtgccagct caggagaaga acttcaccac agctcccgcc 3240
atctgccacg atggcaaggc ccactttcct cgggagggcg tgttcgtgag caacggaacc 3300
cactggtttg tgacacagcg caacttctac gagccacaga tcatcaccac agacaacaca 3360
ttcgtgtccg gcaactgtga cgtggtcatc ggaatcgtga acaacaccgt gtacgatcct 3420
ctgcagccag agctggactc ttttaaggag gagctggata agtacttcaa gaaccacacc 3480
agccctgacg tggatctggg cgacatctct ggaatcaacg ccagcgtggt gaacatccag 3540
aaggagatcg accggctgaa cgaggtggct aagaacctga acgagtccct gatcgatctg 3600
caggagctgg gcaagtacga gcagtacatc aagtggccct ggtacatctg gctgggcttc 3660
taa 3663
<210> 12
<211> 27
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 12
ccgctcgaga tgttcgtgtt tctggtg 27
<210> 13
<211> 28
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 13
ctagctagct taggtgtagt gcatcttc 28
<210> 14
<211> 27
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 14
ccgctcgaga tgttcgtgtt tctggtg 27
<210> 15
<211> 30
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 15
ctagctagct taacagcagc ttccacaaga 30
<210> 16
<211> 27
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 16
ccgctcgaga tgttcgtgtt tctggtg 27
<210> 17
<211> 31
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 17
ctagctagct tacttcaggc aggaacagca g 31
<210> 18
<211> 27
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 18
ccgctcgaga tgttcgtgtt tctggtg 27
<210> 19
<211> 32
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 19
ctagctagct tagaagccca gccagatgta cc 32
Claims (13)
1.双报告基因骨架载体，其特征在于：包括选自慢病毒遗传物质的双报告基因骨架载体，所述双报告基因骨架载体的双报告基因选自荧光素酶和荧光蛋白，荧光素酶报告基因的上游是调节转录的启动子,其中所述启动子优选自hEF1a启动子；所述荧光蛋白报告基因的上游是增强型转录启动子E-IRES；所述双报告基因骨架载体命名为pDLV19-mGFP-Fluc。
2.如权利要求1所述双报告基因骨架载体，其特征在于：所述双报告基因包括荧光蛋白报告基因和荧光素酶报告基因，所述的荧光蛋白报告基因选自绿色荧光蛋白或红色荧光蛋白；所述荧光素酶报告基因选自萤火虫荧光素酶或海肾荧光素酶。
3.如权利要求2所述双报告基因骨架载体，其特征在于：所述荧光蛋白报告基因选择修饰的绿色荧光蛋白mGFP基因，所述绿色荧光蛋白mGFP基因的序列为SEQ ID NO：1；所述荧光素酶报告基因选自密码子优化的萤火虫荧光素酶Fluc基因，所述萤火虫荧光素酶Fluc基因的序列为SEQ ID NO：2。
4.如权利要求3所述双报告基因骨架载体，其特征在于：所述萤火虫荧光素酶Fluc基因的上游是hEF1a启动子，所述hEF1a启动子基因序列为SEQ ID NO：3，所述绿色荧光蛋白mGFP基因的上游是ECMV-IRES增强启动子，所述ECMV-IRES增强启动子的基因序列为SEQ ID NO：4。
5.如权利要求3或4任一所述双报告基因骨架载体，其特征在于：所述绿色荧光蛋白mGFP基因位于荧光素酶Fluc基因的下游，所述绿色荧光蛋白mGFP基因起始密码子序列之前添加了Kozak序列。
6.如权利要求1所述双报告基因骨架载体，其特征在于：所述pDLV19-mGFP-Fluc双报告基因骨架载体具有SEQ ID NO：5的序列。
7.一种四质粒假病毒包装系统，其特征在于：由权利要求1～6中任一双报告基因骨架载体pDLV19-mGFP-Fluc、3种真核表达包装质粒组成；其中所述3种真核表达包装质粒分别整合慢病毒gag-pol基因、慢病毒rev基因以及含有目的病毒的包膜蛋白基因。
8.如权利要求7所述四质粒假病毒包装系统，其特征在于：所述3种真核表达包装质粒选自pCAGGS，质粒pCAGGS中CMV启动子的序列优选替换为：
(1)EF1a启动子；
(2)包含有人巨细胞病毒主要即刻早期蛋白基因增强型启动子。
9.如权利要求8所述四质粒假病毒包装系统，其特征在于：所述含有慢病毒gag-pol的真核表达包装质粒具有SEQ ID NO：6序列，所述包装质粒命名为psl4-gag-pol；所述含有慢病毒rev基因的真核表达包装质粒具有SEQ ID NO：7序列，所述包装质粒命名为psl5-rev。
10.如权利要求8所述四质粒假病毒包装系统，其特征在于：所述pCAGGS真核表达包装质粒在宿主细胞水平表达目的病毒的包膜蛋白，所述pCAGGS真核表达包装质粒细胞水平表达的包膜蛋白选自冠状病毒、疱疹病毒、痘病毒、嗜肝病毒、丝状病毒、棒状病毒、流感病毒、副粘病毒、黄病毒、弹状病毒、副粘病毒、黄病毒、包膜病毒、布尼亚病毒或逆转录病毒的一种病毒或多种病毒。
11.权利要求10所述四质粒假病毒包装系统，其特征在于：所述冠状病毒优选为SARS-CoV-2或SARS；所述嗜肝病毒优选为乙肝病毒或丙肝病毒；所述丝状病毒优选为Ebola病毒；所述弹状病毒优选为狂犬病毒；所述副粘病毒优选为麻疹病毒或呼吸道合胞病毒；所述黄病毒优选为寨卡病毒或登革热病毒。
12.权利要求10所述四质粒假病毒包装系统，其特征在于：所述真核表达包装质粒表达的包膜蛋白选自冠状病毒SARS-CoV-2，所述双报告基因四质粒假病毒包装系统用于制备新冠假病毒时，所述冠状病毒包膜蛋白选自：(1)刺突蛋白S全长氨基酸，即SEQ ID NO：8序列、(2)刺突蛋白S的3’端缺失19个氨基酸，即SEQ ID NO：9序列、(3)刺突蛋白S的3’端缺失28个氨基酸，即SEQ ID NO：10序列或(4)刺突蛋白S的3’端缺失53个氨基酸且同时融合VSV包膜蛋白的胞内段，即SEQ ID NO：11序列；对应的所述pCAGGS真核表达包装质粒在宿主细胞水平表达的囊膜蛋白的质粒命名分别为psl3-S、S19、S28、S53C。
13.一种包装新冠肺炎假病毒，其特征在于：包括权利要求7～12任一所述双报告基因四质粒假病毒包装系统、宿主细胞，所述宿主细胞选自293、293T、293sus、HEK293、HEK293T或HEK293FT细胞。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010191574.7A CN111593073B (zh) | 2020-03-18 | 2020-03-18 | 双报告基因骨架载体、四质粒假病毒包装系统、包装新冠肺炎假病毒 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010191574.7A CN111593073B (zh) | 2020-03-18 | 2020-03-18 | 双报告基因骨架载体、四质粒假病毒包装系统、包装新冠肺炎假病毒 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111593073A true CN111593073A (zh) | 2020-08-28 |
| CN111593073B CN111593073B (zh) | 2022-03-08 |
Family
ID=72187255
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010191574.7A Active CN111593073B (zh) | 2020-03-18 | 2020-03-18 | 双报告基因骨架载体、四质粒假病毒包装系统、包装新冠肺炎假病毒 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111593073B (zh) |
Cited By (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111926041A (zh) * | 2020-10-16 | 2020-11-13 | 广州吉妮欧生物科技有限公司 | 一种SARS-CoV-2假病毒小鼠体内包装系统及其制备方法 |
| CN112301043A (zh) * | 2020-10-13 | 2021-02-02 | 中国医学科学院病原生物学研究所 | 一种新型冠状病毒SARS-CoV-2复制子、其构建方法及应用 |
| CN112592923A (zh) * | 2020-12-11 | 2021-04-02 | 中国科学院深圳先进技术研究院 | Ires序列、ires序列的应用和多顺反子表达载体 |
| CN112746059A (zh) * | 2020-09-15 | 2021-05-04 | 清华大学 | 基于病毒结构蛋白遗传互补的冠状病毒细胞模型 |
| CN113073113A (zh) * | 2021-04-06 | 2021-07-06 | 中国人民解放军军事科学院军事医学研究院 | 一种监测新冠病毒刺突蛋白S1特异性CTLs功能的可视化小鼠模型的构建方法及应用 |
| CN113156113A (zh) * | 2021-01-27 | 2021-07-23 | 中日友好医院(中日友好临床医学研究所) | 用于体外评价新型冠状病毒ade效应的系统及其构建方法 |
| CN113444745A (zh) * | 2021-05-25 | 2021-09-28 | 安徽省立医院(中国科学技术大学附属第一医院) | 病毒s蛋白突变体假病毒盘的构建及其疫苗评价应用 |
| CN113817802A (zh) * | 2021-08-26 | 2021-12-21 | 首都医科大学附属北京地坛医院 | 新冠病毒包膜蛋白突变体侵入能力非诊断评价方法 |
| WO2021254341A1 (zh) * | 2020-06-16 | 2021-12-23 | 睿丰康生物医药科技(浙江)有限公司 | 一种冠状病毒假病毒包装系统及其包装方法、冠状病毒假病毒在评估消杀效力中的应用 |
| CN113930437A (zh) * | 2020-06-29 | 2022-01-14 | 四川大学华西医院 | 一种病毒报告基因及其在抗SARS-CoV-2药物筛选中的用途 |
| CN114134175A (zh) * | 2021-11-01 | 2022-03-04 | 中国医学科学院输血研究所 | 一种同时表达RFP和luciferase的SARS-CoV-2假病毒系统的包装和检测方法 |
| CN114231544A (zh) * | 2020-09-09 | 2022-03-25 | 复旦大学 | 基于细菌人工染色体的新冠病毒SARS-CoV-2亚基因组复制子克隆、构建及应用 |
| WO2022120819A1 (zh) * | 2020-12-11 | 2022-06-16 | 中国科学院深圳先进技术研究院 | Ires序列、ires序列的应用和多顺反子表达载体 |
| CN114657213A (zh) * | 2022-05-23 | 2022-06-24 | 华南农业大学 | 一种猪急性腹泻综合征冠状病毒人工染色体重组载体及其构建方法和应用 |
| CN114913920A (zh) * | 2022-07-19 | 2022-08-16 | 中科南京生命健康高等研究院 | 双报告基因假病毒检测系统的建立与应用 |
| WO2024067770A1 (zh) * | 2022-09-28 | 2024-04-04 | 云舟生物科技(广州)股份有限公司 | 一种慢病毒包装试剂盒 |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1552851A (zh) * | 2003-06-06 | 2004-12-08 | 北京大学 | Sars冠状病毒的假病毒及其制备方法和用途 |
| CN102083462A (zh) * | 2007-08-03 | 2011-06-01 | 巴斯德研究院 | 慢病毒基因转移载体及其医学应用 |
| CN104830908A (zh) * | 2015-06-02 | 2015-08-12 | 中国食品药品检定研究院 | 假病毒包装系统及其用途 |
| CN105602991A (zh) * | 2015-12-31 | 2016-05-25 | 四川农业大学 | 一种乙脑假病毒及其制备方法 |
| CN106754757A (zh) * | 2016-12-23 | 2017-05-31 | 中国疾病预防控制中心病毒病预防控制所 | 高滴度双报告基因的可视化模拟病毒及其应用 |
-
2020
- 2020-03-18 CN CN202010191574.7A patent/CN111593073B/zh active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1552851A (zh) * | 2003-06-06 | 2004-12-08 | 北京大学 | Sars冠状病毒的假病毒及其制备方法和用途 |
| CN102083462A (zh) * | 2007-08-03 | 2011-06-01 | 巴斯德研究院 | 慢病毒基因转移载体及其医学应用 |
| CN104830908A (zh) * | 2015-06-02 | 2015-08-12 | 中国食品药品检定研究院 | 假病毒包装系统及其用途 |
| CN105602991A (zh) * | 2015-12-31 | 2016-05-25 | 四川农业大学 | 一种乙脑假病毒及其制备方法 |
| CN106754757A (zh) * | 2016-12-23 | 2017-05-31 | 中国疾病预防控制中心病毒病预防控制所 | 高滴度双报告基因的可视化模拟病毒及其应用 |
Non-Patent Citations (10)
| Title |
|---|
| SCOTT R. WITTING ET AL.: "Characterization of a 3rd Generation Lentiviral Vector Pseudotyped With Nipah Virus Envelope Proteins For Endothelial Cell Transduction", 《GENE THER.》 * |
| 孙克宁等: "慢病毒载体的构建及其应用于转基因动物的研究进展", 《中国畜牧兽医》 * |
| 张昕等: "表达萤光素酶的HIV药物筛选细胞模型的建立", 《生物技术通讯》 * |
| 张蕊等: "慢病毒载体的研究进展及应用", 《中国畜牧兽医》 * |
| 樊全荣等: "携带Fluc和ZsGreen双报告基因小鼠", 《中国癌症杂质》 * |
| 王国富: "基因治疗中病毒载体的研究进展", 《大理学院学报》 * |
| 王旭娜等: "假病毒技术在医学中的应用研究进展", 《健康之路》 * |
| 管洁等: "含分泌型萤光素酶和绿色荧光蛋白双报告基因的慢病毒载体的制备与表达分析", 《生物技术通讯》 * |
| 阮峥等: "第三代慢病毒包装系统优化及 Rev表达质粒对慢病毒包装效率作用初探", 《生物技术通讯》 * |
| 黄睿等: "假病毒在丙型肝炎病毒研究中的应用", 《军事医学科学院院刊》 * |
Cited By (19)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021254341A1 (zh) * | 2020-06-16 | 2021-12-23 | 睿丰康生物医药科技(浙江)有限公司 | 一种冠状病毒假病毒包装系统及其包装方法、冠状病毒假病毒在评估消杀效力中的应用 |
| CN113930437A (zh) * | 2020-06-29 | 2022-01-14 | 四川大学华西医院 | 一种病毒报告基因及其在抗SARS-CoV-2药物筛选中的用途 |
| CN114231544A (zh) * | 2020-09-09 | 2022-03-25 | 复旦大学 | 基于细菌人工染色体的新冠病毒SARS-CoV-2亚基因组复制子克隆、构建及应用 |
| CN112746059A (zh) * | 2020-09-15 | 2021-05-04 | 清华大学 | 基于病毒结构蛋白遗传互补的冠状病毒细胞模型 |
| CN112301043A (zh) * | 2020-10-13 | 2021-02-02 | 中国医学科学院病原生物学研究所 | 一种新型冠状病毒SARS-CoV-2复制子、其构建方法及应用 |
| CN111926041A (zh) * | 2020-10-16 | 2020-11-13 | 广州吉妮欧生物科技有限公司 | 一种SARS-CoV-2假病毒小鼠体内包装系统及其制备方法 |
| CN111926041B (zh) * | 2020-10-16 | 2020-12-25 | 广州吉妮欧生物科技有限公司 | 一种SARS-CoV-2假病毒小鼠体内包装系统及其制备方法 |
| WO2022120819A1 (zh) * | 2020-12-11 | 2022-06-16 | 中国科学院深圳先进技术研究院 | Ires序列、ires序列的应用和多顺反子表达载体 |
| CN112592923A (zh) * | 2020-12-11 | 2021-04-02 | 中国科学院深圳先进技术研究院 | Ires序列、ires序列的应用和多顺反子表达载体 |
| CN113156113A (zh) * | 2021-01-27 | 2021-07-23 | 中日友好医院(中日友好临床医学研究所) | 用于体外评价新型冠状病毒ade效应的系统及其构建方法 |
| CN113073113A (zh) * | 2021-04-06 | 2021-07-06 | 中国人民解放军军事科学院军事医学研究院 | 一种监测新冠病毒刺突蛋白S1特异性CTLs功能的可视化小鼠模型的构建方法及应用 |
| CN113444745A (zh) * | 2021-05-25 | 2021-09-28 | 安徽省立医院(中国科学技术大学附属第一医院) | 病毒s蛋白突变体假病毒盘的构建及其疫苗评价应用 |
| CN113817802A (zh) * | 2021-08-26 | 2021-12-21 | 首都医科大学附属北京地坛医院 | 新冠病毒包膜蛋白突变体侵入能力非诊断评价方法 |
| CN114134175A (zh) * | 2021-11-01 | 2022-03-04 | 中国医学科学院输血研究所 | 一种同时表达RFP和luciferase的SARS-CoV-2假病毒系统的包装和检测方法 |
| CN114657213A (zh) * | 2022-05-23 | 2022-06-24 | 华南农业大学 | 一种猪急性腹泻综合征冠状病毒人工染色体重组载体及其构建方法和应用 |
| CN114657213B (zh) * | 2022-05-23 | 2022-08-02 | 华南农业大学 | 一种猪急性腹泻综合征冠状病毒人工染色体重组载体及其构建方法和应用 |
| CN114913920A (zh) * | 2022-07-19 | 2022-08-16 | 中科南京生命健康高等研究院 | 双报告基因假病毒检测系统的建立与应用 |
| CN114913920B (zh) * | 2022-07-19 | 2022-12-27 | 中科南京生命健康高等研究院 | 双报告基因假病毒检测系统的建立与应用 |
| WO2024067770A1 (zh) * | 2022-09-28 | 2024-04-04 | 云舟生物科技(广州)股份有限公司 | 一种慢病毒包装试剂盒 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111593073B (zh) | 2022-03-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111593073B (zh) | 双报告基因骨架载体、四质粒假病毒包装系统、包装新冠肺炎假病毒 | |
| AU2018229561B2 (en) | Recombinant adenoviruses and use thereof | |
| US11667890B2 (en) | Engineered artificial antigen presenting cells for tumor infiltrating lymphocyte expansion | |
| CN111295449B (zh) | 腺病毒载体及其用途 | |
| US6361972B1 (en) | Compositions and methods for non-targeted activation of endogenous genes | |
| US20030119104A1 (en) | Chromosome-based platforms | |
| ES2805045T3 (es) | Vectores lentivirales | |
| ES2388527T3 (es) | Vacunas de VIH basadas en Env de múltiples clados de VIH | |
| KR20220125332A (ko) | Pcsk9의 표적화를 위한 조성물 및 방법 | |
| KR102516697B1 (ko) | 조작된 캐스케이드 구성성분 및 캐스케이드 복합체 | |
| WO2004067743A1 (en) | Vaccine production using transposon based vectors | |
| US11129890B1 (en) | Non-integrating HIV-1 comprising mutant RT/IN proteins and the SARS-CoV-2 spike protein | |
| PT1984512T (pt) | Sistema de expressão génica utilizando excisão-união em insetos | |
| WO2008151633A2 (en) | Vectors for vaccines against lentivirus infections | |
| CN111094569A (zh) | 光控性病毒蛋白质、其基因及包含该基因的病毒载体 | |
| CN113692225B (zh) | 经基因组编辑的鸟类 | |
| DK3063278T3 (en) | PREPARATION OF VIRAL VECTORS | |
| KR20240037192A (ko) | 게놈 통합을 위한 방법 및 조성물 | |
| US6740503B1 (en) | Compositions and methods for non-targeted activation of endogenous genes | |
| CA2522166C (en) | Lambda integrase mutein for use in recombination | |
| RU2774631C1 (ru) | Сконструированные компоненты cascade и комплексы cascade | |
| CN110819657B (zh) | 一种减毒棒状病毒的制备方法及应用 | |
| KR20240029020A (ko) | Dna 변형을 위한 crispr-트랜스포손 시스템 | |
| KR20230153437A (ko) | 코로나바이러스를 방지하는 백신 생산을 위한 완전 합성 장쇄 핵산 | |
| CN115768470A (zh) | 用于疫苗生产以防范冠状病毒的全合成的长链核酸 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20200828 Address after: 311200 4th floor, building 2, ChuanHua Kechuang building, ningwei street, Xiaoshan District, Hangzhou City, Zhejiang Province Applicant after: Huijun biomedical technology (Hangzhou) Co.,Ltd. Address before: Second, 150 teaching building, No. 215000 benevolence Road, Suzhou Industrial Park, Suzhou, Jiangsu Applicant before: SUZHOU ULTRAIMMUNE Co.,Ltd. |
|
| TA01 | Transfer of patent application right | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20201123 Address after: Office 1201-1, innovation building, 1000 Linjiang East Road, bailongqiao Town, Wucheng District, Jinhua City, Zhejiang Province 321000 Applicant after: Ruifengkang biomedical technology (Zhejiang) Co.,Ltd. Address before: 311200 4th floor, building 2, ChuanHua Kechuang building, ningwei street, Xiaoshan District, Hangzhou City, Zhejiang Province Applicant before: Huijun biomedical technology (Hangzhou) Co.,Ltd. |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: Double reporter gene skeleton vector, four plasmid pseudovirus packaging system, packaging COVID-19 pseudovirus Effective date of registration: 20221220 Granted publication date: 20220308 Pledgee: Bank of Jinhua Limited by Share Ltd. Wucheng branch Pledgor: Ruifengkang biomedical technology (Zhejiang) Co.,Ltd. Registration number: Y2022980028346 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20231221 Granted publication date: 20220308 Pledgee: Bank of Jinhua Limited by Share Ltd. Wucheng branch Pledgor: Ruifengkang biomedical technology (Zhejiang) Co.,Ltd. Registration number: Y2022980028346 |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: Double reporter gene skeleton vector, four plasmid pseudovirus packaging system, packaging COVID-19 pseudovirus Granted publication date: 20220308 Pledgee: Bank of Jinhua Limited by Share Ltd. Wucheng branch Pledgor: Ruifengkang biomedical technology (Zhejiang) Co.,Ltd. Registration number: Y2024980002578 |