CN114395579B - 一种基因i型与基因iii型日本脑炎病毒感染性克隆及其构建方法与应用 - Google Patents
一种基因i型与基因iii型日本脑炎病毒感染性克隆及其构建方法与应用 Download PDFInfo
- Publication number
- CN114395579B CN114395579B CN202111276598.3A CN202111276598A CN114395579B CN 114395579 B CN114395579 B CN 114395579B CN 202111276598 A CN202111276598 A CN 202111276598A CN 114395579 B CN114395579 B CN 114395579B
- Authority
- CN
- China
- Prior art keywords
- jev
- gene
- vector
- type
- tar
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 91
- 208000015181 infectious disease Diseases 0.000 title claims abstract description 39
- 230000002458 infectious effect Effects 0.000 title claims abstract description 35
- 238000010276 construction Methods 0.000 title claims abstract description 18
- 241000710842 Japanese encephalitis virus Species 0.000 title claims abstract description 17
- 239000013598 vector Substances 0.000 claims abstract description 54
- 239000013612 plasmid Substances 0.000 claims abstract description 38
- 239000012634 fragment Substances 0.000 claims abstract description 24
- 230000006801 homologous recombination Effects 0.000 claims abstract description 7
- 238000002744 homologous recombination Methods 0.000 claims abstract description 7
- 238000000034 method Methods 0.000 claims description 21
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 16
- 239000002299 complementary DNA Substances 0.000 claims description 13
- 108010006025 bovine growth hormone Proteins 0.000 claims description 7
- 230000008488 polyadenylation Effects 0.000 claims description 7
- 238000002156 mixing Methods 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 5
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 claims description 4
- 238000012408 PCR amplification Methods 0.000 claims description 4
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 claims description 4
- 230000002950 deficient Effects 0.000 claims description 4
- 239000002773 nucleotide Substances 0.000 claims description 4
- 125000003729 nucleotide group Chemical group 0.000 claims description 4
- 108091080980 Hepatitis delta virus ribozyme Proteins 0.000 claims description 3
- 239000011248 coating agent Substances 0.000 claims 1
- 238000000576 coating method Methods 0.000 claims 1
- 238000012258 culturing Methods 0.000 claims 1
- 241000700605 Viruses Species 0.000 abstract description 28
- 238000000338 in vitro Methods 0.000 abstract description 15
- 108020000999 Viral RNA Proteins 0.000 abstract description 13
- 241000235342 Saccharomycetes Species 0.000 abstract description 12
- 230000003612 virological effect Effects 0.000 abstract description 6
- 238000012217 deletion Methods 0.000 abstract description 4
- 230000037430 deletion Effects 0.000 abstract description 4
- 238000003780 insertion Methods 0.000 abstract description 4
- 230000037431 insertion Effects 0.000 abstract description 4
- 230000035772 mutation Effects 0.000 abstract description 4
- 230000008878 coupling Effects 0.000 abstract description 3
- 238000010168 coupling process Methods 0.000 abstract description 3
- 238000005859 coupling reaction Methods 0.000 abstract description 3
- 238000005215 recombination Methods 0.000 abstract description 3
- 230000009466 transformation Effects 0.000 abstract description 3
- 210000004027 cell Anatomy 0.000 description 20
- 108020004414 DNA Proteins 0.000 description 17
- 230000003321 amplification Effects 0.000 description 10
- 230000002068 genetic effect Effects 0.000 description 10
- 238000003199 nucleic acid amplification method Methods 0.000 description 10
- 230000035897 transcription Effects 0.000 description 10
- 238000013518 transcription Methods 0.000 description 10
- 238000011144 upstream manufacturing Methods 0.000 description 9
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 7
- 229960005486 vaccine Drugs 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 239000002609 medium Substances 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 5
- 238000003556 assay Methods 0.000 description 5
- 238000003776 cleavage reaction Methods 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 108091008146 restriction endonucleases Proteins 0.000 description 5
- 230000007017 scission Effects 0.000 description 5
- 102000053642 Catalytic RNA Human genes 0.000 description 4
- 108090000994 Catalytic RNA Proteins 0.000 description 4
- 101710144111 Non-structural protein 3 Proteins 0.000 description 4
- 239000008194 pharmaceutical composition Substances 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- 108091092562 ribozyme Proteins 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 239000012096 transfection reagent Substances 0.000 description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 208000037262 Hepatitis delta Diseases 0.000 description 3
- 241000724709 Hepatitis delta virus Species 0.000 description 3
- 239000012124 Opti-MEM Substances 0.000 description 3
- 241000282887 Suidae Species 0.000 description 3
- 108700005077 Viral Genes Proteins 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 239000012091 fetal bovine serum Substances 0.000 description 3
- 238000010166 immunofluorescence Methods 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000011535 reaction buffer Substances 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 230000005030 transcription termination Effects 0.000 description 3
- 238000001890 transfection Methods 0.000 description 3
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 206010014596 Encephalitis Japanese B Diseases 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 101150017040 I gene Proteins 0.000 description 2
- 102100034343 Integrase Human genes 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 201000005807 Japanese encephalitis Diseases 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 101710172711 Structural protein Proteins 0.000 description 2
- 239000011543 agarose gel Substances 0.000 description 2
- 229940031567 attenuated vaccine Drugs 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 230000000120 cytopathologic effect Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 241001493065 dsRNA viruses Species 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 229940031551 inactivated vaccine Drugs 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 239000013605 shuttle vector Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 210000005253 yeast cell Anatomy 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 108020004635 Complementary DNA Proteins 0.000 description 1
- 206010010904 Convulsion Diseases 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 102000007260 Deoxyribonuclease I Human genes 0.000 description 1
- 108010008532 Deoxyribonuclease I Proteins 0.000 description 1
- 101150013191 E gene Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000710781 Flaviviridae Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 206010020741 Hyperpyrexia Diseases 0.000 description 1
- 101150098499 III gene Proteins 0.000 description 1
- 101710203526 Integrase Proteins 0.000 description 1
- 229910009891 LiAc Inorganic materials 0.000 description 1
- 208000003773 Meningism Diseases 0.000 description 1
- -1 NS2B Proteins 0.000 description 1
- 101800001030 Non-structural protein 2A Proteins 0.000 description 1
- 101800001020 Non-structural protein 4A Proteins 0.000 description 1
- 101800001019 Non-structural protein 4B Proteins 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 108091036407 Polyadenylation Proteins 0.000 description 1
- 108020005161 RNA Caps Proteins 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 101710141795 Ribonuclease inhibitor Proteins 0.000 description 1
- 229940122208 Ribonuclease inhibitor Drugs 0.000 description 1
- 102100037968 Ribonuclease inhibitor Human genes 0.000 description 1
- 101710137500 T7 RNA polymerase Proteins 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 206010000210 abortion Diseases 0.000 description 1
- 231100000176 abortion Toxicity 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000036461 convulsion Effects 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 210000003754 fetus Anatomy 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 208000000509 infertility Diseases 0.000 description 1
- 231100000535 infertility Toxicity 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- XIXADJRWDQXREU-UHFFFAOYSA-M lithium acetate Chemical compound [Li+].CC([O-])=O XIXADJRWDQXREU-UHFFFAOYSA-M 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 201000005737 orchitis Diseases 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 230000011514 reflex Effects 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 239000003161 ribonuclease inhibitor Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000002381 testicular Effects 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 238000011426 transformation method Methods 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000029812 viral genome replication Effects 0.000 description 1
- 206010048282 zoonosis Diseases 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives 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
- 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
- C12N2770/24011—Flaviviridae
- C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
- C12N2770/24121—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
- C12N2770/24011—Flaviviridae
- C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
- C12N2770/24122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- 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
- C12N2770/24011—Flaviviridae
- C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
- C12N2770/24151—Methods of production or purification of viral material
- C12N2770/24152—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Engineering & Computer Science (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Mycology (AREA)
- Wood Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Plant Pathology (AREA)
- Gastroenterology & Hepatology (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Virology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一种基因I型与基因III型日本脑炎病毒感染性克隆及其构建方法与应用，属于分子生物学技术领域。本发明通过设计四对保守引物，分四段一次扩增出涵盖基因I型与基因III型JEV基因组全长的片段，基于DNA片段末端的同源性，利用酵母菌中的转化偶联重组技术，将分段的病毒基因组片段与线性化载体进行同源重组，一次性分别构建出携带基因I型与基因III型JEV基因组全长的重组质粒。重组质粒线性化处理后，利用T7启动子经体外转录出病毒RNA，RNA转染BHK‑21细胞后即可获得感染性克隆病毒。本发明可在酵母菌中稳定传代10代以上，不存在基因组本身序列的突变、缺失或外源序列的插入。
Description
技术领域
本发明属于分子生物学领域，涉及两种基因型(I型与III型)日本脑炎病毒的全长基因组感染性克隆的一步构建方法及其应用。
背景技术
日本脑炎(Japanese encephalitis，JE)是由日本脑炎病毒(Japaneseencephalitis Virus，JEV)引起的一种以蚊虫为传播媒介的急性人兽共患病，主要流行于亚洲地区，其中我国是JE的高发区之一。自然界中JEV具有广泛的宿主范围，其主要的侵害对象为人与猪。人主要十岁以下儿童易感，感染后临床症状表现为高热、抽搐、意识障碍、病理反射和脑膜刺激征，危重病例出现死亡或愈后留有不同程度的神经后遗症；猪感染后主要表现为繁殖性障碍，妊娠母猪发生流产、产生死胎或木乃伊胎，公猪则出现睾丸炎、单侧睾丸肿大而不育。近年JEV在我国宁夏、广西、浙江等地相继出现流行，对我国养猪业的持续发展以及我国公共卫生安全造成了巨大的威胁。
JEV是单股正链RNA病毒，属于黄病毒科黄病毒属，基因组大小约为11kb，5’端具I型帽子结构，3’端无poly(A)尾巴，基因组中间含有一大的开放性阅读框，可编码三种结构蛋白(C，PrM，E)和七种非结构蛋白(NS1，NS2A，NS2B,NS3，NS4A，NS4B，NS5)。根据E基因序列，JEV分为五个基因型(I，II，III，IV，V)。1940年我国首次分离到基因III型毒株，随后多年我国一直以基因III型毒株流行为主。但是，最近二十年，基因I型替代基因III型，成为我国主要的流行毒株，而基因型转换的原因目前还没有得到明确的解答。至今我国呈现以基因I型为主，基因I型与III型混合流行的局面。
我国存在基因I型与III型两种毒株混和流行的局面。目前疫苗免疫仍是有效防控JEV的重要手段之一，我国现有的JEV商品化疫苗(弱毒苗与灭活苗)均是基于基因III型毒株研制，可有效的防控基因III型野毒株的感染。但是，现有的基因III型JEV灭活苗或弱毒苗对基因I型JEV感染的交叉保护作用较差，并且近几年基因I型JEV毒株不断在临床中分离到，使得基因I型JEV具有潜在爆发的危害性。因此，研制安全、有效、具有交叉保护力的新型特异性疫苗已成为我国JEV防治的重点。
RNA病毒感染性克隆是通过体外构建病毒cDNA克隆，在DNA水平上对病毒基因进行人工操作，在通过体外拯救的方法获得感染性的病毒颗粒。但JEV反向遗传操作平台的建立存在一定的难度，原因在于JEV基因组的全长克隆在宿主菌如大肠杆菌中存在遗传不稳定现象，病毒基因序列在宿主菌中克隆复制的过程易出现本身序列的突变、缺失或外源序列的插入。另外，基因I型与基因III型JEV全基因组核苷酸序列的同源性在88％左右，若利用传统感染性克隆的构建方法，受到酶切位点的限制，仅能分别进行单个基因型的构建，极大的限制了工作效率。因此，需要寻找一种高效、稳定的基因I型与基因III型JEV感染性克隆的构建方法。一种高效、稳定的基因I型与基因III型JEV感染性克隆的构建及遗传操作，可用于病毒复制和治病机制的研究，同时也是JEV新型基因工程疫苗研制的基础。目前，同时达到高效、稳定且可一步完成基因I型与基因III型JEV毒株反向遗传操作平台构建的方法至今未有报道。
发明内容
发明目的：针对JEV感染性克隆在宿主菌中不稳定的科学问题，本发明提供了一种高效、稳定的基因I型与基因III型日本脑炎病毒感染性克隆的一步构建方法。同时，针对目前我国缺乏基因I型JEV疫苗的实际问题，本发明的基因I型JEV感染性克隆平台，可用于研制安全有效的新型基因I型JEV基因工程疫苗。
技术方案：本发明的原理是通过设计四对保守引物，分四段一次扩增出涵盖基因I型与基因III型JEV基因组全长的片段，基于DNA片段末端的同源性，利用酵母菌中的转化偶联重组技术，将分段的病毒基因组片段与线性化载体进行同源重组，一次性分别构建出携带基因I型与基因III型JEV基因组全长的重组质粒。重组质粒线性化处理后，利用T7启动子经体外转录出病毒RNA，RNA转染BHK-21细胞后即可获得感染性克隆病毒。可在酵母菌中稳定传代10代以上，不存在基因组本身序列的突变、缺失或外源序列的插入。
一种基因I型与基因III型日本脑炎病毒感染性克隆的构建方法，包含以下步骤：
(1)线性化载体TAR vector的获得，本发明优选的载体是基于一种即用型、9.3kb线性化BAC/YAC穿梭载体pYES1L。经过基因合成和同源重组等方法在pYES1L载体上游插入T7启动子序列，在下游插入丁型肝炎病毒核酶(Hepatitis D virus ribozyme)序列与牛生长激素多聚腺苷酸信号(Bovine Growth Hormone(BGH)polyadenylation signal)转录终止序列，获得的重组载体命名为TAR vector。设计载体TAR的扩增引物，利用PCR方法扩增线性化的TAR载体，用于基因I型JEV毒株YZ-1与基因III型毒株HSY-1感染性克隆的构建。
线性化载体TAR vector的扩增引物为(5’-3’)：
上游引物：AACACAGGATCTGGCCGGCATGGTCCCAGCCTCCTCGCTG，下游引物：CTTCTCCTATAGTGAGTCGTATTAAGCTCTGCTTATATAG
ACCTCCCACCG。
(2)构建分别携带基因I型与基因III型JEV基因组全长的重组质粒，分析基因I型毒株YZ-1与基因III型毒株HSY-1的全基因组序列的同源性，设计四对保守的扩增引物，分四段(F1，F2，F3，F4)一次扩增出基因I型与III型JEV的基因组片段。保证扩增出的线性化载体与目的片段间，以及相邻目的片段间具有20bp-30bp的同源臂序列。将线性化载体分别与两种基因型病毒的四个基因组片段混和，加入到MaV203酵母感受态菌中，利用醋酸锂转化法转化酵母细胞，涂于色氨酸(Trp)缺陷型的筛选平板上，30℃培养2天，利用菌落PCR方法鉴定阳性克隆，可成功构建重组质粒TAR-rGI与TAR-rGIII。将两种重组质粒在酵母中连续传代10代以上，测序分析病毒全基因组序列的遗传稳定型。
JEV片段的扩增引物为：
F1上游引物：CAGAGCTTAATACGACTCACTATAGGAGAAGTTTATCTGTGTGAACTTCT，
F1下游引物：TTGTGTGATCCAAGACATTCCCCCAAAGAG；
F2上游引物：CTCTTTGGGGGAATGTCTTGGATCACACAA，
F2下游引物：TGGAACACCGGGATCATCAATCAAGTGAAA；
F3上游引物：TTTCACTTGATTGATGATCCCGGTGTTCCA，
F3下游引物：GGCTTGTCAGCGTTCTTGATGAGAGTCCA；
F4上游引物：TGGACTCTCATCAAGAACGCTGACAAGCC，
F4下游引物：GACCATGCCGGCCAGATCCTGTGTTCTTCCTCACCACCAG；
(3)感染性克隆病毒的拯救，本发明通过转染体外转录获得的病毒RNA来制备感染性克隆病毒，将上诉重组载体TAR-rGI与TAR-rGIII，利用限制性内切酶Bsu36I进行线性化处理，利用T7启动子进行体外转录获得病毒RNA，基因I型与基因III型病毒的RNA分别与转染试剂混合后直接转染BHK-21细胞，37℃培养箱中培养细胞3-5天，观察细胞病变情况。当细胞病变80％左右时，收取细胞上清，离心去掉沉淀，继续接种BHK-21细胞进行传代或扩大培养。本发明均能够成功拯救获得rGI与rGIII感染性克隆病毒。
上述基因I型与基因III型日本脑炎病毒感染性克隆的构建方法得到的基因I型与基因III型日本脑炎病毒克隆在本发明的保护范围之内。
上述基因I型与基因III型日本脑炎病毒感染性克隆的构建方法得到的基因I型YZ-1病毒克隆在本发明的保护范围之内。
上述基因I型与基因III型日本脑炎病毒感染性克隆的构建方法得到的基因III型HSY-1病毒克隆在本发明的保护范围之内。
本发明的目的是通过以下技术方案实现的：
(1)设计保守引物，分四段分别扩增出涵盖基因I型毒株JEV YZ-1与基因III型毒株JEV HSY-1基因组全长的片段，以及扩增出线性化载体TAR vector，具体引物序列见表1，由南京金斯瑞生物科技有限公司合成。
(2)利用酵母同源重组系统，分别将YZ-1基因组的四个片段与HSY-1基因组的四个片段，分别重组在载体TAR vector上，获得重组质粒TAR-rGI与TAR-rGIII。
(3)将上诉质粒在酵母菌中连续传代10代以上，测序分析基因组的遗传稳定型。
(4)将重组质粒TAR-rGI与TAR-rGIII利用限制性内切酶Bsu36I进行线性化处理，线性化的质粒利用T7启动子体外转录出病毒RNA，将基因I型与基因III型的病毒RNA分别与高效转染试剂混合，转染BHK-21细胞中，得到感染性克隆病毒。
(5)通过间接免疫荧光(IFA)方法对拯救的感染性克隆病毒进行鉴定。
有益效果：
1.本发明提供的感染性克隆构建方法不仅适用于基因I型毒株YZ-1与基因III型毒株HSY-1，也适用于其他基因I型毒株和基因III型毒株。
2.本发明提供的感染性克隆构建方法具有高效、快速的特点。利用一步同源重组的方法即可完成两种不同基因型病毒的全长基因组克隆，极大的提高了效率。获得的重组质粒5’端携带有T7启动子序列，3’端加有丁型肝炎病毒核酶(Hepatitis D virusribozyme，HDV)序列与牛生长激素多聚腺苷酸信号(Bovine Growth Hormone(BGH)polyadenylation signal)转录终止序列，经T7启动子体外转录的病毒RNA在转染细胞36h内即可观察到明显的细胞病变并成功拯救出病毒，是一种快速、高效、稳定的JEV遗传操作平台。
3.本发明所构建的两种基因型JEV感染性克隆具有稳定的遗传特性，在酵母菌中稳定传代10代以上，不存在基因组本身序列的突变、缺失或外源序列的插入，克服了JEV基因组的全长克隆在宿主菌中遗传不稳定的现象。
4.本发明所构建的JEV感染性克隆平台，可用于研制安全、有效、具有交叉保护力的新型特异性疫苗，有利于我国JEV的防控。
附图说明
图1是本发明实例中对TAR载体进行改造的示意图及线性化载体PCR扩增结果图(M：Marker；TAR vector：线性化载体)；
图2是本发明实例中基因I型JEV毒株YZ-1与基因III型JEV毒株HSY-1的全基因组片段PCR扩增结果图(M:Marker；毒株YZ-1的四个片段：F1rGI,F2rGI,F3rGI和F4rGI；毒株HSY-1的四个片段：F1rGIII,F2rGIII,F3rGIII和F4rGIII)；
图3是本发明实例中基因I型JEV毒株YZ-1与基因III型JEV毒株HSY-1基因组全长cDNA连接示意图；
图4是本发明实例中感染性克隆TAR-rGI与TAR-rGIII酶切鉴定结果图(M：Marker；1:TAR-rGI质粒Bsu36I单酶切鉴定；2：TAR-rGI质粒SacII与XmaI双酶切鉴定；3：TAR-rGIII质粒Bsu36I单酶切鉴定；4：TAR-rGIII质粒BsrGI与BbvCI双酶切鉴定)；
图5是本发明实例中体外转录获得的病毒RNA转染BHK-21细胞36h的细胞病变图。
图6是本发明实例中利用JEV NS3蛋白特性抗体通过间接免疫荧光法检测被拯救的rGI与rGIII感染性克隆病毒的结果图。
具体实施方式
下面结合实施例对本发明作进一步详细的描述，但本发明的实施方式不限于此。
1.材料
1.1病毒、质粒、细胞
基因I型毒株YZ-1(NCBI登录号：MZ540901)与基因III型毒株HSY-1(NCBI登录号:MZ540902)为本实验室保存；载体质粒pYES1L购自ThermoFisher Scientific由本实验保存；BHK-21细胞系由本实验保存。
1.2主要试剂
抗JEV NS3蛋白抗体购自Genetex公司；Q5高保真DNA聚合酶、dNTP MIX(100mM)、限制性内切酶(Bsu36I,SacII,XmaI,BsrGI,BbvCI)、T7体外转录试剂盒、m7G(5’)ppp(5’)ARNA帽子结构模拟物均购自NEB公司；胶回收试剂盒、RNA提取试剂(TRIzol)购自南京诺威赞公司；Lipofectamine3000转染试剂、M-MLV逆转录试剂盒、MaV203感受态酵母细胞、酵母菌色氨酸合成缺陷型培养基(CSM-Trp Agar Yeast Media minus Glucose)购自ThermoFisher Scientific公司；羊抗鼠FITC标记的二抗购自Abcam公司；质粒大提试剂盒购自天根生化科技有限公司；胎牛血清(FBS)购自Sigma公司；DMEM培养基、opti-MEM培养基、胰蛋白酶、青霉素-链霉素溶液(双抗，100×)购自Hyclone公司；酵母培养基(YPDAMedium)购自庄盟生物科技有限公司。
2.方法
2.1TAR载体构建
基于线性化的BAC/YAC穿梭载体pYES1L，如图1所示对载体进行改造，利用同源重组方法将一个T7启动子序列插入到载体的上游，将一个丁型肝炎病毒核酶(Hepatitis Dvirus ribozyme)序列与牛生长激素多聚腺苷酸信号(Bovine Growth Hormone(BGH)polyadenylation signal)转录终止序列插入到载体的下游。重组载体命名为TAR-vector(约10.1kb)，由本实验室保存。
2.2引物的设计和合成
根据基因I型毒株YZ-1与基因III型毒株HSY-1序列的同源性，设计四对保守扩增引物用于扩增病毒的全长基因组cDNA。根据载TAR vector的序列设计引物，用于扩增出线性化载体TAR vector。
表1.PCR扩增线性化TAR vector及病毒基因组全长的引物：
2.3基因I型毒株YZ-1与基因III型毒株HSY-1感染性克隆的构建
2.3.1线性化载体扩增
利用PCR方法以TAR vector为模板扩增出线性化的载体TAR vector。PCR体系为：质粒模板10ng，上游与下游引物(10mM)各1.5μL，5×Q5 Reaction Buffer 10μL，100mMdNTP 1μL，Q5 Polymerase 0.5μL，RNase Free H2O补充至50μL。PCR反应程序为：98℃30s，60℃15s，72℃5min，30个循环。PCR产物经0.8％琼脂糖凝胶进行电泳，电压为100V，时间为50min；如图1所示，在10000与15000bp间出现特异性的条带，证明获得所需的目的条带。
2.3.2YZ-1与HSY-1的全基因组分段扩增
首先制备病毒基因组，将分别感染病毒YZ-1与HSY-1的细胞上清用TRIzol进行裂解，随后用氯仿、异丙醇进行病毒RNA的抽提，最后用75％乙醇沉淀，溶于DEPC处理的灭菌水中。取病毒RNA进行逆转录合成病毒RNA的第一链cDNA，采取20μL体系，反应条件为：病毒RNA模板2μg，100mM dNTP mix 1μL，5×Buffer 4μL，100mM DTT 1μL，50ng/μL随机引物1μL，Ribonuclease抑制剂1μL，M-MLV逆转录酶(200U/μL)1μL，加入DEPC无菌水至20μL。反应程序为：25℃10min，37℃50min，70℃10min，降温至4℃，最后加入RNase H，37℃20min去除RNA。
分别以YZ-1与HSY-1cDNA为模板，以JEV-F1-F和JEV-F1-R，JEV-F2-F和JEV-F2-R，JEV-F3-F和JEV-F3-R，以及JEV-F4-F和JEV-F4-R为配对引物扩增F1，F2，F3，F4片段。PCR体系为：cDNA模板1μL，上游与下游引物(10mM)各1.5μL，5×Q5 Reaction Buffer 10μL，100mMdNTP 1μL，Q5Polymerase 0.5μL，RNase Free H2O补充至50μL。PCR反应程序为：98℃30s，60℃15s，72℃2min，30个循环。PCR产物经1％琼脂糖凝胶进行电泳，电压为100V，时间为30min；如图2所示，在2350bp，2120bp，3170bp，3320bp处出现特异性的条带，证明获得所需的目的条带。
2.3.3重组质粒TAR-rGI与TAR-rGIII的构建与遗传稳定性分析
将上诉扩增的目的基因与线性化载体利用胶回收试剂盒进行纯化，进一步将纯化产物与线性化载体利用酵母菌中的转化偶联重组技术进行重组，构建策略如图3所示。具体步骤：
将JEV毒株YZ-1或HSY-1基因组的四个片段(各100ng)与线性化载体TAR vector(100ng)预混，总体积小于10μL；取出100μL酵母感受态细胞MaV203置于30℃水浴中解冻不超过90s,将目的片段与载体的混合物加入到酵母感受态细胞中，上下轻微颠倒混匀，随后每管感受态中加入600μL PEG/LiAc溶液，颠倒混合均匀后，放置在30℃水浴中孵育30min，每10min上下轻微颠倒混匀一次；孵育后每管加入35.5μL的DMSO，颠倒混合均匀后在42℃的水浴中热激细胞20min；将热激后的酵母感受态，低速(1800rpm；200-400×g)离心5min，弃掉上清，用1mL高压灭菌盐水(0.9％NaCl)重悬酵母菌；将重悬酵母菌涂于色氨酸缺陷的酵母培养板，培养2-3天。
挑取单独的酵母菌落进行培养，使用特异的检测引物进行菌液的PCR检测。选取PCR验证阳性的酵母菌进行过夜扩增培养，参照酵母质粒提取说明书，进行质粒DNA的提取。随后，使用限制性酶切酶SacII与XmaI对质粒TAR-rGI进行双酶切鉴定；使用BsrGI与BbvCI对质粒TAR-rGIII-进行双酶切鉴定，0.8％琼脂糖凝胶电泳，如图4所示，TAR-rGI双酶切的质粒在10479bp、6405bp、4291bp出现特意条带，TAR-rGIII双酶切的质粒在在8633bp、7334bp、5208bp处出现特意条带。将鉴定正确的质粒送测序。
将重组质粒TAR-rGI与TAR-rGIII在酵母菌中进行连续传代扩增，当扩增第10代次时，提取第10代酵母菌中的重组质粒进行测序。重组质粒测序鉴定正确表明重组质粒在酵母菌中的遗传稳定性。
2.4感染性克隆病毒的拯救与验证
首先经体外转录获得病毒RNA，具体步骤：将重组质粒TAR-rGI与TAR-rGIII利用限制性内切酶Bsu36I进行线性化处理并利用胶回收试剂盒进行纯化(图4)；将纯化后的线性质粒利用T7体外转录试剂盒进行病毒基因组的体外转录，体外转录体系：线性化载体(500ng)、10×Reaction Buffer(2.5μL)、ATP(100mM)(2.5μL)、GTP(100mM)(2.5μL)、UTP(100mM)(2.5μL)、CTP(100mM)(2.5μL)、T7 RNA聚合酶(2.5μL)、帽子结构模拟物(40mM)(2.5μL)、ddH2O补充至总体积25μL，反应条件为37℃水浴2h；将体外转录产物利用DNase I 37℃处理15分钟去除DNA模板。
预先使用含10％FBS的DMEM培养基将BHK-21细胞接种于6孔板细胞培养板中，至于37℃培养箱中培养至细胞密度达到80％左右。按照Lipofectamine 3000转染试剂说明对病毒RNA进行细胞转染，具体操作步骤如下：
首先在一无菌EP管中配置RNA预混液:体外转录获得的病毒RNA，P3000 2μL，opti-MEM稀释至50μL；另一无菌EP管中配置Lipofectamine 3000预混液：Lip3000 3μL，opti-MEM稀释至50μL；将上诉俩种预混液混合均匀，室温孵育15min，加入到待转染的BHK-21细胞中，转染后36h内即可观察到明显的细胞病变，转染后细胞病变情况如图5所示。当细胞病变80％时，收取细胞上清，冻存于-80℃备用。
将拯救的病毒接种BHK-21细胞，培养24h后利用多聚甲醛固定，使用抗JEV NS3蛋白特异的抗体进行间接免疫荧光检测，如图6所示可见拯救的病毒rGI与rGIII与自身的亲本毒均出现特异性的绿色荧光，阴性对照组无荧光产生，证实感染性克隆病毒拯救成功。
序列表
<110> 扬州大学
<120> 一种基因I型与基因III型日本脑炎病毒感染性克隆及其构建方法与应用
<160> 13
<170> SIPOSequenceListing 1.0
<210> 1
<211> 10232
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
cctcgccgca gttaattaaa gtcagtgagc gaggaagcgc gtaactataa cggtcctaag 60
gtagcgaatc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat 120
agatcggcaa gtgcacaaac aatacttaaa taaatactac tcagtaataa cctatttctt 180
agcatttttg acgaaatttg ctattttgtt agagtctttt acaccatttg tctccacacc 240
tccgcttaca tcaacaccaa taacgccatt taatctaagc gcatcaccaa cattttctgg 300
cgtcagtcca ccagctaaca taaaatgtaa gctttcgggg ctctcttgcc ttccaaccca 360
gtcagaaatc gagttccaat ccaaaagttc acctgtccca cctgcttctg aatcaaacaa 420
gggaataaac gaatgaggtt tctgtgaagc tgcactgagt agtatgttgc agtcttttgg 480
aaatacgagt cttttaataa ctggcaaacc gaggaactct tggtattctt gccacgactc 540
atctccatgc agttggacga tatcaatgcc gtaatcattg accagagcca aaacatcctc 600
cttaagttga ttacgaaaca cgccaaccaa gtatttcgga gtgcctgaac tatttttata 660
tgcttttaca agacttgaaa ttttccttgc aataaccggg tcaattgttc tctttctatt 720
gggcacacat ataataccca gcaagtcagc atcggaatct agagcacatt ctgcggcctc 780
tgtgctctgc aagccgcaaa ctttcaccaa tggaccagaa ctacctgtga aattaataac 840
agacatactc caagctgcct ttgtgtgctt aatcacgtat actcacgtgc tcaatagtca 900
ccaatgccct ccctcttggc cctctccttt tcttttttcg accgaattaa ttcttaatcg 960
gcaaaaaaag aaaagctccg gatcaagatt gtacgtaagg tgacaagcta tttttcaata 1020
aagaatatct tccactactg ccatctggcg tcataactgc aaagtacaca tatattacga 1080
tgctgttcta ttaaatgctt cctatattat atatatagta atgtcgtgat ctatggtgca 1140
ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac 1200
ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga 1260
ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac 1320
gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt 1380
agacggatcg cttgcctgta acttacacgc gcctcgtatc ttttaatgat ggaataattt 1440
gggaatttac tctgtgttta tttattttta tgttttgtat ttggatttta gaaagtaaat 1500
aaagaaggta gaagagttac ggaatgaaga aaaaaaaata aacaaaggtt taaaaaattt 1560
caacaaaaag cgtactttac atatatattt attagacaag aaaagcagat taaatagata 1620
tacattcgat taacgataag taaaatgtaa aatcacagga ttttcgtgtg tggtcttcta 1680
cacagacaag gtgaaacaat tcggcattaa tacctgagag caggaagagc aagataaaag 1740
gtagtatttg ttggcgatcc ccctagagtc ttttacatct tcggaaaaca aaaactattt 1800
tttctttaat ttcttttttt actttctatt tttaatttat atatttatat taaaaaattt 1860
aaattataat tatttttata gcacgtgatg aaaaggaccc aggtggcact tttcggggaa 1920
atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca 1980
tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc 2040
aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc 2100
acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggacgc gtagtctaga 2160
ccagccagga cagaaatgcc tcgacttcgc tgctacccaa ggttgccggg tgacgcacac 2220
cgtggaaacg gatgaaggca cgaacccagt ggacataagc ctgttcggtt cgtaagctgt 2280
aatgcaagta gcgtatgcgc tcacgcaact ggtccagaac cttgaccgaa cgcagcggtg 2340
gtaacggcgc agtggcggtt ttcatggctt gttatgactg tttttttggg gtacagtcta 2400
tgcctcgggc atccaagcag caagcgcgtt acgccgtggg tcgatgtttg atgttatgga 2460
gcagcaacga tgttacgcag cagggcagtc gccctaaaac aaagttaaac attatgaggg 2520
aagcggtgat cgccgaagta tcgactcaac tatcagaggt agttggcgcc atcgagcgcc 2580
atctcgaacc gacgttgctg gccgtacatt tgtacggctc cgcagtggat ggcggcctga 2640
agccacacag tgatattgat ttgctggtta cggtgaccgt aaggcttgat gaaacaacgc 2700
ggcgagcttt gatcaacgac cttttggaaa cttcggcttc ccctggagag agcgagattc 2760
tccgcgctgt agaagtcacc attgttgtgc acgacgacat cattccgtgg cgttatccag 2820
ctaagcgcga actgcaattt ggagaatggc agcgcaatga cattcttgca ggtatcttcg 2880
agccagccac gatcgacatt gatctggcta tcttgctgac aaaagcaaga gaacatagcg 2940
ttgccttggt aggtccagcg gcggaggaac tctttgatcc ggttcctgaa caggatctat 3000
ttgaggcgct aaatgaaacc ttaacgctat ggaactcgcc gcccgactgg gctggcgatg 3060
agcgaaatgt agtgcttacg ttgtcccgca tttggtacag cgcagtaacc ggcaaaatcg 3120
cgccgaagga tgtcgctgcc ggctgggcaa tggagcgcct gccggcccag tatcagcccg 3180
tcatacttga agctagacag gcttatcttg gacaagaaga agatcgcttg gcctcgcgcg 3240
cagatcagtt ggaagaattt gtccactacg tgaaaggcga gatcaccaag gtagtcggca 3300
aataaccctc gagcattcaa ggcgccttga ttatttgacg tggtttgatg gcctccacgc 3360
acgttgtgat atgtagatga taatcattat cactttacgg gtcctttccg gtgatccgac 3420
aggttacggg gcggcgacct cgcgggtttt cgctatttat gaaaattttc cggtttaagg 3480
cgtttccgtt cttcttcgtc ataacttaat gtttttattt aaaatacctc gcgagtggca 3540
acactgaaaa tacccatgga gcggcgtaac cgtcgcacag gaaggacaga gaaagcgcgg 3600
atctgggaag tgacggacag aacggtcagg acctggattg gggaggcggt tgccgccgct 3660
gctgctgacg gtgtgacgtt ctctgttccg gtcacaccac atacgttccg ccattcctat 3720
gcgatgcaca tgctgtatgc cggtataccg ctgaaagttc tgcaaagcct gatgggacat 3780
aagtccatca gttcaacgga ggtctacacg aaggtttttg cgctggatgt ggctgcccgg 3840
caccgggtgc agtttgcgat gccggagtct gatgcggttg cgatgctgaa acaattatcc 3900
tgagaataaa tgccttggcc tttatatgga aatgtggaac tgagtggata tgctgttttt 3960
gtctgttaaa cagagaagct ggctgttatc cactgagaag cgaacgaaac agtcgggaaa 4020
atctcccatt atcgtagaga tccgcattat taatctcagg agcctgtgta gcgtttatag 4080
gaagtagtgt tctgtcatga tgcctgcaag cggtaacgaa aacgatttga atatgccttc 4140
aggaacaata gaaatcttcg tgcggtgtta cgttgaagtg gagcggatta tgtcagcaat 4200
ggacagaaca acctaatgaa cacagaacca tgatgtggtc tgtcctttta cagccagtag 4260
tgctcgccgc agtcgagcga cagggcgaag ccctcgagtg agcgaggaag caccagggaa 4320
cagcacttat atattctgct tacacacgat gcctgaaaaa acttcccttg gggttatcca 4380
cttatccacg gggatatttt tataattatt ttttttatag tttttagatc ttctttttta 4440
gagcgccttg taggccttta tccatgctgg ttctagagaa ggtgttgtga caaattgccc 4500
tttcagtgtg acaaatcacc ctcaaatgac agtcctgtct gtgacaaatt gcccttaacc 4560
ctgtgacaaa ttgccctcag aagaagctgt tttttcacaa agttatccct gcttattgac 4620
tcttttttat ttagtgtgac aatctaaaaa cttggcacac ttcacatgga tctgtcatgg 4680
cggaaacagc ggttatcaat cacaagaaac gtaaaaatag cccgcgaatc gtccagtcaa 4740
acgacctcac tgaggcggca tatagtctct cccgggatca aaaacgtatg ctgtatctgt 4800
tcgttgacca gatcagaaaa tctgatggca ccctacagga acatgacggt atctgcgaga 4860
tccatgttgc taaatatgct gaaatattcg gattgacctc tgcggaagcc agtaaggata 4920
tacggcaggc attgaagagt ttcgcgggga aggaagtggt tttttatcgc cctgaagagg 4980
atgccggcga tgaaaaaggc tatgaatctt ttccttggtt tatcaaacgt gcgcacagtc 5040
catccagagg gctttacagt gtacatatca acccatatct cattcccttc tttatcgggt 5100
tacagaaccg gtttacgcag tttcggctta gtgaaacaaa agaaatcacc aatccgtatg 5160
ccatgcgttt atacgaatcc ctgtgtcagt atcgtaagcc ggatggctca ggcatcgtct 5220
ctctgaaaat cgactggatc atagagcgtt accagctgcc tcaaagttac cagcgtatgc 5280
ctgacttccg ccgccgcttc ctgcaggtct gtgttaatga gatcaacagc agaactccaa 5340
tgcgcctctc atacattgag aaaaagaaag gccgccagac gactcatatc gtattttcct 5400
tccgcgatat cacttccatg acgacaggat agtctgaggg ttatctgtca cagatttggg 5460
ggtggttcgt cacatttgtt ctgacctact gagggtaatt tgtcacagtt ttgctgtttc 5520
cttcagcctg catggatttt ctcatacttt ttgaactgta atttttaagg aagccaaatt 5580
tgagggcagt ttgtcacagt tgatttcctt ctctttccct tcgtcatgtg acctgatatc 5640
gggggttagt ttgtcatcat tgatgagggt tgattatcac agtttattac tctgaattgg 5700
ctatccgcgt gtgtacctct acctggagtt tttcccacgg tggatatttc ttcttgcgct 5760
gagcgtaaga gctatctgac agaacagttc ttctttgctt cctcgccagt tcgctcgcta 5820
tgctcggtta cacggctgcg gcgagcgcta gtgataataa gtgactgagg tatgtgctct 5880
tcttatctcc ttttgtagtg ttgctcttat tttaaacaac tttgcggttt tttgatgact 5940
ttgcgatttt gttgttgctt tgcagtaaat tgcaagattt aataaaaaaa cgcaaagcaa 6000
tgattaaagg atgttcagaa tgaaactcat ggaaacactt aaccagtgca taaacgctgg 6060
tcatgaaatg acgaaggcta tcgccattgc acagtttaat gatgacagcc cggaggcgag 6120
gaaaataacc cggcgctgga gaataggtga agcagcggat ttagttgggg tttcttctca 6180
ggctatcaga gatgccgaga aagcagggcg actaccgcac ccggatatgg aaattcgagg 6240
acgggttgag caacgtgttg gttatacaat tgaacaaatt aatcatatgc gtgatgtgtt 6300
tggtacgcga ttgcgacgtg ctgaagacgt atttccaccg gtgatcgggg ttgctgccca 6360
taaaggtggc gtttacaaaa cctcagtttc tgttcatctt gctcaggatc tggctctgaa 6420
ggggctacgt gttttgctcg tggaaggtaa cgacccccag ggaacagcct caatgtatca 6480
cggatgggta ccagatcttc atattcatgc agaagacact ctcctgcctt tctatcttgg 6540
ggaaaaggac gatgtcactt atgcaataaa gcccacttgc tggccggggc ttgacattat 6600
tccttcctgt ctggctctgc accgtattga aactgagtta atgggcaaat ttgatgaagg 6660
taaactgccc accgatccac acctgatgct ccgactggcc attgaaactg ttgctcatga 6720
ctatgatgtc atagttattg acagcgcgcc taacctgggt atcggcacga ttaatgtcgt 6780
atgtgctgct gatgtgctga ttgttcccac gcctgctgag ttgtttgact acacctccgc 6840
actgcagttt ttcgatatgc ttcgtgatct gctcaagaac gttgatctta aagggttcga 6900
gcctgatgta cgtattttgc ttaccaaata cagcaatagt aatggctctc agtccccgtg 6960
gatggaggag caaattcggg atgcctgggg aagcatggtt ctaaaaaatg ttgtacgtga 7020
aacggatgaa gttggtaaag gtcagatccg gatgagaact gtttttgaac aggccattga 7080
tcaacgctcc tcaactggtg cctggagaaa tgctctttct atttgggaac ctgtctgcaa 7140
tgaaattttc gatcgcctga ttaaaccacg ctgggagatt agataatgaa gcgtgcgcct 7200
gttattccaa aacatacgct caatactcaa ccggttgaag atacttcgtt atcgacacca 7260
gctgccccga tggtggattc gttaattgcg cgcgtaggag taatggctcg cggtaatgcc 7320
attactttgc ctgtatgtgg tcgggatgtg aagtttactc ttgaagtgct ccggggtgat 7380
agtgttgaga agacctctcg ggtatggcca ggtaatgaac gtgaccagga gctgcttact 7440
gaggacgcac tggatgatct catcccttct tttctactga ctggtcaaca gacaccggcg 7500
ttcggtcgaa gagtatctgg tgtcatagaa attgccgatg ggagtcgccg tcgtaaagct 7560
gctgcactta ccgaaagtga ttatcgtgtt ctggttggcg agctggatga tgagcagatg 7620
gctgcattat ccagattggg taacgattat cgcccaacaa gtgcttatga acgtggtcag 7680
cgttatgcaa gccgattgca gaatgaattt gctggaaata tttctgcgct ggctgatgcg 7740
gaaaatattt cacgtaagat tattacccgc tgtatcaaca ccgccaaatt gcctaaatca 7800
gttgttgctc ttttttctca ccccggtgaa ctatctgccc ggtcaggtga tgcacttcaa 7860
aaagccttta cagataaaga ggaattactt aagcagcagg catctaacct tcatgagcag 7920
aaaaaagctg gggtgatatt tgaagctgaa gaagttatca ctcttttaac ttctgtgctt 7980
aaaacgtcat ctgcatcaag aactagttta agctcacgac atcagtttgc tcctggagcg 8040
acagtattgt ataagggcga taaaatggtg cttaacctgg acaggtctcg tgttccaact 8100
gagtgtatag agaaaattga ggccattctt aaggaacttg aaaagccagc accctgatgc 8160
gaccacgttt tagtctacgt ttatctgtct ttacttaatg tcctttgcta caggccagaa 8220
agcataactg gcctgaatat tctctctggg cccactgttc cacttgtatc gtcggactga 8280
taatcagact gggaccacgg tcccactcgt atcgtcggtc tgattattag tctgggacca 8340
cggtcccact cgtatcgtcg gtctgattat tagtctggga ccacggtccc actcgtatcg 8400
tcggtctgat aatcagactg ggaccacggt cccactcgta tcgtcggtct gattattagt 8460
ctgggaccat ggtcccactc gtatcgtcgg tctgattatt agtctgggac cacggtccca 8520
ctcgtatcgt cggtctgatt attagtctgg aaccacggtc ccactcgtat cgtcagtctg 8580
attattagtc tgggaccacg gtcccactcg tatcgtcggt ctgattatta gtctgggacc 8640
acgatcccac tcgtgttgtc ggtctgatta tcggtctggg accacggtcc cacttgtatt 8700
gtcgatcaga ctatcagcgt gagactacga ttccatcaat gcctgtcaag ggcaagtatt 8760
gacatgtcgt cgtaacctgt agaacggagt aacctcggtg tgcggttgta tgcctgctgt 8820
ggattgctgc tgtgtcctgc ttatccacaa cattttgcgc acggttatgt ggacaaaata 8880
cctggttacc caggccgtgc cggcacgtta accgggctgc atccgatgca agtgtgtcgc 8940
tgtcgacggc ctcctcaccc ggtcacgtga gctcatttaa cccactccac aaaaaggctc 9000
aacaggttgg tggttctcac caccaaaagc accacacccc acgcaaaaac aagtttttgc 9060
tgatttttct ttataaatag agtgttatga aaaattagtt tctcttactc tctttatgat 9120
atttaaaaaa gcggtgtcgg cgcggctaca acaacgcgcc gacaccgttt tgtaggggtg 9180
gtactgacta tttttataaa aaacattatt ttatattagg ggtgctgcta gcggcgcggt 9240
gtgttttttt ataggatacc gctaggggcg ctgctagcgg tgcgtccctg tttgcattat 9300
gaattagtta cgctagggat aacagggtaa tatagaaccc gaacgaccga gcgcagcggc 9360
ggccgcgctg ataccgccgc cgttacataa cttacggtaa atggcccgcc tggctgaccg 9420
cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata 9480
gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta 9540
catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc 9600
gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac 9660
gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga 9720
tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg 9780
ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg 9840
caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctta atacgactca 9900
ctataggaga aaacacagga tctggccggc atggtcccag cctcctcgct ggcgccggct 9960
gggcaacatt ccgaggggac cgtcccctcg gtaatggcga atgggacctg tgccttctag 10020
ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac 10080
tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca 10140
ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag 10200
caggcatgct ggggatgcgg tgggctctat gg 10232
<210> 2
<211> 10965
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
agaagtttat ctgtgtgaac ttcttggctt agtatcgttg agaagaatcg agagattagt 60
gcagtttaaa cagtttttta gaacggaaga acaaccatga ctaaaaaacc aggagggccc 120
ggaaaaaacc gggccatcaa tatgctgaaa cgcggattac cccgcgtatt cccactagtg 180
ggagtgaaga gggtagttat gagcttgttg gacggcagag ggccagtacg atttgtgctg 240
gctcttatca cgttcttcaa gtttacagca ttagccccga ccaaggcgct tttgggccgc 300
tggagagcag tggaaaaaag tgtggcaatg aaacatctta ccagtttcaa acgagaactt 360
ggaacactca ttgacgccgt gaacaagcgg ggcaaaaaac aaaacaaaag aggagggaat 420
gaaagcttga ttatgtggct tgccagcttg gcaatcgtaa cagcctgtgc cggagccatg 480
aagctatcaa actttcaagg aaagcttctg atgaccatca acaacacgga cattgcggat 540
gtcatcgtga tccccacctc aaaaggtgaa aacagatgct gggtacgagc aatcgacgtt 600
ggttacatgt gtgaagacac catcacgtat gaatgtccga agcttgccgt gggcaacgat 660
ccggaagacg tggactgctg gtgcgacaat caggaagtct acgtgcagta tggtcgctgc 720
acacggacca ggcattccaa acgaagcaga agatccgttt cggtccacac gcatggggaa 780
agctcactag tgaacaaaaa agaggcttgg ctggattcaa cgaaggccac gcgatacctc 840
atgaaaacgg agaactggat cataaggaac cctggttatg ctttcctggc ggtggcactt 900
ggatggatgc ttggcagcaa caatggccaa cgtgtggtgt tcactattct cttgctattg 960
gtcgccccgg cttacagttt taactgtctg ggaatgggga atcgggattt catagaagga 1020
gccagtggag ccacttgggt ggatctggtg ttggaaggag atagctgttt gacaattatg 1080
gcaaacgaca aaccaacact agatgtccgc atgatcaaca ttgaagctag ccaacttgct 1140
gaagtcagga gttactgcta tcacgcttca gtcactgaca tttcaacggt ggctcgatgc 1200
cccacgactg gagaagccca caatgaaaaa cgtgctgaca gcagctacgt gtgcaaacaa 1260
ggctttactg accgcggatg gggaaatgga tgtggacttt tcgggaaagg aagcattgac 1320
acatgcgcaa aattttcgtg taccagtaag gccattggaa gaatgatcca accagagaac 1380
atcaagtaca gggttggcat attcgtgcac ggaaccacta cctcggaaaa ccatgggaat 1440
tactcagcgc aagtaggagc gtctcaagca gcaaagttta ctgtaactcc aaatgctcct 1500
tcaataaccc tcaagcttgg tgattatgga gaagtcacac tggattgtga accaaggagt 1560
ggactgaaca ctgaagcgtt ctatgtcatg accgtgggtt cgaagtcatt cttagtccat 1620
agggaatggt tccatgacct ttctcttccc tggacgtccc cctcaagcac ggcatggaga 1680
aacagagaac tcctcatgga atttgaagag gcacatgcca caaaacaatc tgtcgtagct 1740
cttgggtcac aggagggagg cctccatcaa gcgttggcag gagccatcgt ggtggagtac 1800
tcgagctcag tgaaattgac atcaggtcac ctgaaatgca ggctaaaaat ggacaaactg 1860
gctctgaagg gcacgactta tggcatgtgt acagaaaaat tctcgttcgc gaaaaatcca 1920
gcggacacgg gccatggaac agttgtcatt gagctcacat attctggaag tgatggtccc 1980
tgtaaaattc cgattgtctc agtcgctagt ttaaacgaca tgacccctgt ggggaggctg 2040
gtaacagtaa accccttcgt cgcaacatct agctccaact caaaggtgct ggttgagatg 2100
gaacctccct tcggagactc ttatatcgtg gttggaagag gggacaagca gattaaccat 2160
cactggcaca aagctggaag cacgctgggc aaagctttct caacaacttt gaaaggggct 2220
cagagactag cagcgctagg tgacacagcc tgggacttcg gttccattgg aggggtattc 2280
aactccatag ggaaagctgt tcaccaagta tttggcggtg cattcagaac gctctttggg 2340
ggaatgtctt ggatcacaca aggactaatg ggggccttac ttctttggat gggtgtcaac 2400
gcacgagacc ggtcaatcgc cctggctttt ctggccacgg gaggtgtgct cgtgttttta 2460
gcgaccaatg tgcatgccga cactggctgt gccattgaca tcacaagaaa agagatgagg 2520
tgtggaagtg gcatcttcgt gcacaacgac gtagaggctt gggtagatag gtacaaatat 2580
ttgccagaga cgcccagatc cttagcgaag atcgtccaca aagcacatca agaaggagtg 2640
tgcggggtca gatccgtcac tagactggaa caccagatgt gggagtctgt gcgagacgaa 2700
ctgaatgtct tgctcaaaga gaacgcggtg gatctcagtg tggtggtgaa caagcccgtg 2760
gggagatatc gctcagcccc caaacgccta tccatgactc aagaaaagtt tgagatgggc 2820
tggaaagcat ggggaaaaag cattctcttc gcccccgaat tggccaactc cacgttcgtc 2880
gtggatggac ccgagacaaa ggaatgccct gatgagcgca gagcctggaa cagcatgcaa 2940
atcgaagatt tcggcttcgg catcacatca acccgagtgt ggctgaaaat tagagaggag 3000
aacactgatg ggtgtgatgg agcaatcata gggacagctg ttaaagggca tgtggcagtt 3060
catagtgact tgtcatactg ggtcgagagc cgtctcaatg acacctggaa acttgagagg 3120
gctgtcttcg gggaggtgaa atcttgcact tggcccgaga cacacactct ttggggtgac 3180
ggtgttgagg agagcgagct tatcatccca cacaccatag ctggaccgag aagcaagcac 3240
aaccggagag aagggtataa aacacaaaac cagggaccct gggatgagaa cggcatcgtt 3300
cttgactttg actattgtcc aggaacaaaa gtcaccatca cagaggactg tggcaagagg 3360
ggtccctcaa tcagaaccac tactgacagt ggaaagctga tcaccgattg gtgctgccgc 3420
agctgttctc taccgccttt gcggttccgg acagaaaatg gttgctggta tgggatggaa 3480
atcagacctg ttaggcatga cgaaacaaca ctcgttaggt cacaggttga cgctttcaac 3540
ggcgaaatga ttgacccttt tcagctgggc cttctggtga tgtttctggc cacccaggag 3600
gtccttcgca agaggtggac ggccagattg acgattcctg cggttttggg ggctctactt 3660
gtgctgatgc ttgggggcat cacttacact gacctggcaa gatatgtggt gctagttgct 3720
gcggctttcg cggaggccaa cagtggagga gatgttctgc acctcgctct gatagccgtc 3780
ttcaagatcc aaccagcttt tctggtcatg aacatgctta gcgcgagatg gacgaaccaa 3840
gagaacgtgg ttctggtcct gggggcggct tttttccaac tagcttcagt ggatttacag 3900
atcggagtcc acggaatcct gaacgctgca gccatagcat ggatgatcgt tcgagcgatc 3960
acatttccca caacttctac cgttgccatg ccaatcttag cgctcctaac tccgggaatg 4020
agggctttgt atctggacac ttatagaatc attcttcttg tcataggaat ttgttccctg 4080
ctgcaagaga ggagaaagac catggcgaag aagaaaggag ccgtgctctt gggcttagcg 4140
ctcacatcca ccggatggtt ttcgcccacc actatcgcag ccggactaat ggtctgcaac 4200
ccaaacaaga agagggggtg gccagccacc gagttccttt cagcggttgg gttgatgttt 4260
gccattgtgg gaggtctagc cgagttggac atcgaatcta tgtcaatacc cttcatgctg 4320
gcagggctca tggcagtgtc ctacgtggta tcaggaaagg caaccgacat gtggctggac 4380
cgggctgccg atatcagctg ggagatggag gctgcaatca caggaagtag ccggaggcta 4440
gatgttaagt tggatgacga cggcgacttt cacttgattg atgatcccgg tgttccatgg 4500
aaggtctggc ttctgcgcat gtcttgtatc ggtttagccg ctctgacacc ctgggctatc 4560
gttcccgccg cttttggcta ttggctcact ctgaaaacaa caaaaagagg gggcgtgttc 4620
tgggacacgc catctccaaa gccttgctta aagggggaca ccaccacagg agtttaccga 4680
attatggcca gggggattct aggcacttac caggccggag ttggagtcat gtatgagaat 4740
gttttccaca cattgtggca cacaactaga ggagcagcca tcatgagtgg agaaggaaag 4800
ctaacgccat actggggtag tgtgaaggaa gaccgcataa gctatggagg cccgtggagg 4860
tttgaccgga agtggaatgg aacagatgat gtgcaagtga ttgtggtgga accagggaaa 4920
cctgcagtaa acatccagac aaaaccggga gtgtttcgca cccctttcgg ggaagttgga 4980
gcagtcagct tggactaccc acggggaaca tccggctcac ccatcctaga ttccaatgga 5040
gacatcatag gcttgtatgg caatggggtt gaactcggcg atggatcgta tgtcagcgcc 5100
attgtgcagg gcgaccgtca agaggaacca gttccagatg cctacactcc aagcatgctg 5160
aaaaagagac agatgactgt gctggacctg cacccaggtt cgggaaaaac caggaagatc 5220
ctaccccaaa taattaagga cgccatccag cagcgcttga gaacagccgt gctggcaccc 5280
acacgagtgg tagcagcaga aatggcggaa gctttgagag gactcccagt acgatatcaa 5340
acctcggcag tgcagaggga gcatcaggga aatgaaatag tagacgtaat gtgccatgcc 5400
actctgaccc atagactaat gtcaccaaac agggtgccca actacaattt attcgtgatg 5460
gatgaggctc acttcactga cccagctagc atcgccgctc ggggttatat tgcaaccaag 5520
gtggaactgg gggaggcagc agccattttt atgacggcga ccccgcccgg gaccactgat 5580
ccctttcccg actcaaatgc cccaattcat gacctgcagg atgagatccc agacagggca 5640
tggagcagtg gatacgaatg gatcacggac tatgcgggaa aaactgtgtg gttcgtggca 5700
agtgtgaaaa tggggaatga gatcgcaatg tgcctccaaa gagcggggaa aaaggtcatc 5760
cagctcaatc gtaagtcata tgacacagaa tacccaaaat gcaaaaatgg agattgggac 5820
tttgttatca ccactgacat ctctgagatg ggggccaatt ttggtgcgag cagggtcatt 5880
gactgcagaa agagtgtgaa acccaccatc ctagaggagg gagaaggtag agtcattctt 5940
ggaaacccat cccccataac cagtgcaagt gcagcccaac ggagaggtag agtgggcagg 6000
aatcctaacc aagttggaga tgaataccat tacggggggg ccaccagtga agatgacagc 6060
aacctagccc actggacaga ggcaaaaatt atgctagaca acatacatat gcctaatgga 6120
ttagtggctc agctgtacgg gccagagagg gaaaaggctt tcacaatgga tggagagtac 6180
cgactcagag ttgaggagaa gaagaacttc ttggagctgc ttagaacggc tgacctccca 6240
gtatggctag cctacaaggt ggcgtccaat ggcattcagt acactgacag aaaatggtgc 6300
tttgatggac cacgcacaaa tgccatacta gaagataaca ctgaggtgga gatagttacc 6360
cgaatgggtg agagaaagat cctcaagccg agatggctcg atgcgagggt ttatgcagac 6420
caccaggccc tcaagtggtt taaggatttt gcagcgggca agagatcagc cgtcagtttc 6480
atagaggtgc tcggtcgcat gcctgagcat ttcatgggaa agacacggga agccttagac 6540
acaatgtatc tggtggcaac agctgagaaa ggtggaaagg cacaccgcat ggctcttgaa 6600
gaactgcccg acgcattgga gaccatcaca ctcatcgttg ccatcactgt gatgacagga 6660
ggattcttcc tgctcatgat gcagagaaag ggtataggaa aaatgggtct aggggctcta 6720
gtgcttacgc tggctacctt tttcctatgg gcggcagagg ttcctggaac caaaatagcg 6780
ggcaccctac tggtcgccct gttgctaatg gtggtcctca tcccggaacc agaaaaacag 6840
aggtcacaga cagacaacca gttggcagtg tttcttatct gcgtcctgac cgtggtcgga 6900
gtggtggcag caaatgagta cggaatgctg gaaaaaacca aagcagacct taagagcatg 6960
tttggcggaa ggacgcaagc accaggactg accggattgc ctagcatggc actggacttg 7020
cgcccagcca cagcttgggc gctgtatggg gggagcacag ttgtgttaac ccctctcctg 7080
aagcatctaa tcacctcaga atatgtcacc acatcgttag cttcaatcag ttcacaagcg 7140
ggttcgctgt ttgttttgcc gcgaggcgtg cctttcactg acttggatct aaccgttggc 7200
cttgtctttc ttggctgttg gggccaaatc accctcacca cgttcctaac agctatggtg 7260
ctagtgacac tccactatgg atacatgctc cctgggtggc aagcagaggc actcagagct 7320
gctcagagaa gaacagcggc tggcataatg aagaatgccg tcgtggacgg aatggtcgcc 7380
accgatgtgc ccgaactgga aagaaccact cccttgatgc aaaagaaagt cggccaagtg 7440
ctcctcatag gggtcagcgt ggcggcgttt ctcgtcaacc ccaatgtcac caccgtgaga 7500
gaggcaggtg tgttggtgac ggcagccaca ctcaccttgt gggacaatgg ggccagtgcc 7560
gtctggaatt ccaccaccgc tacggggctt tgccatgtca tgcgaggcag ctacctagct 7620
ggcggctcca ttgcctggac tctcatcaag aacgctgaca agccctcctt aaaaaggggg 7680
aggcctggag gcaggacgct aggggagcag tggaaggaaa agttgaatgc tatgagcagg 7740
gatgagttct tcaaatacag aagagaggcc ataattgagg tggaccgcac tgaagcacgc 7800
agggctaggc gcgagaacaa catagtggga ggacacccag tctcgcgagg gtcagcaaag 7860
ctccgctggc tcgtggaaaa aggatttgtc tcgccaatag gaaaagtcat agatctggga 7920
tgcgggcgcg gaggctggag ctactacgca gcaactctga aaaaagttca ggaagtcaaa 7980
gggtacacga aaggtggggc gggacacgaa gaaccgatgc tcatgcagag ttacggttgg 8040
aacctggtct cgttaaagag tggggtggac gtattctaca aaccctcgga gcctagtgac 8100
accctgttct gtgacatagg agaatcttcc ccaagtccag aggtggagga acaacgcacg 8160
ctgcgcgtcc tagaaatgac atctgactgg ttacatcggg gacctagaga gttctgcata 8220
aaagtgctct gcccttatat gcctaaagtt atagaaaaga tggaagtcct gcaacgtcgt 8280
ttcggtggcg ggttggtgcg ccttcccctg tctcgaaact ccaaccatga gatgtactgg 8340
gtgagtggag ctgctggcaa tgtggtgcat gcggtcaaca tgaccagcca agtgctgcta 8400
gggcgaatgg atcgcacagt gtggagaggg ccaaagtacg aagaggatgt caacctgggc 8460
agcgggacga gagctgtggg gaagggagag gtccatagcg accagaaaaa aattaggaag 8520
agaatccaga aacttagaga ggaattcgct acaacctggc acaaagaccc agagcatcca 8580
taccgaactt ggacctatca tggaagctac gaagtgaagg ccactggctc agcaagctct 8640
ctcgtcaatg gggtggtaaa gctcatgagc aaaccctggg acgccatcgc caatgtcacc 8700
acaatggcca tgactgacac cacccccttt ggccaacaga gggtcttcaa agagaaggtt 8760
gacacgaagg ctccagagcc accagcagga gtcaaggaag tgctcaacga gaccaccaac 8820
tggctgtggg cccacttgtc acgggagaaa cgaccccgct tgtgcactaa ggaagaattc 8880
ataaagaaag tcaacagcaa cgcagctctc ggagcagtgt tcgctgaaca aaaccaatgg 8940
agcacggcgc gggaagccgt gggcgaccct ctgttctggg agatggtcaa tgaagaaagg 9000
gaaaaccatt tgcgagggga gtgccacacg tgcgtttaca acatgatggg aaaaagagag 9060
aaaaaacctg gagagttcgg aaaggctaaa gggagtaggg ctatttggtt catgtggctc 9120
ggagctcggt acctagagtt cgaagcccta ggatttctaa atgaagacca ttggctgagc 9180
cgagagaatt caggaggtgg ggtggaaggt tcaggcgtcc aaaagctggg atacattctc 9240
cgtgacatag cagggaagca aggaggtaaa atgtatgccg atgacaccgc cgggtgggac 9300
accagaatca ctagaaccga cttggaaaat gaagccaaag tgctggagct tttggatggt 9360
gaacatcgca tgctcgcccg agccataatt gaactaacgt acaggcacaa agtggtcaag 9420
gttatgaggc ctgcagcagg aggaaagaca gtgatggacg tgatatcacg agaagaccaa 9480
agggggagtg ggcaggtggt gacctacgct ctcaacacat tcacgaacat tgctgtccag 9540
cttgtccgct tgatggaggc tgagggggtc attggaccac aacacttgga acagctgccc 9600
aggaaaaaca aaatagctgt taggacctgg ctttttgaga atggagagga gagagtgact 9660
aggatggcga tcagtggaga cgactgcgtt gtcaagccgc tggatgacag attcgccacg 9720
gctctccatt tcctcaacgc aatgtcgaag gtcagaaaag atatccaaga atggaagcct 9780
tcgcatggtt ggcacgactg gcagcaggtt cccttttgct ccaatcattt tcaggagatt 9840
gtgatgaaag atggaaggag catagtcgtc ccgtgcagag ggcaggatga gctgattggc 9900
agggcgcgca tctccccagg agctggatgg aatgtgaagg acacagcttg cctggccaaa 9960
gcgtatgcac agatgtggct gcttctatac ttccatcgga gggatctacg ccttatggca 10020
aatgcaatct gctcagcagt tccagtggac tgggtgccca caggcagaac atcctggtca 10080
atacactcaa aaggagagtg gatgaccact gaagacatgc tgcaagtctg gaacagggta 10140
tggattgaag aaaatgaatg gatgatggac aagaccccaa tcacaagctg gacagacgtt 10200
ccgtacgtgg gaaagcgtga ggacatctgg tgtggcagtc tcatcggaac gcgatccagg 10260
gcaacatggg ctgagaacat ctacgcggca ataaaccaag tgagggccat cattggaaaa 10320
gaaaattatg ttgattacat gacttccctc agaagatatg aggatgtatt gatccaggag 10380
gatagggtca tttagacatg ataaagtcat gtgtgtaatg tgagacaaga aaatgtgcat 10440
gtggagtcag gccagcaaaa gctgccaccg gatactgagt agacggtgct gcctgcgtct 10500
cagtcccagg aggactgggt taacaaatct gacaacggaa ggtgggaaag ccctcagaac 10560
cgtctcggaa gcaggtccct gctcaccgga agttgaaaga ccaacgtcag gccacaattt 10620
tgtgccactc cgctggggag tgcggcctgc gcagccccag gaggactggg ttaacaaagc 10680
cgttgaggcc cccacggccc aagcctcgtc taagatgcaa tagactaggt gtaaggacta 10740
gaggttagag gagaccccgt ggaaacaaca ttatgcggcc caagccccct cgaagctgta 10800
gaggaggtgg aaggactaga ggttagagga gaccccgcat ttgcatcaaa acagcatatt 10860
gacacctggg aatagactgg gagatcttct gctctatctc aacatcagct actaggcaca 10920
gagcgccgaa gtatgtagct ggtggtgagg aagaacacag gatct 10965
<210> 3
<211> 10977
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
agaagtttat ctgtgtgaac ttcttggctt agtatcgttg agaagaatcg agagattagt 60
gcagtttaaa cagtttttta gaacggaaga taaccatgac taaaaaacca ggagggcccg 120
gtaaaaaccg ggctatcaat atgctgaaac gcggcctacc ccgcgtattc ccactagtgg 180
gagtgaagag ggtagtaatg agcttgttgg acggcagagg gccagtacgt ttcgtgctgg 240
ctcttatcac gttcttcaag tttacagcat tagccccgac caaggcgctt ttaggccgat 300
ggaaagcagt ggaaaagagt gtagcaatga aacatctcac tagtttcaaa cgagaacttg 360
gaacactcat tgacgccgtg aacaagcggg gcagaaagca aaacaaaaga ggaggaaatg 420
aaggctcaat catgtggctc gcgagcttgg cagttgtcat agcttgtgca ggagccatga 480
agttgtcaaa tttccagggg aagcttttga tgaccattaa caacacggac attgcagacg 540
ttatcgtgat tcccacctca aaaggagaga acagatgctg ggtccgggca atcgacgtcg 600
gctacatgtg tgaggacact atcacgtacg aatgtcctaa gcttaccatg ggcaatgatc 660
cagaggatgt ggattgctgg tgtgacaacc aagaagtcta cgtccaatat ggacggtgca 720
cgcggaccag gcattccaag cgaagcagga gatccgtgtc ggtccaaaca catggggaga 780
gttcactagt gaataaaaaa gaggcttggc tggattcaac gaaagccaca cgatatctca 840
tgaaaactga gaactggatc ataaggaatc ctggctatgc tttcctggcg gcggtacttg 900
gctggatgct tggcagtaac aacggtcaac gcgtggtatt caccatcctc ctgctgctgg 960
tcgctccggc ttacagtttt aattgtctgg gaatgggcaa tcgtgacttc atagaaggag 1020
ccagtggagc cacttgggtg gacttggtgc tagaaggaga tagctgcttg acaattatgg 1080
caaacgacaa accaacattg gacgtccgca tgatcaacat cgaagctagc caacttgctg 1140
aggtcagaag ttactgttat catgcttcag tcactgacat ctcgacggtg gctcggtgcc 1200
ccacgactgg agaagcccac aacgagaagc gagctgatag tagctatgtg tgcaaacaag 1260
gcttcactga tcgtgggtgg ggcaacggat gtggactttt cgggaaggga agcattgaca 1320
catgtgcaaa attctcctgc accaggaaag cgattgggag aacaatccag ccagaaaaca 1380
tcaaatacga agttggcatt tttgtgcatg gaaccaccac ttcggaaaac catgggaatt 1440
attcagcgca agttggggcg tcccaggcgg caaagtttac agtaacaccc aatgctcctt 1500
cgacaaccct caaacttggt gactacggag aagtcacact ggactgtgag ccaaggagtg 1560
gactgaacac tgaagcgttt tacgtcatga ccgtggggtc aaagtcattt ctggtccata 1620
gggaatggtt tcatgacctc gctctcccct ggacgtcccc ttcgagcaca gcgtggagaa 1680
acagagaact cctcatggag tttgaagggg cgcacgccac aaaacagtcc gttgttgctc 1740
ttgggtcaca ggaaggaggc ctccatcagg cgttggcagg agccatcgtg gtggagtact 1800
caagctcagt gaagttaaca tcaggccacc tgaaatgtag gctgaaaatg gacaaactgg 1860
ctctgaaagg cacaacctat ggcatgtgca cagaaaaatt ctcgttcgcg aaaaatccgg 1920
cggacactgg tcacggaaca gttgtcattg aactctccta ctctgggagt gatggcccct 1980
gcaaaattcc gattgtctcc gttgcgagcc tcaatgacat gacccccgtt gggcggctgg 2040
tgacagtgaa ccccttcgtc gcgacttcca gtgccaattc aaaggtgctg gtcgagatgg 2100
aacccccctt cggagactcc tacatcgtag ttggaagggg agacaagcag atcaaccacc 2160
attggcacaa agctggaagc acgctgggca aagccttttt aacaactctg aagggagctc 2220
agagactggc agcgttgggt gacacagcct gggactttgg ctccattgga ggggtcttca 2280
actccatagg aaaagccgtt caccaagtgt ttggtggtgc cttcagagca ctctttgggg 2340
gaatgtcttg gatcacacaa gggctaatgg gtgccctact actctggatg ggcgtcaacg 2400
cacgagaccg atcaattgct ttggccttct tagccacagg aggtgtgctc gtgttcttag 2460
cgaccaatgt gcatgctgac actggatgtg ccattgacat cacaagaaaa gagatgaggt 2520
gtggaagtgg catcttcgtg cacaacgacg tggaagcctg ggtggatagg tataaatatt 2580
tgccagaaac gcccagatcc ctagcgaaga tcgtccacaa agcgcacaag gaaggcgtgt 2640
gcggagtcag atctgtcact agactggagc atcaaatgtg ggaagccgta cgggatgaat 2700
tgaacgtcct gctcaaagag aatgcagtgg acctcagtgt ggtcgtgaac aagcccgtgg 2760
ggagatatcg ctcagcccct aaacgcctat ccatgacgca agagaagttt gaaatgggct 2820
ggaaagcatg gggaaaaagc attctctttg ccccggaatt ggctaactcc acatttgtcg 2880
tagatggacc tgagacaaag gaatgccctg atgagcacag agcttggaac agcatgcaaa 2940
tcgaagactt cggctttggc atcacatcaa cccgtgtgtg gctgaaaatt agagaggaga 3000
gcactgacga gtgtgatgga gcgatcatag gtacggctgt caaaggacat gtggcagtcc 3060
atagtgactt gtcgtactgg attgagagtc gctacaacga cacatggaaa cttgagaggg 3120
cagtctttgg agaggttaaa tcttgcactt ggccagagac acacacccta tggggagatg 3180
gtgttgagga aagtgaactc atcattccgc ataccatagc cggaccaaaa agcaagcaca 3240
atcggaggga agggtataag acacaaaacc agggaccttg ggacgagaat ggcatagtct 3300
tggactttga ctattgccca gggacaaaag tcaccattac agaggattgt ggcaagagag 3360
gcccttcggt cagaaccact actgacagtg gaaagttgat cactgactgg tgctgtcgca 3420
gttgctccct tccgccccta cgattccgga cagaaaatgg ctgctggtac ggaatggaaa 3480
tcagacctgt taggcatgat gaaacaacac tcgtcagatc gcaggttgat gcttttaatg 3540
gtgaaatggt tgaccctttt cagctgggcc ttctggtgat gtttctggcc acccaggagg 3600
tccttcgcaa gaggtggacg gccagattga ccattcctgc ggttttgggg gccctacttg 3660
tgctgatgct tgggggcatc acttacactg atttggcgag gtatgtggtg ctagtcgctg 3720
ctgctttcgc agaggccaac agtggaggag acgtcctgca ccttgctttg attgccgttt 3780
ttaagatcca accagcattt ttagtgatga acatgcttag cacgagatgg acgaaccaag 3840
aaaacgtggt tctggtccta ggggctgcct ttttccaatt ggcctcagta gatctgcaaa 3900
taggagttca cggaatcctg aatgccgccg ctatagcatg gatgattgtc cgggcgatca 3960
ccttccccac aacctcctcc gtcaccatgc cagtcttagc gcttctaact ccgggaatga 4020
gggctctata cctagatact tacagaatca tcctcctcgt catagggatt tgctctctgc 4080
tgcaagagag gaaaaagacc atggcaaaaa agaaaggagc tgtactcttg ggcttagcgc 4140
tcacatccac tggatggttt tcgcccacca ctatagctgc cggactaatg gtctgcaacc 4200
caaacaagaa gagagggtgg ccagctactg agtttttgtc ggcagttgga ttgatgtttg 4260
ccatcgtagg tggtttggcg gagttggata ttgaatccat gtcaataccc ttcatgctgg 4320
caggtctcat ggcagtgtcc tacgtggtgt caggaaaagc aacagatatg tggcttgaac 4380
gggccgccga catcagctgg gagatggatg ctgcaatcac aggaagcagt cggaggctgg 4440
atgtgaagct ggatgatgac ggagattttc acttgattga tgatcccggt gttccatgga 4500
aggtctgggt cctgcgcatg tcttgcattg gcttagccgc cctcacgcct tgggccattg 4560
ttcccgccgc ttttggttat tggctcactt taaaaacaac aaaaagaggg ggcgtgtttt 4620
gggacacgcc atccccaaaa ccttgctcaa aaggagacac cactacagga gtttaccgca 4680
ttatggctag agggattctt ggcacttacc aggccggcgt cggagtcatg tacgagaatg 4740
ttttccacac actatggcac acaactagag gagcagccat tatgagtgga gaaggaaaat 4800
tgacgccata ctggggtagt gtgaaagaag accgcatagc ttacggaggc ccatggaggt 4860
ttgatcgaaa atggaatgga acagatgacg tgcaagtgat cgtggtagaa ccggggaagg 4920
ctgcagtaaa catccagaca aaaccaggag tgtttcggac tcccttcggg gaggttgggg 4980
ctgttagtct ggattacccg cgaggaacat ccggctcacc cattctggat tccaatggag 5040
acatcatagg cctgtacggc aatggagttg agcttggcga tggctcatac gtcagcgcca 5100
tcgtgcaggg tgaccgtcag gaggaaccag tcccagaagc ttacacccca aacatgttga 5160
gaaagagaca gatgactgta ctagatttgc accctggttc agggaaaacc aggaaaattc 5220
tgccacaaat aattaaggac gctatccagc agcgcctaag aacagctgtg ttggcaccga 5280
cgcgggtggt agcagcagaa atggcagaag ctttgagagg gctcccagta cgatatcaaa 5340
cttcagcagt gcagagagag caccaaggga atgaaatagt ggatgtgatg tgccacgcca 5400
ctctgaccca tagactgatg tcaccgaaca gagtgcccaa ctacaaccta tttgtcatgg 5460
atgaagctca tttcaccgac ccagccagta tagccgcacg aggatacatt gctaccaagg 5520
tggaattagg ggaggcagca gccatcttta tgacagcgac cccgcctgga accacggatc 5580
cttttcctga ctcaaatgcc ccaatccatg atttgcaaga tgagatacca gacagggcgt 5640
ggagcagtgg atacgaatgg atcacagaat atgcgggaaa gaccgtgtgg tttgtggcaa 5700
gcgtaaaaat ggggaatgag attgcaatgt gcctccaaag agcggggaaa aaggtcatcc 5760
aactcaaccg caagtcctat gacacagaat acccaaaatg taagaatgga gactgggatt 5820
ttgtcatcac caccgacatc tctgaaatgg gggccaactt cggtgcgagc agggtcatcg 5880
actgtagaaa gagcgtgaag cccaccatct tagaagaggg agaaggcaga gtcatcctcg 5940
gaaacccatc tcccataacc agtgcaagcg cagctcaacg gaggggcaga gtaggcagaa 6000
accccaacca ggttggagat gaataccact atgggggggc caccagtgaa gatgacggta 6060
acctagccca ttggacagag gcaaagatca tgttagacaa catacacatg cccaatggac 6120
tggtggccca gctctatgga ccagagaggg aaaaggcctt cacaatggat ggcgaatacc 6180
gtctcagagg cgaagaaaag aaaaacttct tagagctgct taggacggct gacctcccgg 6240
tgtggctggc ctacaaggtg gcgtccaatg gcattcagta caccgacaga aagtggtgtt 6300
ttgatgggcc gcgtacgaat gccatactgg aggacaacac cgaggtagag atagtcaccc 6360
ggatgggtga gaggaaaatc ctcaagccga gatggcttga tgcaagagtt tatgcagatc 6420
accaagccct caagtggttc aaagacttcg cagcaggaaa gagatcagcc gttagcttca 6480
tagaggtgct cggtcgtatg cctgagcatt tcatgggaaa gacgcgggaa gctttagaca 6540
ccatgtactt ggttgcaacg gctgagaaag gtgggaaagc acaccgaatg gctctcgaag 6600
agctgccaga tgcactggaa accattacac ttattgttgc tatcactgtg atgacaggag 6660
gattctttct actcatgatg cagcgaaagg gtatagggaa gatgggtctt ggagctctag 6720
tgctcacgct agctaccttc ttcctgtggg cggcagaggt tcctggaacc aaaatagcag 6780
ggaccctgct gatcgccctg ctgcttatgg tggttctcat cccagaaccg gaaaaacaga 6840
ggtcacagac agataaccaa ctggcggtgt ttctcatctg tgtcttgacc gtggttggag 6900
tggtggcagc aaacgagtac gggatgctag aaaaaaccaa agcagacctc aagagcatgt 6960
ttggcggaaa gacgcaggca tcaggactga ctggattgcc aagcatggca ctggacctgc 7020
gtccagccac agcttgggca ctgtatgggg ggagcacagt cgtgctaacc cctcttctga 7080
agcacctgat cacgtcggaa tacgtcacca catcgctagc ctcaattaac tcacaagctg 7140
gctcattatt tgtcttgcca cgaggcgtgc cttttaccga cctagacttg accgttggcc 7200
tcgtcttcct tggctgttgg ggtcaaatca ccctcacaac gtttttgaca gccatggttc 7260
tggcgacact tcactatggg tacatgctcc ctggatggca agcagaagca ctcagggctg 7320
cccagagaag gacagcggct ggaataatga agaatgccgt tgttgacgga atggtcgcca 7380
ctgatgtgcc tgaactggaa aggaccactc ctctgatgca aaagaaagtt ggacaggtgc 7440
tcctcatagg ggtaagcgtg gcagcgttcc tcgtcaaccc caatgtcacc actgtgagag 7500
aagcaggggt gttggtgacg gcggctacgc tcactttgtg ggacaatgga gccagtgccg 7560
tttggaattc caccactgcc acgggactct gccatgtaat gcgaggtagc tacctggctg 7620
gaggctccat tgcttggact ctcatcaaga acgctgacaa gccctccttg aaaaggggaa 7680
ggcctggggg caggacgcta ggggagcagt ggaaggaaaa actaaatgcc atgagcagag 7740
aagagttttt taaataccgg agagaggcca taatcgaggt ggaccgcact gaagcacgca 7800
gggctagacg tgaaaataac atagtgggag gacatccggt ttcgcgaggc tcagcaaaac 7860
tccgttggct cgtggagaaa ggatttgtct cgccaatagg aaaagtcatt gatctagggt 7920
gtgggcgtgg aggatggagc tactacgcag caaccctgaa gaaggtccag gaagtcagag 7980
gatacacgaa aggtggggcg ggacatgaag aaccgatgct catgcagagc tacggctgga 8040
acctggtctc cctgaagagt ggagtggacg tgttttacaa accttcagag tccagtgaca 8100
ctctgttctg cgacataggg gaatcctccc caagtccaga agtagaagaa caacgcacac 8160
tacgcgtcct agagatgaca tctgactggt tgcaccgagg acctagagag ttctgcataa 8220
aagttctttg cccctacatg cccaaggtta tagaaaaaat ggaagttctg cagcgccgct 8280
tcggaggtgg gctagtgcgt ctccccctgt cccgcaactc caatcacgag atgtattggg 8340
ttagtggagc cgctggcaat gtggtgcacg ctgtgaacat gaccagccag gtactactgg 8400
ggcgaatgga tcgcacagtg tggagagggc caaagtatga ggaagatgtc aacctaggga 8460
gcggaacaag agccgtggga aagggagaag tccatagcaa tcaggagaaa atcaagaaga 8520
gaatccagaa gcttaaagaa gaattcgcca caacgtggca caaagaccct gagcatccat 8580
accgcacttg gacataccac ggaagctatg aagtgaaggc tactggctca gctagctctc 8640
tcgtcaacgg agtggtgaag ctcatgagca agccttggga cgccattgcc aacgtcacca 8700
ccatggccat gactgacacc accccttttg gacagcaaag agttttcaag gagaaagttg 8760
acacgaaggc tcctgagcca ccagctggag ccaaggaagt gctcaacgag accaccaact 8820
ggctgtgggc ctacttgtca cgggaaaaaa gaccccgctt gtgcaccaag gaagaattca 8880
taaagaaagt caatagcaac gcggctcttg gagcagtgtt cgctgaacag aatcaatgga 8940
gcacggcgcg tgaggctgtg gatgacccgc ggttttggga gatggttgat gaagagaggg 9000
aaaaccatct gcgaggagag tgtcacacat gtatctataa catgatggga aaaagagaga 9060
agaagcctgg agagttcgga aaagctaaag gaagcagggc catttggttc atgtggcttg 9120
gagcacggta tctagagttt gaagctttgg ggttcctgaa tgaagaccat tggctgagcc 9180
gagagaattc aggaggtgga gtggaaggct caggcgtcca aaagctggga tacatcctcc 9240
gtgatatagc aggaaagcaa ggagggaaaa tgtacgctga tgataccgcc gggtgggaca 9300
ctagaattac cagaactgat ttagaaaatg aagctaaggt gctggagctt ctagacggtg 9360
aacaccgcat gctcgcccga gccataattg aattgactta caggcacaaa gtggtcaagg 9420
tcatgagacc tgcagcagaa ggaaagaccg tgatggacgt gatatcaaga gaagatcaaa 9480
gggggagtgg acaggtggtc acttatgctc ttaacacttt cacgaacatc gctgtccagc 9540
tcgtcaggct gatggaggct gagggggtca ttggaccaca acacttggaa cagctaccta 9600
gaaaaaacaa gatagctgtc aggacctggc tctttgagaa tggagaggag agagtgacca 9660
ggatggcgat cagcggagac gactgtgtcg tcaagccgct ggacgacaga ttcgccacgg 9720
ccctccactt cctcaacgca atgtcaaagg tcagaaaaga catccaggaa tggaagcctt 9780
cgcatggctg gcacgattgg cagcaagttc ccttctgctc taaccatttt caggagattg 9840
tgatgaaaga tggaaggagt atagttgtcc cgtgcagagg acaggatgag ctgataggca 9900
gggctcgcat ctccccagga gctggatgga atgtgaagga cacagcttgt ctggccaaag 9960
catatgcaca gatgtggcta ctcctatact tccatcgtag ggacttgcgt ctcatggcaa 10020
atgcgatttg ctcagcagtg ccagtggatt gggtgcccac gggcaggaca tcctggtcga 10080
tacactcgaa aggagagtgg atgaccacag aagacatgct gcaggtctgg aacagagtct 10140
ggattgaaga aaatgaatgg atgatggaca agactccaat cacaagctgg acagacgttc 10200
cgtacgtggg aaagcgtgag gacatctggt gtggcagcct cattggaacg cgatccagag 10260
caacctgggc tgagaacatc tacgcggcga taaaccaggt tagagctgtc attgggaaag 10320
aaaattatgt tgactacatg acctcactca ggagatacga agacgtcttg atccaggaag 10380
acagggtcat ctagtgtgat ttaaggtaga aaagtagact atgtaagtaa tgtaaatgag 10440
aaaatgcata catatggagt caggccagca aaagctgcca ccggatactg ggtagacggt 10500
gctgcctgcg tctcagtccc aggaggactg ggttaacaaa tctgacaaca gaaagtgaga 10560
aagccctcag aaccgtctcg gaagcaggtc cctgctcact ggaagttgaa ggaccaacgt 10620
caggccacaa atttgtgcca ctccgctggg gagtgcggcc tgcgcagccc caggaggact 10680
gggttaccaa agccgttgag gcccccacgg cccaagcctc gtctaggatg caatagacga 10740
ggtgtaagga ctagaggtta gaggagaccc cgtggaaaca acaacatgcg gcccaagccc 10800
cctcgaagct gtagaggagg tggaaggact agaggttaga ggagaccccg catttgcatc 10860
aaacagcata ttgacacctg ggaatagact gggagatctt ctgctctatc tcaacatcag 10920
ctactaggca cagagcgccg aagtatgtag ctggtggtga ggaagaacac aggatct 10977
<210> 4
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
aacacaggat ctggccggca tggtcccagc ctcctcgctg 40
<210> 5
<211> 51
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
cttctcctat agtgagtcgt attaagctct gcttatatag acctcccacc g 51
<210> 6
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
cagagcttaa tacgactcac tataggagaa gtttatctgt gtgaacttct 50
<210> 7
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
ttgtgtgatc caagacattc ccccaaagag 30
<210> 8
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
ctctttgggg gaatgtcttg gatcacacaa 30
<210> 9
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
tggaacaccg ggatcatcaa tcaagtgaaa 30
<210> 10
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
tttcacttga ttgatgatcc cggtgttcca 30
<210> 11
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
ggcttgtcag cgttcttgat gagagtcca 29
<210> 12
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
tggactctca tcaagaacgc tgacaagcc 29
<210> 13
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
gaccatgccg gccagatcct gtgttcttcc tcaccaccag 40
Claims (1)
1.一种基因I型与基因III型日本脑炎病毒感染性克隆的构建方法，其特征在于，包括如下步骤：
（1）构建线性化载体TAR vector：利用同源重组在pYES1L载体上插入一段携带有T7启动子、丁型肝炎病毒核酶和牛生长激素基因多聚腺苷酸加尾序列的核苷酸序列，获得的重组载体TAR vector；利用PCR方法扩增TAR vector载体，得到线性化的TAR vector；
（2）构建分别携带基因I型与基因III型JEV基因组全长的重组质粒：获得基因I型JEV的cDNA与基因III型JEV的 cDNA，分别以基因I型与基因III型JEV 的cDNA为模板，以JEV-F1-F和JEV-F1-R、JEV-F2-F和JEV-F2-R、JEV-F3-F和JEV-F3-R以及JEV-F4-F和JEV-F4-R为为引物，通过PCR扩增分别得到四个基因片段；
（3）将步骤（2）得到的基因I型与基因III型JEV 的四个基因片段分别与线性化载体TARvector混合，将混合物转化酵母感受态细胞MaV203，然后将转化后的酵母感受态细胞涂布于色氨酸缺陷的酵母培养板，培养2-3天，对得到的转化子进行验证，得到含有基因I型JEV的cDNA的TAR-rGI重组质粒与含有基因III型JEV的cDNA的TAR-rGIII重组质粒，即基因I型与基因III型日本脑炎病毒感染性克隆；
步骤（1）中，所述的重组载体TAR vector的构建方法如下：利用SEQ ID NO.：1所示的线性化片段与线性化pYES1L载体进行同源重组，得到重组载体TAR vector；
步骤（1）中，所述利用PCR方法扩增TAR vector载体，其使用的引物如SEQ ID NO.：4~5所示；
步骤（2）中，所述的基因I型JEV的cDNA的核苷酸序列如SEQ ID NO.：2所示；
步骤（2）中，所述基因III型JEV的 cDNA的核苷酸序列如SEQ ID NO.：3所示；
步骤（2）中，所述JEV-F1-F和JEV-F1-R的序列如SEQ ID NO.：6~7所示；
所述JEV-F2-F和JEV-F2-R的序列如SEQ ID NO.：8~9所示；
所述JEV-F3-F和JEV-F3-R的序列如SEQ ID NO.：10~11所示；
所述JEV-F4-F和JEV-F4-R的序列如SEQ ID NO.：12~13所示。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111276598.3A CN114395579B (zh) | 2021-10-29 | 2021-10-29 | 一种基因i型与基因iii型日本脑炎病毒感染性克隆及其构建方法与应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111276598.3A CN114395579B (zh) | 2021-10-29 | 2021-10-29 | 一种基因i型与基因iii型日本脑炎病毒感染性克隆及其构建方法与应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114395579A CN114395579A (zh) | 2022-04-26 |
| CN114395579B true CN114395579B (zh) | 2024-04-19 |
Family
ID=81225348
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111276598.3A Active CN114395579B (zh) | 2021-10-29 | 2021-10-29 | 一种基因i型与基因iii型日本脑炎病毒感染性克隆及其构建方法与应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114395579B (zh) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115725622A (zh) * | 2022-09-19 | 2023-03-03 | 扬州大学 | 一株表达asfv p30蛋白的重组gi型日本脑炎病毒及其构建方法与应用 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2001299355A (ja) * | 2000-04-27 | 2001-10-30 | National Institute Of Infectious Diseases | 日本脳炎ウイルス遺伝子をコードするｃＤＮＡを含む発現ベクターおよびそれを用いたワクチン |
| CN103088049A (zh) * | 2012-12-26 | 2013-05-08 | 中国人民解放军军事医学科学院生物工程研究所 | 日本脑炎病毒sa14-14-2株基于dna的感染性克隆、其构建方法及应用 |
| CN111206003A (zh) * | 2020-02-20 | 2020-05-29 | 扬州大学 | 弱后酸化瑞士乳杆菌sh2-5-66及其应用 |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2952724A1 (en) * | 2014-06-20 | 2015-12-23 | Inserm (Institut National De La Sante Et De La Recherche Medicale) | Method for rapid generation of an infectious rna virus |
-
2021
- 2021-10-29 CN CN202111276598.3A patent/CN114395579B/zh active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2001299355A (ja) * | 2000-04-27 | 2001-10-30 | National Institute Of Infectious Diseases | 日本脳炎ウイルス遺伝子をコードするｃＤＮＡを含む発現ベクターおよびそれを用いたワクチン |
| CN103088049A (zh) * | 2012-12-26 | 2013-05-08 | 中国人民解放军军事医学科学院生物工程研究所 | 日本脑炎病毒sa14-14-2株基于dna的感染性克隆、其构建方法及应用 |
| CN111206003A (zh) * | 2020-02-20 | 2020-05-29 | 扬州大学 | 弱后酸化瑞士乳杆菌sh2-5-66及其应用 |
Non-Patent Citations (4)
| Title |
|---|
| Hao Zheng等.A simple method for developing an infectious cDNA clone of Japanese encephalitis virus.Virus Genes.2017,第53卷摘要，对比文件1第5页右栏最后一段第6页左栏第一段，图1，第6页右栏最后一段第7页左栏第一段. * |
| Jefferson José da Silva Santos等.Construction and characterisation of a complete reverse genetics system of dengue virus type 3.Mem Inst Oswaldo Cruz..2013,第108卷(第8期),摘要，第988页左栏第2段. * |
| 冷生玲 ; 黄荣 ; 冯亚岚 ; 唐丽萍 ; 周仲辉 ; 陈大斌 ; 杨健 ; .乙脑病毒野毒株SA14感染性克隆的构建及病毒拯救.中国病原生物学杂志.2018,(第09期),全文. * |
| 王晓杜 ; 赵凡凡 ; 代兵 ; 邵东华 ; 蒋春英 ; 周圻 ; 马志永 ; .猪日本乙型脑炎病毒DNA复制子载体的构建及外源基因表达分析.畜牧兽医学报.2015,(第01期),全文. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114395579A (zh) | 2022-04-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2015100929A1 (zh) | 利用CRISPR/Cas9系统构建真核基因敲除文库的方法 | |
| Becker | Cancer a Comprehensive Treatise 2: Etiology: Viral Carcinogenesis | |
| KR20190098147A (ko) | 신생항원의 바이러스성 전달 | |
| CN110295149B (zh) | 一种突变株3型鸭甲肝病毒ch-p60-117c株及构建方法 | |
| CN112029736B (zh) | 一种预防非洲猪瘟重组伪狂犬病病毒活疫苗及其制备方法 | |
| CN112961219B (zh) | 重组腺相关病毒、其突变体及其构建方法和应用 | |
| CN108949794B (zh) | 一种tale表达载体及其快速构建方法和应用 | |
| CN114395579B (zh) | 一种基因i型与基因iii型日本脑炎病毒感染性克隆及其构建方法与应用 | |
| CN113293145A (zh) | 一种麻疹病毒活载体新冠疫苗 | |
| Yokomizo et al. | Rabies virus glycoprotein expression in Drosophila S2 cells. I. Functional recombinant protein in stable co‐transfected cell line | |
| KR20230046313A (ko) | 다중에피토프 백신 카세트 | |
| CN109321571A (zh) | 一种利用CRISPR/Cas9制备重组猪伪狂犬病毒的方法 | |
| CN101260408B (zh) | 一种双色荧光报告载体的构建方法和应用 | |
| Bishop et al. | The molecular biology of RNA tumor viruses | |
| CN110295180B (zh) | 一种3型鸭甲肝病毒突变基因isa-a117c-c4334a及构建方法 | |
| CN109055375A (zh) | 一种crispr辅助反式增强子激活基因表达的方法及其应用 | |
| CN110684781B (zh) | 一种3型鸭甲肝病毒突变基因isa-a117c-t1142a及构建方法 | |
| CN109897825A (zh) | 一种简单高效产生乙型肝炎病毒重组cccDNA的细胞系统 | |
| CN114395017B (zh) | SARS-CoV-2病毒样颗粒的制备方法及其应用 | |
| CN114591922A (zh) | 一种表达狂犬病毒g蛋白的重组黑猩猩源腺病毒及其制备方法 | |
| CN109371167A (zh) | 用于检测CRISPR/Cas9基因编辑系统切割基因产生移码突变的基因元件及应用 | |
| CN110484546B (zh) | 一种3型鸭甲肝病毒突变基因isa-c4334a及构建方法 | |
| CN110283835B (zh) | 一种3型鸭甲肝病毒突变基因isa-t1142a-c4334a及构建方法 | |
| CN117025675B (zh) | 一种提高Admax重组腺病毒包装系统的外源基因表达量的方法及应用 | |
| CN110295148A (zh) | 一种快速构建3型鸭甲肝病毒反向遗传株的方法 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |