CN111733170A - 一种表达荧光素酶的重组犬麻疹病毒 - Google Patents
一种表达荧光素酶的重组犬麻疹病毒 Download PDFInfo
- Publication number
- CN111733170A CN111733170A CN202010618800.5A CN202010618800A CN111733170A CN 111733170 A CN111733170 A CN 111733170A CN 202010618800 A CN202010618800 A CN 202010618800A CN 111733170 A CN111733170 A CN 111733170A
- Authority
- CN
- China
- Prior art keywords
- nluc
- virus
- measles virus
- nucleic acid
- rcmv
- 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.)
- Pending
Links
- 241000282465 Canis Species 0.000 title claims abstract description 16
- 241000712079 Measles morbillivirus Species 0.000 title claims abstract description 15
- 108060001084 Luciferase Proteins 0.000 title claims abstract description 14
- 239000005089 Luciferase Substances 0.000 title claims abstract description 14
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 21
- 150000007523 nucleic acids Chemical group 0.000 claims abstract description 16
- 238000003776 cleavage reaction Methods 0.000 claims abstract description 8
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 8
- 229910052698 phosphorus Inorganic materials 0.000 claims abstract description 8
- 230000007017 scission Effects 0.000 claims abstract description 8
- 238000011144 upstream manufacturing Methods 0.000 claims abstract description 8
- 101150084044 P gene Proteins 0.000 claims abstract description 3
- 102000004169 proteins and genes Human genes 0.000 claims description 4
- 230000003834 intracellular effect Effects 0.000 claims 1
- 241000700605 Viruses Species 0.000 abstract description 26
- 239000000758 substrate Substances 0.000 abstract description 7
- HTBLMRUZSCCOLL-UHFFFAOYSA-N 8-benzyl-2-(furan-2-ylmethyl)-6-phenylimidazo[1,2-a]pyrazin-3-ol Chemical compound OC1=C(CC2=CC=CO2)N=C2N1C=C(N=C2CC1=CC=CC=C1)C1=CC=CC=C1 HTBLMRUZSCCOLL-UHFFFAOYSA-N 0.000 abstract description 5
- 238000000034 method Methods 0.000 abstract description 5
- 238000005516 engineering process Methods 0.000 abstract description 2
- 230000002068 genetic effect Effects 0.000 abstract description 2
- 238000003780 insertion Methods 0.000 abstract description 2
- 230000037431 insertion Effects 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 20
- 238000003757 reverse transcription PCR Methods 0.000 description 8
- 108700026244 Open Reading Frames Proteins 0.000 description 7
- 239000002299 complementary DNA Substances 0.000 description 7
- 239000013612 plasmid Substances 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 6
- 208000015181 infectious disease Diseases 0.000 description 4
- 238000004020 luminiscence type Methods 0.000 description 4
- 230000000174 oncolytic effect Effects 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- 239000012124 Opti-MEM Substances 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000012921 fluorescence analysis Methods 0.000 description 3
- 230000008014 freezing Effects 0.000 description 3
- 238000007710 freezing Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 108020004707 nucleic acids Proteins 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
- 244000309459 oncolytic virus Species 0.000 description 3
- 238000010257 thawing Methods 0.000 description 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 101001065501 Escherichia phage MS2 Lysis protein Proteins 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 241000712045 Morbillivirus Species 0.000 description 2
- 241000711504 Paramyxoviridae Species 0.000 description 2
- 101710172711 Structural protein Proteins 0.000 description 2
- 230000000120 cytopathologic effect Effects 0.000 description 2
- 239000013613 expression plasmid Substances 0.000 description 2
- 210000001165 lymph node Anatomy 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 210000002741 palatine tonsil Anatomy 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- APKFDSVGJQXUKY-KKGHZKTASA-N Amphotericin-B Natural products O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1C=CC=CC=CC=CC=CC=CC=C[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-KKGHZKTASA-N 0.000 description 1
- 241000712083 Canine morbillivirus Species 0.000 description 1
- 102000053642 Catalytic RNA Human genes 0.000 description 1
- 108090000994 Catalytic RNA Proteins 0.000 description 1
- 241000252203 Clupea harengus Species 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000282324 Felis Species 0.000 description 1
- 108700007698 Genetic Terminator Regions Proteins 0.000 description 1
- 108060003393 Granulin Proteins 0.000 description 1
- 208000037262 Hepatitis delta Diseases 0.000 description 1
- 241000724709 Hepatitis delta virus Species 0.000 description 1
- 101150062031 L gene Proteins 0.000 description 1
- 239000012097 Lipofectamine 2000 Substances 0.000 description 1
- 241000282339 Mustela Species 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 206010058874 Viraemia Diseases 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- APKFDSVGJQXUKY-INPOYWNPSA-N amphotericin B Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/C=C/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-INPOYWNPSA-N 0.000 description 1
- 229960003942 amphotericin b Drugs 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 238000005415 bioluminescence Methods 0.000 description 1
- 230000029918 bioluminescence Effects 0.000 description 1
- 230000002051 biphasic effect Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 239000012930 cell culture fluid Substances 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 238000012761 co-transfection Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 239000012531 culture fluid Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000001079 digestive effect Effects 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 108091006047 fluorescent proteins Proteins 0.000 description 1
- 102000034287 fluorescent proteins Human genes 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 235000019514 herring Nutrition 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000005210 lymphoid organ Anatomy 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 210000004779 membrane envelope Anatomy 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 210000004877 mucosa Anatomy 0.000 description 1
- 208000020470 nervous system symptom Diseases 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 108091092562 ribozyme Proteins 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
Images
Classifications
-
- 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
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0069—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
-
- 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
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/18011—Paramyxoviridae
- C12N2760/18411—Morbillivirus, e.g. Measles virus, canine distemper
- C12N2760/18421—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
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/18011—Paramyxoviridae
- C12N2760/18411—Morbillivirus, e.g. Measles virus, canine distemper
- C12N2760/18422—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Immunology (AREA)
- Biophysics (AREA)
- Virology (AREA)
- Medicinal Chemistry (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biomedical Technology (AREA)
- Gastroenterology & Hepatology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明的目的是提供一种表达
荧光素酶NLuc的重组犬麻疹病毒，其中用于拯救表达NLuc的重组CMV核酸片段的结构为5′‑N‑P‑NLuc‑M‑F‑H‑L‑3′；其中，N、P、M、F、H、L为病毒的结构基因；NLuc为
荧光素酶的ORF，该ORF上游包含M基因起始序列和Not I酶切位点，下游包含Pme I酶切位点及P基因终止序列。本发明通过反向遗传技术拯救的rCMV‑NLuc，在细胞内对于NLuc的表达水平较高，在发光底物furimazine作用下，可发出肉眼可见的淡蓝色荧光。外源基因的插入并未显著影响病毒的生长动力学，且外源基因在病毒传代过程中相对较为稳定。
Description
技术领域
本发明属于重组病毒构建技术领域，具体涉及一种表达
荧光素酶的重组犬麻疹病毒。
背景技术
犬麻疹病毒(canine morbillivirus,CMV)，旧称“犬瘟热病毒(canine distempervirus)”，属于副粘病毒科(Paramyxoviridae)、麻疹病毒属(Morbillivirus)成员。CMV的宿主广泛，犬科动物、鼬科动物、猫科动物等均易感染。病毒侵入机体24h内，主要在局部组织的巨噬细胞内增殖，而后扩散到扁桃体和支气管淋巴结单核细胞中，引起扁桃体和支气管淋巴结肿大；在感染后2～4d内持续复制，随后扩散到其他淋巴器官。患病动物的临床症状通常是呼吸系统、胃肠消化系统、表皮黏膜和中枢神经系统症状，伴随着双相热、全身不适和病毒血症。
CMV基因组为单股、负链、线性的RNA，全长15 690nt，编码六个结构蛋白(N、P、M、F、H、L蛋白)和两个非结构蛋白(C、V蛋白)。病毒经纯化后电镜下观察形状不定，但多呈球形或椭球形，外披脂质囊膜，囊膜外表面嵌有铆钉状纤突。囊膜内表面紧邻基质蛋白，其单体之间相互作用构成了整个病毒的外骨架，内部则含有鲱鱼骨状螺旋型核衣壳。CMV具有溶瘤活性，是一种理想的溶瘤病毒。溶瘤病毒的溶瘤活性研究可借助于小动物体内发光影像，使其溶瘤效果的观察更为直观。因此构建一种表达荧光素酶(而非荧光蛋白)的重组溶瘤病毒，将为其下游溶瘤活性的研究奠定良好的基础。
发明内容
本发明目的是提供一种表达
荧光素酶(NLuc)的重组犬麻疹病毒(rCMV-NLuc)，从而弥补现有技术的不足。
本发明提供一种用于拯救表达NLuc的重组CMV核酸片段(rCMV-NLuc cDNAclone)，所述核酸片段的结构为：5′-N-P-NLuc-M-F-H-L-3′。其中，N、P、M、F、H、L为病毒的结构基因；NLuc为
荧光素酶的ORF，该ORF上游包含M基因起始序列(M GS)和Not I酶切位点，下游包含Pme I酶切位点及P基因终止序列(P GE)。
作为实施例的一种具体记载，所述核酸片段的序列为SEQ ID NO:1；
本发明所提供的核酸片段用于在细胞内拯救能够表达NLuc的重组犬麻疹病毒；
所述的细胞为BSR-T7/5细胞和Vero-Dog-SLAM(VDS)细胞，两者分别用于重组病毒的拯救和传代；
本发明再一个方面提供一种能表达NLuc的重组犬麻疹病毒，所述的病毒是使用表达NLuc的重组CMV核酸片段的重组质粒在细胞内拯救获得的。
本发明通过反向遗传技术拯救的rCMV-NLuc，在细胞内对于NLuc的表达水平较高，在发光底物furimazine作用下，可发出肉眼可见的淡蓝色荧光。外源基因的插入并未显著影响病毒的生长动力学，且外源基因在病毒传代过程中相对较为稳定。
附图说明
图1：rCMV-NLuc cDNA clone模式图。
图2：四质粒共转染72h后荧光分析。
图3：rCMV-NLuc盲传第二代诱导的合胞体病变。
图4：第五代rCMV-NLuc的RT-PCR鉴定。
图5：第五代rCMV-NLuc的试纸条检测。
图6：第五代rCMV-NLuc的荧光分析。
图7：第七代rCMV-NLuc及野生型病毒于不同感染时间诱导的细胞病变。
图8：rCMV-NLuc及野生型病毒生长曲线的比较。
图9：rCMV-NLuc各代次荧光分析。
图10：RT-PCR鉴定不同代次rCMV-NLuc。
具体实施方式
本发明构建了一种含NLuc的rCMV-NLuc cDNA clone，将其与分别表达CMV N、P、L蛋白的真核表达质粒共转染BSR-T7/5细胞，成功拯救了rCMV-NLuc，进而对重组病毒进行鉴定及特性分析。
以下通过实施例来进一步阐明本发明，但是应理解，所述实施例只是举例说明目的，本发明的保护范围并不仅限于说明书的具体实施例的记载。
实施例1、rCMV-NLuc cDNAclone的构建
本实施例所构建的用于拯救表达NLuc的重组CMV核酸片段rCMV-NLuc cDNA clone呈“5′-N-P-NLuc-M-F-H-L-3′”分布(图1)；其中，N、P、M、F、H、L为病毒的结构基因；本实施例所选N、P、M、F、H、L基因为CMV毒株5804P(GenBank:AY386316.1)的相关基因；NLuc为
荧光素酶的ORF，该ORF上游包含M GS和Not I酶切位点，下游包含Pme I酶切位点及P GE。
为获得理想的拯救效率，本发明将外源的NLuc基因插入到P和M基因之间；同时，为了提高NLuc的表达量，在NLuc ORF上游插入了Kozak序列。
其中一种rCMV-NLuc cDNA clone的核苷酸序列为SEQ ID NO:1。
将核苷酸序列为SEQ ID NO:1的rCMV-NLuc cDNA clone的全长序列进行核酸合成，亚克隆至pBR322质粒。整个cDNA clone上游受T7启动子序列调控，下游受丁肝病毒核酶及T7终止子序列调控。
实施例2、rCMV-NLuc的拯救
复苏BSR-T7/5细胞，在DMEM培养基中传代2次，DMEM培养基包含penicillin(100U/mL)、streptomycin(100μg/mL)、amphotericin B(0.25μg/mL)、G418(500μg/mL)及10％FBS(fetal bovine serum)。转染前一天，在6孔板中接种BSR-T7/5细胞，次日待细胞达到70％汇合度，将rCMV-NLuc cDNA clone质粒及分别表达CMV N、P、L蛋白的真核表达质粒共转染BSR-T7/5细胞，四者每孔的转染量分别为4μg、2μg、1μg、1μg。按每孔加150μL Opti-MEM和16μL Lipofectamine 2000的比例配制溶液I。再按每孔加150μL Opti-MEM和8μg质粒配制溶液II。将溶液I和II轻柔混匀，室温放置15min以形成脂质体-DNA复合物。吸弃6孔板培养基，每孔加1mL Opti-MEM，逐滴加入脂质体-DNA混合液。将6孔板置于CO2培养箱转染6h，吸弃转染液，加入DMEM，置于培养箱中培养72h。收集细胞液冻融1次，与发光底物furimazine混合(V:V＝100:1)，同时设未转染细胞作为对照，置于Viber发光成像仪内观察。结果表明，四质粒共转染BSR-T7/5细胞可拯救rCMV-NLuc，表达的荧光素酶可催化发光底物发出荧光(图2)。
实施例3、rCMV-NLuc的盲传及鉴定
将拯救的rCMV-NLuc在VDS细胞内进行盲传，第二代(P2)便可观察到合胞体细胞病变(图3)。继续盲传至第五代(P5)，病毒液冻融一次，收集上清提取总RNA，用于病毒的RT-PCR检测。上游引物序列：5′-gatcaaaagtatcacacatgcttaa-3′，下游引物序列：5′-gatcgaagtcgtacacctcagtcat-3′，扩增799bp片段。RT-PCR反应采用High Fidelity OneStep RT-PCR Kit(Takara)，反应条件设定如下，45℃10min,94℃2min,经历30个循环：98℃(10s),55℃(15s),68℃(10s)。为消除残留质粒污染，另设PCR对照。PCR反应采用2×PrimeSTAR Max Premix(Takara)，反应条件设定为30个循环：98℃(10s),55℃(5s),72℃(10s)。RT-PCR和PCR反应结束后，同时进行琼脂糖凝胶电泳，结果如图4所示，仅RT-PCR扩增出了特异性条带。将该条带切下，核酸经回收后进行Sanger测序，结果与对应序列相符。
P5代病毒细胞培养液，冻融1次，离心收集上清，进行试纸条检测，同时设无病毒感染的细胞培养液作为对照。如图5所示，rCMV-NLuc检测结果为阳性，对照为阴性。P5代病毒细胞培养液，冻融1次，与发光底物furimazine混合(V:V＝100:1)，同时设野生型CMV(wt-CMV)作为对照，置于Viber发光成像仪内观察，结果显示，只有rCMV-NLuc培养液发出明亮的荧光(图6)。
实施例4、rCMV-NLuc的生长动力学
将VDS细胞接种10个35-mm细胞培养皿(2×106细胞/皿)，37℃培养2h。其中5个培养皿接种rCMV-NLuc，另5个培养皿接种wt-CMV，MOI值皆为0.001，置于37℃培养。感染细胞后0、24、48、72h、96h，分别取出对应培养皿，观察细胞形态(图7)，冻融一次，离心收集上清，测定病毒滴度。将测得结果绘制生长曲线，结果如图8所示，rCMV-NLuc和wt-CMV生长动力曲线略有差异，后者稍强于前者。
实施例5、rCMV-NLuc的传代稳定性
将rCMV-NLuc在VDS细胞中连续传代(48～72h/代)，共计15代，第5至15代病毒上清液与发光底物混合。结果如图9，除wt-CMV对照，每代rCMV-NLuc感染细胞皆发出荧光。分别提取第7、9、11、13、15代病毒培养液的总RNA，进行RT-PCR分析，方法同实施例3。结果如图10所示，五个代次病毒皆扩增出亮度均一的799bp目的条带。将所有条带切下，核酸经回收后进行Sanger测序，结果与预期一致。
上述结果表明本发明方法制备的重组CMV，插入的外源NLuc基因并未显著影响病毒的生长动力学，且外源基因在病毒传代过程中稳定存在。
尽管参考其示例性实施方案，对本发明进行具体地显示和描述，但是本领域的普通技术人员应该理解，在不背离本发明说明书具体技术思路的情况下，可以在其中进行各种形式和细节的变化，可以进行各种实施方案的任意组合。
序列表
<110> 青岛农业大学
<120> 一种表达荧光素酶的重组犬麻疹病毒
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 16332
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
accagacaaa gttggctaag gatagataaa ttattgaata ttttattaaa aacttagggt 60
caatgatcct accttagggg acaaggtcag ggttcagacc taccgatatg gccagccttc 120
ttaagagcct cacattgttc aagaggactc gggaccaacc cccacttgcc tcgggctccg 180
gaggagcaat aagagggata aagcatgtca ttatagtcct aatcccgggt gattcgagca 240
ttgttacaag atctcgacta ttggatagac ttgttagatt ggtcggtgat ccggaaatca 300
acggacctaa attaactggg attttaatca gtatcctctc cttgttcgtg gagtcccctg 360
gacagttgat ccagaggatc atagacgacc ctgatgtaag catcaagtta gtagaggtca 420
tcccaagcat caactctgtt tgcggtctta catttgcatc cagaggagca agtttggatt 480
ctgaggcaga tgagttcttc aaaattgtag acgaagggtc gaaagctcaa ggacaattag 540
gctggttgga gaataaggat attgtagaca tagaagttga tgatgctgag caattcaata 600
tcttgctagc ttccatcttg gctcaaactt ggatcctgct cgctaaagca gtgactgctc 660
ctgatactgc agccgactcg gagatgagaa ggtggattaa gtatacccaa cagagacgtg 720
tggtcgggga atttagaatg aacaaaatct ggcttgatat tgttagaaac aggattgctg 780
aggacttatc tttgaggcgg ttcatggtag cactcatctt ggatatcaaa cgatccccag 840
ggaacaagcc tagaattgct gaaatgattt gtgatataga taactacatt gtggaagctg 900
gattagctag tttcatctta actatcaaat ttggcattga aactatgtat ccggctcttg 960
ggttgcatga gttttctgga gagttaacaa ctattgaatc ccttatgatg ctataccaac 1020
agatgggtga aacagcaccg tacatggtta ttctggaaaa ttctgttcag aacaaattta 1080
gtgcaggatc ctacccattg ctctggagtt atgccatggg agttggtgtt gaacttgaaa 1140
actccatggg agggttaaat ttcggtagat cctactttga tccagcttat ttcaggctcg 1200
ggcaagaaat ggtcagaaga tctgccggca aagtgagctc tgcacttgcc gccgagcttg 1260
gcatcaccaa ggaagaggct cagctagtgt cagaaatagc atccaagaca acggaggacc 1320
ggacaattcg cgctgctggt cccaagcaat ctcaaatcac ttttctgcac tcagaaagat 1380
ccgaagtcac taatcaacaa cccccagcca tcaacaagag gtccgagaac caaggaggag 1440
acaaataccc catccacttc agtgatgaac ggtttccggg gtatacccca gatgtcaacg 1500
gctccgaatg gagtgaatca cgctatgata cccaaactat tcaagatgat ggaaacgacg 1560
atgacaggaa atcaatggaa gcaatcgcca agatgagaat gcttaccaag atgctcagtc 1620
aacctgggac tagcgaaggg ggttctcccg tctataatga tagagagcta ctcaattaaa 1680
tattcaagac cagtgttaca tcagtcaaca attatccttc taaattcatt ataaaaaact 1740
taggacccag gtccaacaaa cccgatcaat cattcatccg accacccgtt ctattcctaa 1800
atggcagagg agcaggccta tcatgtcagc aaagggctgg aatgcctcaa agccctcaga 1860
gagaatcctc ctgaccttga ggagattcaa gaggtcagca gcatcagaga ccaaacccgc 1920
aacccaggcc aggagaatgg aaccgcaagc atgcaggaag aggaggtctc tcaggatctc 1980
gatgaatcac acgagccagc aaaaggatca aactatgtcg gccatgtact ccaaaataat 2040
ccgggaagtg gaaagggcaa cactgcgctt gtggaggcag agcagcccgc taaagatgtc 2100
atccaaccag gacctggaat acgatgttat catgtttatg atcacagcgg tgaagaggtt 2160
aagggaaccg aagatgctga cagtctcgtg gtacctgcag gcgctgtcag taatcgagga 2220
ttcgagagag gagaaggaag ccttgatgat agcactgagg attctggcga agattattcc 2280
gagggaaatg cttcatctaa ctggggatat tctttcggcc ttaaaccaga cagagcagct 2340
gatgtgagca tgctgatgga agaggaattg agtgctctgc tcaagacaag cagaaatgta 2400
gggattcaga aaagggatgg aaagactctg cagttcccac acaatcccga aggtaagaca 2460
ggggatccgg agtgtgggtc cattaaaaag ggcacaggag agaggtcagc ctcacatgga 2520
atggagatag ttgctggatc gacaaatggt gcaacccaat ctgcactcaa gtcaactggg 2580
ggatcatcag ggccaagtgt gtctgcggag aatgtccgcc aacctgcagt gagtgcaaag 2640
atgacccaga aatgcaaacc cgagtctggt acgcaactcc ctcccaagac ctcaaatgag 2700
gctgaatctg acagtgagta cgatgatgag cttttctctg aaatacaaga gattcgatct 2760
gccattacta aactaactga agataatcaa gcaatacttt ctaaactgga taccttatta 2820
ctgcttaaag gagagactga ttcaattaag aaacaaatta gcaaacaaaa tatcgctatt 2880
tccacgattg aggggcatct atcaagcatt atgatagcta tacctggttt tgggaaggac 2940
actggagacc ctacggcaaa tgtcgacatt aacccagagc tccgccctat aatagggaga 3000
gattcaggaa gagcactggc ggaagttctc aagcaacccg catcatcccg cggtaatcgg 3060
aaggacagcg gtattgcctt gggctcaaaa ggtcaactat taagagacct ccagctgaaa 3120
cctattgaca gagagtctag ctcagcaatc ggatacaaac cgaaggatac cgcaccttcc 3180
aaagctgtgc ttgcatcatt gattagatca agcagagttg atcaaagtca caaacacaac 3240
atgctggcgc ttcttaaaaa tatcaaaggg gatgacaatc taaacgagtt ctaccagatg 3300
atcaaaagta tcacacatgc ttaagctgta gcatttacta atcttttaac aggcgcagaa 3360
ctgctttcac tatcgcttaa aagcaattat aaaaaactta ggacacaaga gcctaagtcc 3420
tctccaaaaa agcggccgca ccatggtctt cacactcgaa gatttcgttg gggactggcg 3480
acagacagcc ggctacaacc tggaccaagt ccttgaacag ggaggtgtgt ccagtttgtt 3540
tcagaatctc ggggtgtccg taactccgat ccaaaggatt gtcctgagcg gtgaaaatgg 3600
gctgaagatc gacatccatg tcatcatccc gtatgaaggt ctgagcggcg accaaatggg 3660
ccagatcgaa aaaattttta aggtggtgta ccctgtggat gatcatcact ttaaggtgat 3720
cctgcactat ggcacactgg taatcgacgg ggttacgccg aacatgatcg actatttcgg 3780
acggccgtat gaaggcatcg ccgtgttcga cggcaaaaag atcactgtaa cagggaccct 3840
gtggaacggc aacaaaatta tcgacgagcg cctgatcaac cccgacggct ccctgctgtt 3900
ccgagtaacc atcaacggag tgaccggctg gcggctgtgc gaacgcattc tggcgtaagt 3960
ttaaacgctg tagcatttac taatctttta acaggcgcag aactgctttc actatcgctt 4020
aaaagcaatt ataaaaaact taggacacaa gagcctaagt cctctccaaa aaaatgactg 4080
aggtgtacga cttcgatcag tcttcatggg acaccaaagg ctcattggcc cccattttgc 4140
ccaccaccta tcctgatggt aggttagtac ctcaggtcag agtgatagat ccaggactcg 4200
gcgatcgaaa agatgaatgc ttcatgtata tttttctact gggtataata gaagacaacg 4260
atggcctcgg acccccaatt ggaagaacat ttggatcgct gcctttaggt gttggacgca 4320
ctactgccag acctgaggaa ttattgaagg aagccaccct gttggacatt gtggtaaggc 4380
gaactgcagg tgtcaaggaa caactggtgt tttacaataa taccccattg cacatcttaa 4440
ctccgtggaa gaaggtcctt acgagtggga gtgtgttcag tgcaaaccaa gtctgtaacg 4500
cagttaattt aataccgtta gacatagcac agagatttag agtggtatat atgagcatca 4560
ctcgactatc agacgatgga agttacagaa ttcctcgagg gatgtttgaa ttccgctcta 4620
ggaatgcttt agcatttaat attttagtca ccattcaagt tgagggagat gtctgttcga 4680
accgaggtaa tttgagcatg ttcaaagatc atcaagtgac attcatggtg catatcggca 4740
acttcagccg taagaagaac caagcttact ctgctgatta ctgtaaactg aaaattgaaa 4800
agatgggatt agtgtttgct ctaggaggga tagggggaac cagtcttcac atacgatgta 4860
ctggtaagat gagcaaggct ttgaatgccc agctagggtt caagaaaatc ctgtgttacc 4920
cgctcatgga gatcaatgaa gatttgaatc gatttctatg gagattagag tgcaaaatag 4980
taagaatcca agcagtcttg caaccatcag tcccacaaga tttcaggatt tataatgatg 5040
ttatcatcag cgatgatcag ggtcttttca aaattctcta aatcattggt tcatgaacta 5100
aaaatcaaat gccttggtgg cattgtccag gatctcttaa tcccctcaga caagaattga 5160
ggctacaagt atcaactgtc tcgatgtcgc tcctgcactt taagcgtgtt ctataggttt 5220
ctaaactgct ctttcgtgcc tactactctg gtggctctgc aatatgaaga cagctgaatc 5280
aaaccaattc atgcctaaga gtaggttgat cattatcgga ccaagaaatt tatggatgct 5340
tggggttttg aacttcgcct ctaggaatct cactttaaca attatacttc cacgcacttg 5400
cccgagctca aactatcact agtagtcctg tttcacgaaa ttgtgactgt ctatctttct 5460
atcaccaatc gttaataatt aatcaaaact tagggtccag gacgtagcaa gccaacaggc 5520
caaccaagtc catcaatccg aggccgggca ggaaccccca cgagcagaca agccccatgc 5580
acaacaaaat ccccaaagga tccaaaaccc caacacccac ccaacaagac ccccccccac 5640
aacatagtat caaatccgcc gagaccaaga cctcccaagc acgatacaac ataacatcgg 5700
ctcagcaatc cacacgccat ggtcctcgaa catcggatag gccagttcac tacatcatga 5760
acaggaccag gtcttgcaag caagccagct acagatcaga taacatcccg gctcacggag 5820
accacgaggg tattatccat cacacaccag agagtgtctc ccaaggggtg agatcccggt 5880
tcaaaaggcg gcaatccaat gcaaccagct caggctctca gtgcacctgg ttagtcctgt 5940
ggtgcatcgg agtagccagt ctctttcttt gttccaaggc tcagatacat tggaataatt 6000
tgtcaactat tgggattatc gggactgaca gtgtccatta taagatcatg actaggccta 6060
gtcaccagta cttggtcata aaactaatgc ctaatgtttc acttatagat aattgtacca 6120
aagcagaatt aggtgagtat gagaaattat taagttcagt cctcgagcca atcaaccaat 6180
ctttgactct aatgaccaag aacgtgaagc ccctacagtc agtagggtca ggtaggagac 6240
aaaggcgttt tgcaggagtg gtgcttgcag gtgcagcttt aggagtggcc acagctgcac 6300
aaatcactgc agggatagct ttacatcaat ccaacctcaa tgctcaagca atccaatctc 6360
tgagaactag ccttgaacag tccaacaagg ctatagaaga aattagggag gcaacccagg 6420
aaaccatcat tgccgttcag ggagtccagg attatgttaa taatgaactt gtccctgcta 6480
tgcaacatat gtcgtgtgaa ttagttgggc agagattagg tttaaaactg cttaggtatt 6540
ataccgagtt gttgtcgata tttggcccga gtctacgtga ccctatttca gccgagatat 6600
caattcaagc actgagttat gctcttgggg gagaaattca taagatactt gagaagttgg 6660
ggtattctgg aaatgatatg attgcaattt tggagagtcg agggataaaa acaaaaataa 6720
ctcatgttga tattcccggg aaactcatca tcttaagtat ctcataccca actttatcag 6780
aagtaaaggg ggttatagtc cacagattgg aagcagtttc ttataacata gggtcacagg 6840
agtggtacac cactgtcccg aggtatgttg caactaatgg ctatttaata tctaactttg 6900
atgagtcatc ctgtgtattc gtctcagaat cagccatttg tagccagaac tccttatacc 6960
ccatgagccc gcttctacaa cagtgcatta ggggcgacac ttcatcctgt gctcggacct 7020
tggtgtctgg gactatgggc aacaagttta ttctgtcaaa aggtaatatc gtcgcaaatt 7080
gtgcttctat actatgtaag tgttatagca caagcacaat tatcaatcag agtcctgata 7140
agttactgac atttattgcc tccgatactt gcccactggt tgaaatagat ggtgtaacta 7200
tccaggttgg agggaggcaa tatcctgata tggtatatga aagcaaagtt gccttaggcc 7260
ctgctatatc acttgagagg ttagatgtag gtacaaattt agggaacgcc cttaagaaac 7320
tggatgatgc taagatattg atagactcct ctaaccagat ccttgagacg gttaggcgct 7380
cttcctttaa ttttggcagt ctcctcagcg ttcccatatt aatctgtaca gccctggctt 7440
tgttgtttct gatttactgt tgtaaaagac gctaccaaca gacactcaag cagcatatta 7500
aagtcgatcc gacatttaaa cctgatttga ctggaacttc gaaatcatat gtaagatcac 7560
tctgaagtac tctggtcaca cgtcttaccc gattgtcagg attgaaatct ataaatctcg 7620
ccctattttc tttaaaagct atcaaactgc aacaaatagt ggagaggact gactacgatt 7680
atcgaaatta aagaaaactt agggctcagg tagtccaaca atgctctcct accaagacaa 7740
ggtgggtgcc ttctataagg ataatgcaag agctaattca tccaagctgt ccttagtgac 7800
agaagagcaa gggggcagga gaccacccta tttgctgttt gtccttctca tccttctggt 7860
tggaatcatg accttgcttg ctatcactgg agttcgattt caccaagtat caactagcaa 7920
tatggaattt agcagattgc tgaaagagga tatggagaaa tcagaggccg tacatcacca 7980
agtcatagat gtcttgacac cgctcttcaa aattattgga gatgaggttg ggttacagtt 8040
gccacaaaaa ctaaacgaga tcaaacaatt tatccttcaa aagacaaact tcttcaatcc 8100
gaacagggaa ttcgacttcc gcgatctcca ctggtgcatt aatccaccta gtaagatcaa 8160
ggtgaatttt actaattact gcgatacaat tgggatcaaa aagtctattg catcggcagc 8220
aaatcccatc cttttatcag cactctccgg aggcagaggt gacatattcc caccatacag 8280
atgcagtgga gctactactt cagtgggcag agttttcccc ctatcagtat cattgtccat 8340
gtctttgatc tcaaaaacat cagagataac cagtatgcta actgctatct cagacggagt 8400
gtatggtaaa acctatttgc tagtgcctga ttacattgaa ggggagttcg acacgcaaaa 8460
gattcgagtc tttgagatag ggttcatcaa acggtggctg aataacatgc cattactcca 8520
gacaaccaac tacatggtcc tcccggagaa ttccaaagcc aaggtatgta ctatagcagt 8580
gggcgagttg acactggctt ccttgtgtgt agatgagagc accgtattgt tatatcatga 8640
cagcaatggt tcacaagatg gtattctagt agtgacgctg gggatatttg gagcaacacc 8700
tatggatcaa gttgaagaag tgatacctgt cgctcaccca tcagtagaaa aaatacatat 8760
aacaaatcac cgtgggttca taaaagattc aatagcaacc tggatggtgc ctgcattggt 8820
ctctgagaaa caagaggaac aaaaaaattg tctggagtcg gcttgtcaaa gaaaaactta 8880
ccctatgtgc aaccaaacgt catgggaacc ctttggaggg ggacagttgc catcttatgg 8940
gcggttgaca ttacctctag atccaagcat tgaccttcaa cttaacatat catttacata 9000
cggtccggtt atactgaatg gagacggtat ggattattat gaaagcccac tttcggactc 9060
cggatggctt accattcctc ccaggaacgg aacagtcctt ggattgataa acaaagcaag 9120
tagaggagac cagttcactg taatccccca tgtgttgaca tttgcgccca gggaatcaag 9180
tggaaattgt tatttaccta ttcaaacatc ccagattatg gataaagatg tccttactga 9240
gtccaattta gtggtgttgc ctacacagaa ttttagatat gtcatagcaa catatgatat 9300
atcccggggc gatcatgcaa ttgtttatta tgtttatgac ccaatccgga cgatttctta 9360
tacgcaccca tttagactaa ccaccaaggg tagacctgat ttcctaagga ttgaatgttt 9420
tgtgtgggat gacgatttgt ggtgtcacca attttaccga tttgaggctg acatcaccaa 9480
ctctacaacc agtgttgaga atttagtccg tataagattc tcatgtaacc gttcaaaacc 9540
ttgacagtat gatgatacac atctcaattg gacttagata tgatgactga ggtgagaaat 9600
cccttaccga cgattgaatt aaaccatctc cagcattata aaaaaactaa ggatccagga 9660
tcctttcagt catggactct gtttcagtaa atcagattct ataccctgag gtccatctag 9720
atagcccaat tgtaaccaat aagctagtag ctatcttaga gtatgcacga attagacata 9780
actatcaact ccttgataca acattagtgc gtaatatcaa agagagaatt tcagaagggt 9840
tatcaaacca gatgatcatt aactgtatcg aaattgggag cattgttaat cagaccttgt 9900
tatcttatcc caaacataac catgtgatgt atccaaattg caacaaactt ttgtttcatg 9960
cacaggatcg agtcatctct ctgaggttga gaaatatatt caaaagaggg aacagcatct 10020
atagtaaaat aacagacggg gtcaaaagat gtttgaacga tattaatctt agtattggtt 10080
taggagatgc actggacaag actattgggg ccaaaattga cgaagcaggc ataattatgc 10140
aaagctcaca atggttcgaa cctttccttc tatggtttac aattaaaaca gaaatgagat 10200
cagtgattaa atcctctact cacaactgtc gcaaacgaag gcagaatcct gtctttgtaa 10260
aaggtgaatc atttaatgtg ttagtgtctc gggatcttgt atgtatcatt gacctcacca 10320
gtcacaatgt ttattaccta acatttgaaa tggtcctgat gtattgtgat gtgatagaag 10380
ggagactcat gactgatact gctatggcaa ttgatcaacg ttactcaact ttgcatgtca 10440
gaatcaggta tctttggaat ctaattgacg gatttttccc agacttagga aattcaacct 10500
atcaattggt agctttacta gagcctcttt cattggctta cttgcaatta aaagacatca 10560
ccttctctct caggggtgct tttctgaatc actgctttgc cgaaattcag gagattttac 10620
aggacaatgg cttctatact gaagagacgt tccaaacctt aacccaggct ctagacttcg 10680
ttttcatcac agaggatata catataacag gagaaatctt ttcctttttt aggagtttcg 10740
gtcacccaag gttagaagca ataaccgcag cagagaatgt acggaaacac atgaaccagc 10800
ccaaagttat ctcctatgag accatgatga agggacacgc tatattctgt gggataatta 10860
ttaacggtta tcgggataga catggaggta catggcctcc aatggacctt cctgtccatg 10920
catctcctat catcaggaat gctcatgcct caggagaggg gatcacctat agtcaatgta 10980
tagaaaactg gaaatccttt gcaggaattc gatttaaatg ctttatgccc cttagcctag 11040
acagtgattt gaccatgtat ttgaaagata aggctttggc agcccttaag aaagagtggg 11100
attcagtgta cccaaaagaa ttcctcaggt acaacccacc tcgctccact gagtctcgga 11160
gacttgtcaa tgtatttcta gaggactctc agtttgaccc ttataacatg attatgtacg 11220
ttatctcagg tcaatatcta gaagatcctg acttcaacct atcatacagc cttaaggaga 11280
aagagattaa agaggtgggg aggctattcg ctaaaatgac ctacaaaatg cgagcctgtc 11340
aagtcatagc agaaaacttg atatccaatg gaattgggaa gtacttcaag gacaatggga 11400
tggcaaagga tgaacacgat ctcactaaag cactgcacac tctggctgtg tccggggttc 11460
cgaaagacaa gaaagactcc catcgtggcc tctctactca gcgtaagtct ataaaacctg 11520
caccttatcg gggggccctt cactccgtct cttccccaag tagtagatat atagacacaa 11580
atccaaattt ttgcaccagt agaagagaag acaatgacat agggatctat gaaaccgtaa 11640
gtgcatttat aactacagat ctcaaaaagt actgtctgaa ttggcgatat gagaccatta 11700
gtatatttgc tcagagatta aatgaaatct acggtctccc ctcatttttt caatggttgc 11760
acagaagatt ggaacagtcg atcctatacg taagtgaccc ccactgcccc ccagatctcg 11820
atcgccatgt ggacttgaac acagccccta actctcaaat attcatcaaa tacccaatgg 11880
ggggagtgga gggatactgt cagaagttat ggactattag cactatacct tatctgtact 11940
tggcagcaca tgaaagcggt gtcagaattg catcacttgt ccaaggtgat aaccaaacca 12000
ttgctgtcac taagagagtt ccaagcacct ggtcctatgc cttgaagaag tctgaagcca 12060
gtcgagtcac cacagaatac tttatagcct tgagacagag attacatgat gtcggacatc 12120
atttgaaagc aaatgaaaca ataatatctt cccacttttt tgtatattca aaaggaatct 12180
attatgatgg aatgttaatt tcacaatccc tgaaaagtat agctagatgt gtattttggt 12240
cagaaacaat agtggacgag acccgagccg catgcagcaa catatcaaca acactggcaa 12300
aagccattga gaaagggttt gacaggtact tagcctatgc gctgaatatt ttaaagatca 12360
ttcaacaagt attaatttca ttaggattca ctatcaattc agctatgaca cgggatgtga 12420
tagaacccct tttacaagat cactgtctct tgaccaagat ggcaatcctt cccgcaccta 12480
taggtggtct taattacctc aatatgagta ggctctttgt caggaacatc ggggatcccg 12540
tgacatcttc tattgctgac ctcaaacgaa tgatccgatc aggccttctc ggagtagaga 12600
ttctacatca agtcatgacc caataccctg gtgactcttc gtacttagat tgggcaagtg 12660
acccttattc tgccaatctg ccctgtgtcc agagcataac ccgactcctt aaaaatatca 12720
cggccaggca tgtccttatc aacagtccaa atcctatgct gaaaggattg ttccatgatg 12780
aaagtcagga agaggatgaa gctttagcag ctttcttgat ggataggaaa attattatcc 12840
caagagctgc acatgaaatt ctagataaca cgatcacggg tgcaagggaa gcaattgctg 12900
gaatgctaga taccacaaag gggttgataa gagcaagcat gaaaagaggg ggtctaaccc 12960
ctagaataat aaaccgtttg tcaacttatg attatgagca atttagggca ggtatcagac 13020
tgttgtcagg gaaagggcat gatccgctca tcgatcaaga ctcatgttcc gtccagttag 13080
cgagagcatt aaggaaccac atgtgggcta agttggcgaa gggtcgtccg atttatggtc 13140
tagaagtccc ggatatcctt gaatcaatga agggttatat gataagaaga catgagtctt 13200
gtttgctttg cgcatcgggc tcccataact atggttggtt ttttgtaccg gcaaattgcc 13260
aattagatag tattacagag ggaacatctg cactgagggt accatacatt gggtccacaa 13320
cagaagaaag aacagacatg aaattagcat tcgtcaagtc ccctagtagg tctctaaaat 13380
cagcagtgag aatagcaact gtgtactcat gggcctatgg tgatgatgac gaatcttggc 13440
aagaggcttg gactttggca aaacagagag cgaacatctc acttgaagaa ttacggatga 13500
ttaccccaat ttctacttct actaatctag ctcaccgact aagagacaag agtactcaag 13560
tcaaatactc agggacatct cttatcagag tagcacgtta tgcaacaatc tcgaatgata 13620
acctttcttt tgtgatagct gacaagaaag tggacacgaa ctttatttat cagcaaggta 13680
tgctcctggg cctggggatt ctcgagcact tatttagact ttcttcaacc actggcgact 13740
ctaacaccgt actgcattta catgtcgaaa cagattgttg tgtaataccc atgagcgacc 13800
atccaagggt cccaggtctc agaaaggtcg ttataccaag aaatagttgt acaaatcctt 13860
tgatctacga cagtaaccct attattgaga aagacgcagt cagactttat aaccagagtc 13920
acaggaagca cattgtggag tttgtcacat ggacaacggg gcagctttat catgtactag 13980
ctaaatctac tgctatgtct atggttgaga tgattacaaa gtttgaaaaa gaccacctaa 14040
atgaggtctc cgctttaatt ggcgacgatg atatcaatag ctttatcact gagtttcttc 14100
tagttgagcc taggttattt actgtgtatc taggtcaatg tgctgcaatc aactggggct 14160
ttgaaattca ttatcaccgg ccttctggaa agtaccaaat gggtgaattg ttgttttctt 14220
tcttgagtag aatgagtaaa ggagtcttca aaattttaac caatgcattg agtcatccca 14280
aagtatatag acgattttgg gacagtggga tgattgaacc tgttcacggt ccctctcttg 14340
actcccaaaa cctacacata actgtatgca acctgatcta taactgttac atgatttacc 14400
tagaccttct gttaaatgat gagttagatg atttctcatt cattttatgc gaaagtgatg 14460
aggacgtcat acctgaaaga tttgacaaca tacaagctag gcacctatgc atcctatctg 14520
acctttattg taaccctcgt gattgtcccc agattcgtgg gttgacacca acacagaaat 14580
gtgctgtatt atcgaggtac ttaaaatcaa aagctctaga gtcccatgtt ggtctgacat 14640
ggaatgacaa acctatcttg atagatcagt attcatgttc cctgacatat ctaagaagag 14700
gctcaatcaa gcagataaga ttgagagtgg accccgggtt catcactgat gctgttggat 14760
gcttagaaaa acaatctcta aggaaaagtc ctatctctaa ggcctcagaa ttgaaatcag 14820
aatttgatcc accgaaagat gatctggcta cactcctgag ccagctatca acaaggacac 14880
acaacttacc tattacagga ttaggagtcc gaaactatga ggttcactca ttcagaagaa 14940
ttgggatcaa ctctactgca tgttacaagg cagttgagat agtttctgtt attaagaacg 15000
aatttacgtc tgaagaacac ggtttattcc taggagaggg ttcaggtgca atgctgacag 15060
tatataaaga gctattgaaa ttgtcaagat gttattataa cagtggtgtg tcggtagagt 15120
ctagaactgg acaacgagag atttcacctt acccttctga ggtcagtctt gtggaacatc 15180
aattaggact cgataaattg gtgactgtgc tgttcaatgg gagaccagag gtaacttggg 15240
ttgggagtgt tgattgttac aagtatatac taagccagat atctgctagc agtcttggat 15300
tgattcactc ggatatcgag tcattacctg ataaagacat aattgaaaaa ttggaagagc 15360
tgtctgccat attatcaatg actttgatat tagggaaggt agggtcagtg ttagtaatca 15420
agatcatgcc cgttagtggc gactgggttc aaggatttat tttgtatgca ctcccacatt 15480
ttcttcgaag tttcatagtt tacccaagat acagcaattt tgtgtcaaca gaggcctacc 15540
tcgtttttac cggtcttaga gcagggagac tagtcaatcc ggaggggatt aaacaacaga 15600
ttttgcgagt cggtatccga acttcacccg ggttagtagg gcacatcctt tcatcaaagc 15660
agacagcatg tgtgcagtct ttgcatggac ctccatttca ggctaaatct tttaatcctt 15720
acctccaggg tttaacaagt attgagaagg ttttgatcaa ttgtgggctt acaattaacg 15780
gtcttaaagt atgcaaaaac ctgcttcacc atgatatctc gtcaggcgag gaagggctaa 15840
aaggatctat cacgatcctt tatagggaac tcgcacggtt caaggataac caccaatttt 15900
cacatggaat gttccatgca tacccagtgt taatcgcaag tcaggagagg gagcttgtat 15960
ctatcattgc aaggaagtat tgtggctata ttttgcttta ctcgggagat ttatacgaaa 16020
ttaccaggat agttcgaaat ctaaaagcca gccacataat ttttgactta caccgtaatt 16080
tatttatgga taacctgtcc agatctgacc ggtctctcat cctgacaaca atccccaaaa 16140
agaattggct cttccaactt gagacaaaag agataaaaga gtggttcaaa ctgttagggt 16200
atagtgcact gattagaaac cactgacaga ctggtccgac tcctaaccct ctgctattca 16260
ctgctattaa atttaattat acgaaaaaaa acaacggtta tcaataagtt atcataccca 16320
gctttgtctg gt 16332
Claims (6)
1.一种用于拯救表达NLuc的重组犬麻疹病毒的核酸片段，其特征在于，所述核酸片段的结构为5′-N-P-NLuc-M-F-H-L-3′，其中N、P、M、F、H、L为犬麻疹病毒的结构基因；NLuc为
荧光素酶的ORF，该ORF上游包含M基因起始序列和Not I酶切位点，下游包含PmeI酶切位点及P基因终止序列。
2.如权利要求1所述的核酸片段，其特征在于，所述核酸片段的序列为SEQ ID NO:1。
3.权利要求1或2所述的核酸片段在细胞内拯救重组犬麻疹病毒的应用。
4.如权利要求3所述的应用，其特征在于，所述的细胞为BSR-T7/5细胞和Vero-Dog-SLAM细胞。
5.一种能表达NLuc蛋白的重组犬麻疹病毒，其特征在于，所述的重组犬麻疹病毒是使用权利要求1或2所述的核酸片段在细胞内拯救获得的。
6.一种用于检测权利要求5所述的重组犬麻疹病毒的引物对，其特征在于，所述的引物对中，上游引物序列为5′-gatcaaaagtatcacacatgcttaa-3′，下游引物序列为5′-gatcgaagtcgtacacctcagtcat-3′。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010618800.5A CN111733170A (zh) | 2020-07-01 | 2020-07-01 | 一种表达荧光素酶的重组犬麻疹病毒 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010618800.5A CN111733170A (zh) | 2020-07-01 | 2020-07-01 | 一种表达荧光素酶的重组犬麻疹病毒 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111733170A true CN111733170A (zh) | 2020-10-02 |
Family
ID=72653978
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010618800.5A Pending CN111733170A (zh) | 2020-07-01 | 2020-07-01 | 一种表达荧光素酶的重组犬麻疹病毒 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111733170A (zh) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113025631A (zh) * | 2021-03-13 | 2021-06-25 | 青岛农业大学 | 一种表达双报告标签的重组副流感病毒5型 |
| CN113416750A (zh) * | 2021-07-05 | 2021-09-21 | 青岛农业大学 | 一种表达犬细小病毒2a型VP2的重组犬瘟热病毒 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1455816A (zh) * | 2000-06-23 | 2003-11-12 | 美国氰胺公司 | 从cDNA中挽救犬热病病毒 |
| US20040121308A1 (en) * | 1999-09-06 | 2004-06-24 | Yoshiyuki Nagai | Paramyxoviruses comprising modified transcription start sequence |
| CN111094569A (zh) * | 2017-07-11 | 2020-05-01 | 国立大学法人东京大学 | 光控性病毒蛋白质、其基因及包含该基因的病毒载体 |
-
2020
- 2020-07-01 CN CN202010618800.5A patent/CN111733170A/zh active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040121308A1 (en) * | 1999-09-06 | 2004-06-24 | Yoshiyuki Nagai | Paramyxoviruses comprising modified transcription start sequence |
| CN1455816A (zh) * | 2000-06-23 | 2003-11-12 | 美国氰胺公司 | 从cDNA中挽救犬热病病毒 |
| CN111094569A (zh) * | 2017-07-11 | 2020-05-01 | 国立大学法人东京大学 | 光控性病毒蛋白质、其基因及包含该基因的病毒载体 |
Non-Patent Citations (4)
| Title |
|---|
| CHRISTOPHER L PARKS等: "Expression of a foreign gene by recombinant canine distemper virus recovered from cloned DNAs", 《VIRUS RESEARCH》 * |
| KENTARO FUJITA等: "Host range and receptor utilization of canine distemper virus analyzed by recombinant viruses: Involvement of heparin-like molecule in CDV infection", 《VIROLOGY》 * |
| 刘松梅: "《抗魏氏梭菌病转基因植物研究》", 31 August 2012, 黑龙江大学出版社 * |
| 石晓玲等: "反向遗传学技术在麻疹病毒属病毒中的研究进展", 《西北民族大学学报(自然科学版)》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113025631A (zh) * | 2021-03-13 | 2021-06-25 | 青岛农业大学 | 一种表达双报告标签的重组副流感病毒5型 |
| CN113416750A (zh) * | 2021-07-05 | 2021-09-21 | 青岛农业大学 | 一种表达犬细小病毒2a型VP2的重组犬瘟热病毒 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1367839A (zh) | 含有已改变的转录起始序列的副粘病毒 | |
| CN111733170A (zh) | 一种表达荧光素酶的重组犬麻疹病毒 | |
| CN1447700A (zh) | 用于将基因引入肾细胞的病毒载体 | |
| CN112458064A (zh) | 盖他病毒全长感染性克隆、复制子系统及其制备和应用 | |
| CN109321535A (zh) | 一种热稳定的新城疫病毒弱毒疫苗候选株 | |
| CN105200085A (zh) | 重组人成纤维细胞生长因子-18的生产方法及其用途 | |
| CN110951778B (zh) | 犬瘟热病毒CDV-3株感染性cDNA克隆及其构建方法和应用 | |
| CN108018264B (zh) | 一种利用多拷贝基因共表达杆状病毒外源蛋白的构建和表达方法 | |
| CN113817753B (zh) | 表达SARS-CoV-2纤突蛋白或其变异体SΔ21的假型化VSV病毒构建和应用 | |
| CN112680443B (zh) | 一种启动子pCalm1及其应用 | |
| CN104130977A (zh) | 一种抗肿瘤药物筛选细胞模型及其应用 | |
| CN114292823A (zh) | 携带基因VII型新城疫病毒F和HN基因的重组LaSota疫苗株及其构建方法和应用 | |
| CN114574452B (zh) | Hn基因易位构建重组新城疫疫苗候选株vii-hnf的方法及用途 | |
| CN112301042B (zh) | A型塞内卡病毒全长感染性cDNA克隆及其构建方法及应用 | |
| CN109295095A (zh) | 一种热稳定的基因viii型新城疫弱毒株外源基因表达载体 | |
| CN110904056B (zh) | 一种传染性支气管炎病毒rH120-YZS1Δ5a及其构建方法和应用 | |
| CN107018955A (zh) | 一种抗猪圆环病毒2型的转基因猪 | |
| CN1252274C (zh) | 用于将外源基因导入气道上皮细胞的重组仙台病毒载体 | |
| CN107177630A (zh) | 一种无外源标记基因的抗pcv2转基因猪制备方法 | |
| CN108486115B (zh) | 一种用于淡水螯虾细胞外源基因表达的转染方法及其应用 | |
| ES2275699T3 (es) | Produccion de un papilomavirus humano(hpv) quimerico. | |
| CN111926025B (zh) | 一株经过密码子替换的基因ⅶ型新城疫病毒的拯救方法 | |
| CN112852762B (zh) | 新型冠状病毒肺炎副粘病毒疫苗株及其构建方法 | |
| JPWO2007007921A1 (ja) | 分節ゲノム型組換えモノネガウイルスベクター | |
| CN113025631A (zh) | 一种表达双报告标签的重组副流感病毒5型 |
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 | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20201002 |