肠道病毒D组68型假病毒的构建 : = Construction of enterovirus D68 pseudovirus
目的制备携带萤火虫荧光素酶报告基因的肠道病毒D组68型(enterovirus D68,EV-D68)假病毒。方法分别构建携带绿色荧光蛋白报告基因的EV-D68表达子和携带萤光虫荧光素酶报告基因的EV-D68复制子,共转染HEK-293T细胞后收获EV-D68假病毒;实时逆转录聚合酶链反应(reverse transcription PCR,RT-PCR)及蛋白质印迹法(Western blotting)鉴定EV-D68假病毒;实时荧光定量PCR检测假病毒滴度,荧光强度定量检测假病毒感染活性。结果成功构建EV-D68表达子和复制子;成功制备EV-D68假病毒,RT-PCR和Western blotting结果表明其具有萤火虫荧光素酶报告基因和与活病毒相同的衣壳蛋白,拷贝数为(4～8)×106copies/m L,荧光强度与假病毒感染剂量呈正相关,证明其具有感染活性。结论成功构建了EV-D68假病毒。.
Objective To construct a Enterovirus D68(EV-D68) pseudovirus carrying the firefly luciferase report gene and to develop a feasible identification method. Methods The EV-D68 capsid was inserted into green fluorescent protein and the EV-D68 replicon was inserted into the fluorescent luciferase report gene. The EV-D68 pseudovirus was harvested after co-transfection to HEK-293 T cells with plasmids. The EV-D68 pseudovirus RNA was extracted by using of magnetic beads and luciferase gene fragment was amplified by reverse transcription PCR; cell precipitation of EV-D68 pseudovirus was detected by Western blotting for VP1 protein; pseudovirus titer was detected by real-time quantitative reverse transcription PCR; infective activity of pseudovirus was experimented by the sensitive cells. Results The EV-D68 capsid and replicon were constructed successfully and the EV-D68 pseudovirus was prepared successfully. The RT-PCR and Western blotting showed that the constructed pseudovirus had the firefly luciferase reporter gene and the same capsid protein as the live virus. The copy number was(4-8) ×106 copies/m L. The pseudovirus infection dose was linearly positively correlated with the RLU(relative light unit) values. Conclusion The EV-D68 pseudovirus was successfully constructed in this study..
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2019-11-14 17:19 2019 |
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| Erschienen: |
2019-11-14 17:19 |
| Enthalten in: |
Zur Gesamtaufnahme - year:2019 |
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| Enthalten in: |
Wei sheng wu xue mian yi xue jin zhan - (2019), 06 vom: 14 17:19. Nov., Seite 8-14 Original Letters: Enthalten in 微生物学免疫学进展 (DE-600)2998876-7 (DE-600)2998876-7 甘肃省兰州市 |
| Reihe: |
China Academic Journals (CAJ), E, 医药卫生科技 = Medicine & Public Health China Academic Journals (CAJ), A, 理工A(数学物理力学天地生) = Mathematics/ Physics/ Mechanics/ Astronomy |
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| Sprache: |
Chinesisch |
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| Weiterer Titel: |
Construction of enterovirus D68 pseudovirus |
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| Beteiligte Personen: |
董方玉 [VerfasserIn] |
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| Links: |
oversea.cnki.net [lizenzpflichtig] |
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| Anmerkungen: |
Author info:DONG Fang-yu;CUI Bo-pei;LI Ke-lei;CUI Li-sha;LIU pei;MA Chao;WU Xing;LIANG Zheng-lun;National Institutes for Food and Drug Control,Institute for Biological Product Control,Division of Hepatitis Vaccine |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
CAJ645223123 |
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| 245 | 1 | 0 | |a 肠道病毒D组68型假病毒的构建 |b = Construction of enterovirus D68 pseudovirus |
| 246 | 3 | 1 | |a Construction of enterovirus D68 pseudovirus |
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| 490 | 0 | |a China Academic Journals (CAJ) |a A |a 理工A(数学物理力学天地生) = Mathematics/ Physics/ Mechanics/ Astronomy | |
| 500 | |a Author info:DONG Fang-yu;CUI Bo-pei;LI Ke-lei;CUI Li-sha;LIU pei;MA Chao;WU Xing;LIANG Zheng-lun;National Institutes for Food and Drug Control,Institute for Biological Product Control,Division of Hepatitis Vaccine | ||
| 520 | |a 目的制备携带萤火虫荧光素酶报告基因的肠道病毒D组68型(enterovirus D68,EV-D68)假病毒。方法分别构建携带绿色荧光蛋白报告基因的EV-D68表达子和携带萤光虫荧光素酶报告基因的EV-D68复制子,共转染HEK-293T细胞后收获EV-D68假病毒;实时逆转录聚合酶链反应(reverse transcription PCR,RT-PCR)及蛋白质印迹法(Western blotting)鉴定EV-D68假病毒;实时荧光定量PCR检测假病毒滴度,荧光强度定量检测假病毒感染活性。结果成功构建EV-D68表达子和复制子;成功制备EV-D68假病毒,RT-PCR和Western blotting结果表明其具有萤火虫荧光素酶报告基因和与活病毒相同的衣壳蛋白,拷贝数为(4～8)×106copies/m L,荧光强度与假病毒感染剂量呈正相关,证明其具有感染活性。结论成功构建了EV-D68假病毒。 | ||
| 520 | |a Objective To construct a Enterovirus D68(EV-D68) pseudovirus carrying the firefly luciferase report gene and to develop a feasible identification method. Methods The EV-D68 capsid was inserted into green fluorescent protein and the EV-D68 replicon was inserted into the fluorescent luciferase report gene. The EV-D68 pseudovirus was harvested after co-transfection to HEK-293 T cells with plasmids. The EV-D68 pseudovirus RNA was extracted by using of magnetic beads and luciferase gene fragment was amplified by reverse transcription PCR; cell precipitation of EV-D68 pseudovirus was detected by Western blotting for VP1 protein; pseudovirus titer was detected by real-time quantitative reverse transcription PCR; infective activity of pseudovirus was experimented by the sensitive cells. Results The EV-D68 capsid and replicon were constructed successfully and the EV-D68 pseudovirus was prepared successfully. The RT-PCR and Western blotting showed that the constructed pseudovirus had the firefly luciferase reporter gene and the same capsid protein as the live virus. The copy number was(4-8) ×106 copies/m L. The pseudovirus infection dose was linearly positively correlated with the RLU(relative light unit) values. Conclusion The EV-D68 pseudovirus was successfully constructed in this study. | ||
| 610 | 2 | 4 | |a 中国食品药品检定研究院肝炎病毒疫苗研究室 |
| 610 | 2 | 4 | |a 兰州生物制品研究所有限责任公司第二研究室 |
| 650 | 4 | |a 医学微生物学（病原细菌学、病原微生物学） | |
| 650 | 4 | |a 基础医学 | |
| 650 | 4 | |a 医药、卫生 | |
| 650 | 4 | |a Biology | |
| 650 | 4 | |a Fundamental Medicine | |
| 650 | 4 | |a 医药卫生科技 | |
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| 650 | 4 | |a 理工A(数学物理力学天地生) | |
| 650 | 4 | |a Mathematics/ Physics/ Mechanics/ Astronomy | |
| 650 | 4 | |a 肠道病毒D组68型 | |
| 650 | 4 | |a 假病毒 | |
| 650 | 4 | |a 鉴定方法 | |
| 650 | 4 | |a 急性弛缓性脊髓炎 | |
| 650 | 4 | |a Enterovirus D68 | |
| 650 | 4 | |a Pseudovirus | |
| 650 | 4 | |a Identification method | |
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