Construction and rescue of EGFP-enterovirus type 71 recombinant virus
10.3760/cma.j.cn112866-20191231-00212
- VernacularTitle:肠道病毒71型EGFP标记重组病毒的构建与拯救
- Author:
Hailu ZHANG
1
;
Shaoxia SONG
;
Kai WANG
;
Xin WANG
;
Shuhan LI
;
Li ZHAO
;
Zhiyu WANG
;
Hongling WEN
Author Information
1. 山东大学齐鲁医学院公共卫生学院微生物检验学系 山东省"十三五"高等学校感染性疾病防控重点实验室，济南 250012
- Keywords:
Enterovirus;
Enhanced green fluorescent protein;
Virus rescue
- From:
Chinese Journal of Experimental and Clinical Virology
2020;34(5):511-515
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To construct and rescue enhanced green fluorescent protein(EGFP)-labeled EGFP-EV-A71 recombinant virus.Methods:pMD19T-SDLY107 full-length plasmid was used as a template, and 2A protease recognition sequence was added to the 5′end of the structural protein VP4. EGFP gene was amplified using the pEGFP-N1 plasmid as a template and inserted into the above-mentioned recombinant plasmid. RD cells were transfected to rescue the recombinant virus EGFP-EV-A71 after enzymatic digestion and in vitro transcription. The cell culture infectious dose 50% endpoint (CCID 50) was determined. Real-time fluorescent quantitative PCR (qRT-PCR) was used to detect the virus replication level at different time points, and the virus replication curve was drawn to compare the replication ability of the recombinant virus and the parent virus. Lactate dehydrogenase (LDH) and cell proliferation (CCK-8) experiments were used to determine the cell injury rate and survival rate of infected cells, respectively. Results:Recombinant EV-A71 infectious cDNA clones containing specific restriction sites and EGFP were successfully constructed. Typical cytopathic effect and green fluorescence was observed. The qRT-PCR replication curve showed that the recombinant virus had similar replication kinetics to the parent virus.Conclusions:EGFP-labeled enterovirus type 71 recombinant virus EGFP-EV-A71 was successfully rescued.
