Construction and application of enterovirus D68 infectious clone expressing enhanced green fluorescent protein
10.3760/cma.j.cn112309-20241114-00386
- VernacularTitle:表达绿色荧光蛋白的肠道病毒D68感染性克隆的构建与应用
- Author:
Dong ZHANG
1
;
Fengyu CHI
;
Xu ZHANG
;
Yuting ZHAO
;
Xiaoqian WANG
;
Juan LI
;
Zhenjie ZHANG
;
Jie TONG
;
Yuming LI
Author Information
1. 山东第一医科大学（山东省医学科学院）公共卫生与健康管理学院，济南 250117
- Publication Type:Journal Article
- Keywords:
Enterovirus;
Infectious clone;
Enhanced green fluorescent protein;
Drug evaluation
- From:
Chinese Journal of Microbiology and Immunology
2025;45(11):906-913
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To construct a full-length infectious clone of enterovirus D68（EV-D68）expressing enhanced green fluorescent protein（EGFP）by reverse genetics in order to provide an efficient tool for studying the biological characteristics and screening antiviral drugs for EV-D68.Methods:Gene synthesis and overlap PCR techniques were used to construct the full-length clone plasmid pUC57-EV-D68 of EV-D68. The full-length viral sequence was then transferred into the pCAGGS plasmid to obtain the pCAGGS-EV-D68 plasmid. The EGFP gene was amplified and inserted into the pCAGGS-EV-D68 plasmid to construct the pCAGGS-EGFP-EV-D68 plasmid. Then，the two constructed plasmids were transfected into human rhabdomyosarcoma（RD）cells to rescue recombinant viruses RV-EV-D68 and RV-EGFP-EV-D68. The rescued viruses were identified using PCR，Western blot，and immunofluorescence techniques. The antiviral effect of doxycycline was evaluated using the rescued RV-EGFP-EV-D68. Statistical analysis was performed using the two independent samples t-test. Results:The recombinant virus RV-EGFP-EV-D68 capable of expressing EGFP was successfully rescued. Even after 15 serial passages，the virus retained EGFP expression with no significant difference in viral titers compared to the parental virus，indicating its stable passage in RD cells. Besides，the rescued strains exhibited similar replication characteristics to the parental virus. While at 24 and 36 h after infection，the titers of the rescued strains were significantly lower than that of the parental strain（both P<0.05）. This study demonstrated that doxycycline significantly reduced the fluorescence intensity of RV-EGFP-EV-D68-infected RD cells（ P<0.01）. Meanwhile，a negative correlation was observed between the doxycycline concentration and the fluorescence intensity，indicating that the rescued virus could be used for antiviral drug evaluation. Conclusions:This study successfully constructs an infectious clone of EV-D68 expressing EGFP. The rescued recombinant virus RV-EGFP-EV-D68 has been verified to be applicable for the evaluation of antiviral drugs.
