Rescue of Hantaan virus 84FLi strain minigenomes by RNA polymerase Ⅰ-driven system
10.3760/cma.j.issn.1000-6680.2009.01.003
- VernacularTitle:应用RNA聚合酶Ⅰ反向遗传操作技术拯救汉滩病毒84FLi株微基因组
- Author:
Ye ZHANG
;
Xinhong LI
;
Hong JIANG
;
Changxing HUANG
;
Pingzhong WANG
;
Li SUN
;
Xuefan BAI
- Publication Type:Journal Article
- Keywords:
Hantaan virus;
Genetic techniques;
Minigenome;
DNA-directed RNA polyrnerases
- From:
Chinese Journal of Infectious Diseases
2009;27(1):6-10
- CountryChina
- Language:Chinese
-
Abstract:
Objective To develop a reverse genetics system for Hantaan virus (HTNV) 84FLi strain by using RNA polymerase [ (pol Ⅰ)-mediated transcription. Methods Complementary DNA (cDNA) containing the coding sequence for chloramphenicol acetyhransferase (CAT) was inserted into the 5'-and 3'-terminal untranslated regions of HTNV 84FLi L segment. These chimeric cDNAs (pol Ⅰ expression cassette) were cloned into plasmids and between the human pol Ⅰ promoter and terminator to generate sense and anti-sense RNA pol Ⅰ transcription reporter plasmids. The reporter plasmids were transfeeted into 293T cells or the 1:1 combination of 293T and HTNV infected Vero cells. These cells were cotransfected with expression plasmids encoding Ⅰ. (RNA dependent RNA polymerase) and N (nucleoprotein) viral proteins, Cells were harvested 48 h post-transfection and the CAT activity was detected. The 293T cells were infected with the supernatant to explore the passage ability of CAT activity. ResultsThe reporter plasmids pLvRNA-CAT and pLcRNA-CAT were constructed successfully. CAT activity was detected in transfected cells and could also be serially passaged in the rescued virus minigenomes. Conclusion The RNA polymerase ]-driven reverse genetics system successfully rescues HTNV 84FLi minigenomes.
