- Author:
Jeong Min KIM
1
;
Sang Im YUN
;
Byung Hak SONG
;
Yu Jeong CHOI
;
Jun Sun PARK
;
Young Min LEE
Author Information
- Publication Type:Original Article
- Keywords: Japanese encephalitis virus; Flavivirus; RNA-dependent RNA polymerase; NS5
- MeSH: Animals; Antibody Formation; Asia, Southeastern; Asian Continental Ancestry Group; Australia; Clone Cells; DNA; Encephalitis Virus, Japanese; Encephalitis, Japanese; Encephalitis, Viral; Flavivirus; Genome; Humans; Immune Sera; Immunization; Mice; Proteins; RNA; RNA Replicase; Sprains and Strains; Vero Cells
- From:Journal of Bacteriology and Virology 2009;39(1):53-60
- CountryRepublic of Korea
- Language:Korean
- Abstract: Japanese encephalitis virus (JEV), a member of mosquito-borne flaviviruses, is the leading cause of viral encephalitis in a large geographic area of Southeast Asia and Australia. JEV contains a single-stranded positive-sense RNA genome, which encodes its own RNA-dependent RNA polymerase (NS5) that is required for genomic RNA replication. In this study, we have described a pair of mouse antisera specific to the N- or C-terminal region of the NS5. Initially, two hydrophilic regions corresponding to the N-terminus and C-terminus of the NS5 protein were individually amplified by reverse transcription-PCR from the genomic RNA of JEV K87P39 strain. The amplified DNA fragments were cloned into a prokaryotic expression vector, pGEX-4T-1; the resulting constructs were used for the expression of GST fusion proteins, designated GST/NS5N and GST/NS5C, in E. coli BL-21 strain. Following immunization of three BALB/c mice with each of the purified GST/NS5N and GST/NS5C, we obtained two pools of the antisera, specifically recognizing the ~103-kDa NS5 and several smaller NS5-related proteins in BHK-21 and Vero cells infected with JEV K87P39 strain. Overall, we have successfully expressed the N- and C-terminal regions of JEV NS5 fused to the C-terminus of GST and generated the mouse antisera capable of recognizing the NS5 and its related proteins in JEV-infected cells. This would provide a valuable reagent for the study of JEV NS5 in the viral life cycle.