Amplification and cloning of the full-length genome of Japanese encephalitis virus by a novel long RT-PCR protocol in a cosmid vector.
A novel and rapid full-length long RT-PCR technique was established to produce genome-length cDNA from Japanese encephalitis virus. In vitro positive strand RNA transcripts from the full-length RT-PCR amplicon including T7 promoter sequences at the 5' end were proved to be infectious upon transfection. The full-length amplicon without the T7 promoter was cloned into a cosmid vector under the SP6 promoter. This stable clone, designated as pJEV-1, was characterised further and used as a genetic resource for generation of infectious RNA transcripts, gene manipulation and expression. The 'run-off' transcript from pJEV-1 with vector sequences at the either end of the insert was not infectious, but transcripts of the full-length PCR amplicon from pJEV-1 produced infectious virus upon transfection. A transcript with an engineered Xho I site from two ligated PCR fragments amplified from pJEV-1 was also infectious. Furthermore, the coding region for premembrane and envelope proteins (preM-E) from pJEV-1 was subcloned and expressed in the Drosophila Expression System. The expressed protein showed correct molecular size and was immunoreactive with a Japanese encephalitis virus E protein-specific antibody. The derivation of genome-size cDNA from Japanese encephalitis virus and the stable clone will facilitate investigation of this virus and elucidation of its pathogenesis at the molecular level.
Zhang
F
F
Department of Microbiology, the Fourth Military Medical University, Xi'an, 710032, Shaanxi, People's Republic of China.
Huang
Q
Q
Ma
W
W
Jiang
S
S
Fan
Y
Y
Zhang
H
H
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods; D003360:Cosmids / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics; D004664:Encephalitis Virus, Japanese / Q000235:genetics* / Q000472:pathogenicity; D004672:Encephalitis, Japanese / Q000821:virology; D005784:Gene Amplification; D005822:Genetic Vectors / Q000235:genetics*; D016679:Genome, Viral; D051379:Mice; D008807:Mice, Inbred BALB C; D012367:RNA, Viral / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic
D018076:DNA, Complementary; D012367:RNA, Viral