The presence of RNA splicing signals in the cDNA construct of the E2 gene of classical swine fever virus affected its expression.
E2 is the major neutralizing antigen for classical swine fever virus (CSFV) infection. Previously, we have cloned and sequenced the E2 cDNA of Taiwan strain p97 by the reverse transcription-polymerase chain reaction (RT-PCR) method from CSFV-infected tissue. The presence of RNA splicing donor and acceptor sites were found in the cDNA sequence. In this study, transfection of E2 cDNA into mammalian cells resulted in the production of a spliced RNA. Site-directed mutagenesis of the donor and acceptor sites prevented the RNA splicing event and generated a full length transcript in COS7 cells. Although the spliced E2 transcript has not been reported in natural infection of CSFV, this study suggested that the potential splicing sites affected the E2 gene expression when the plasmid-based E2 gene was introduced into mammalian cells.
Shiu
J S
JS
Graduate Institute of Basic Science, Chang-Gung University School of Medicine, Taoyuan, Taiwan.
Liu
S T
ST
Chang
T J
TJ
Ho
W C
WC
Lai
S S
SS
Chang
Y S
YS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002478:Cells, Cultured; D006692:Classical Swine Fever Virus / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics*; D015870:Gene Expression; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D012326:RNA Splicing; D013552:Swine; D014158:Transcription, Genetic; D014162:Transfection; D014759:Viral Envelope Proteins / Q000235:genetics*
D018076:DNA, Complementary; D014759:Viral Envelope Proteins; C087086:glycoprotein E2, classical swine fever virus