[Molecular cloning and expression of E2 gene of the Chinese classical swine fever virus(shimen strain) and preliminary studies of its DNA vaccine].
A 1.1 bp fragment of E2 gene of Chinese classical swine fever virus(CSFV) Shimen strain, a standard virulent strain, was amplified by RT-PCR from total RNA of cell cultures infected by CSFV, and cloned into pGEM T vector. The nucleotide sequence of this fragment was sequenced by Sanger's method and the amino acid sequence was deduced. Compared with the corresponding region of Alfort, Brescia and C strain of CSFV, the nucleotide sequence homology is 84.7%, 92.6% and 95.2% respectively, and the amino acid sequence 89.4%, 92.6% and 94.6%, respectively, we subcloned 1.1 bp of E2 gene cDNA into baculovirus transfer vector and successfully constructed two recombinant baculoviruses expressing GST-E2 and GST-GFP-E2 fusion protein respectively by homologous recombination in sf-9 cell. Furthermore, we also constructed recombinant eukaryotic expression vector pcE2 containing E2 gene in frame and transfected COS-7 cell by lipofectamine, the indirect immunofluorescence assay (IFA) showed that the expressed E2 protein can be recognized by E2 specific monoclonal antibody the pcE2 DNA was directly injected into BALB/c mice intramuscular(i.m.) and the CSFV E2-specific antibodies was measured by enzyme-linked immunosorbent assay(ELISA) the ELISA results indicated the E2-specific antibodies was induced in inoculated mice and virus neutralization assays also indicate single inoculations of plasmids expressing CSFV E2 glycoprotein raised neutralizing antibody in BALB/c mice. these results will be beneficial to investigate the possibility of DNA vaccine against CSFV.
Zhou
P
P
College of Life Sciences, Peking University, Beijing 100871.
Lu
Y
Y
Chen
J
J
Zhai
Z
Z
Ding
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002460:Cell Line; D006692:Classical Swine Fever Virus / Q000276:immunology*; D003001:Cloning, Molecular; D015870:Gene Expression; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D017385:Sequence Homology; D018411:Spodoptera; D019444:Vaccines, DNA / Q000276:immunology*; D014759:Viral Envelope Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology*; D014765:Viral Vaccines / Q000276:immunology*
D019444:Vaccines, DNA; D014759:Viral Envelope Proteins; D014765:Viral Vaccines; C087086:glycoprotein E2, classical swine fever virus