[Molecular cloning and expression of E2 gene of the Chinese classical swine fever virus(shimen strain) and preliminary studies of its DNA vaccine]

Wei Sheng Wu Xue Bao. 2000 Jun;40(3):243-51.
[Article in Chinese]

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • COS Cells
  • Cell Line
  • Classical Swine Fever Virus / immunology*
  • Cloning, Molecular
  • Gene Expression
  • Mice
  • Mice, Inbred BALB C
  • Molecular Sequence Data
  • Sequence Homology
  • Spodoptera
  • Vaccines, DNA / immunology*
  • Viral Envelope Proteins / biosynthesis
  • Viral Envelope Proteins / genetics*
  • Viral Envelope Proteins / immunology*
  • Viral Vaccines / immunology*

Substances

  • Vaccines, DNA
  • Viral Envelope Proteins
  • Viral Vaccines
  • glycoprotein E2, classical swine fever virus