CPB 697 RESEARCH SEMINAR
Yi-Ning Chen, BVM, MVM
Graduate Student in Molecular Virology
Department of Comparative Pathobiology
Purdue University
“Identification And Characterization Of Turkey Coronavirus Neutralizing Epitope”
Thursday, April 24, 2008
VPTH 112
3:30 pm
Abstract:
Spike (S) protein of coronavirus plays a role in eliciting virus-specific neutralizing antibody. The primary objectives of the present study were to identify and characterize the neutralizing epitope in S protein of turkey coronavirus (TCoV). Spike gene was arranged into 10 fragments to map the linear epitopes. Each fragment was cloned into an expression vector (pTriEx3 or pcDNA3.1). The cloned fragment was transfected to Cos-7 cells. The expression and antigenicity of inserted gene fragments was confirmed by immunofluorescence antibody (IFA) assay using anti-His monoclonal antibody (Mab) or anti-TCoV turkey serum. Two-week-old turkeys were intramuscularly inoculated with the His-positive and TCoV-positive DNA plasmids carrying the S gene fragments or their expressed proteins multiple times in various intervals for a total of 3 months. Sera were collected after the first inoculation for antibody-capture enzyme-linked immunosorbent assay (ELISA), IFA, or virus neutralization (VN) titers to TCoV. DNA plasmid coding for S1 gene (pTriEx3-S1), S gene fragment spanning 3’end of S1 gene and 5’ end of S2 gene (pTriEx3-4F/4R), S fragment covering 3’ end of S1 gene (pTriEx3-Mod4F/Epi4R), or S gene fragment flanking 3’ end of S2 gene (pTriEx3-6F/6R) was transfected to Cos-7 cells and the expressed protein was positively reacted with anti-His Mab and anti-TCoV serum by IFA. The highest serum ELISA optical density (O.D.) value to TCoV of 4.0 was obtained from turkeys inoculated with pTriEx3-4F/4R and boosted with expressed 4F/4R protein, pTriEx3-6F/6R boosted with expressed 6F/6R protein or pTriEx3-Mod4F/Epi4R boosted with Mod4F/Epi4R protein. The highest IFA titer to TCoV was from the antiserum induced by expressed 4F/4R protein, 6F/6R protein, and Mod4F/Epi4R protein were 1:400, 1:200, and 1:200, respectively. The VN titers against TCoV from the antiserum induced by boosting with expressed 4F/4R protein, 6F/6R protein, and Mod4F/Epi4R protein were 1:8, < 1:1, and 1:11, respectively. The results indicated that the protein (4F/4R or Mod4F/Epi4R) coding for 3’end of TCoV S1 gene possesses the neutralizing epitope, while the protein (6F/6R) encoding TCoV S gene fragment flanking 3’end of S2 gene has antigenic epitope, but not neutralizing epitope.