Cloning Expression and Purification of Envelope Proteins E1 and E2 of Western Equine Encephalitis Virus and Potential use of them as Antigens in Immunoassays

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Authors
  1. Hu, W-.G.
  2. Chau, D.
  3. Wong, C.
  4. Masri, S.A.
  5. Fulton, R.E.
  6. Nagata, L.P.
Corporate Authors
Defence R&D Canada - Suffield, Ralston ALTA (CAN);British Columbia Univ, Vancouver BC (CAN) Dept of Microbiology and Immunology
Abstract
The genes encoding envelope proteins El and E2 of western equine encephalitis virus (WEEV) were respectively cloned into a prokaryotic T7 RNA polymerase-regulated expression vector. The recombinant C-terminal 6x-His-tagged WEEV El and E2 were expressed in bacteria as inclusion bodies that were subsequently solubilized with 8 M urea, purified by immobilized metal ion affinity chromatography and finally refolded using an arginine system. The purified 6 x His-tagged proteins showed 50 kDa bands as revealed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, consistent with the expected sizes of WEEV El and E2. The potential of the recombinant WEEV E1 and E2 as antigens for serologic tests to detect anti-WEEV antibodies for diagnosis of WEEV infection was assessed by an enzyme-linked immunosorbent assay with anti-WEEV polyclonal antibodies obtained from the mice infected with WEEV. The anti-WEEV antibodies bound the recombinant WEEV E1 and E2 in a dose dependent manner. On the contrary, antibodies against Venezuelan equine encephalitis virus with a genetic background and a disease spectrum very similar to WEEV, did not bind to the recombinant WEEV E1 and E2. Our results suggest that the recombinant WEEV El and E2 possess predominant antigenicity of WEEV and have the potential to be used as antigens in immunoassays to detect anti-WEEV antibodies for serological diagnosis of WEEV infection so as to eliminate the need for preparation of cell culture-derived viral antigens
Keywords
Western equine encephalitis virus;Recombinant E1E2;Prokaryotic expression;Antigens;Enzyme-linked immunosorbent assay
Report Number
DRDC-SUFFIELD-SL-2007-184 — Scientific Literature
Date of publication
30 Jul 2007
Number of Pages
6
Reprinted from
Veterinary Microbiology, vol 128, 2007, p 374-379
DSTKIM No
CA031444
CANDIS No
530331
Format(s):
Electronic Document(PDF);Hardcopy

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