Cloning, expression and purification of envelope proteins E1 and E2 of western equine encephalitis virus and potential use of them as antigens in immunoassays.
The genes encoding envelope proteins E1 and E2 of western equine encephalitis virus (WEEV) were respectively cloned into a prokaryotic T7 RNA polymerase-regulated expression vector. The recombinant C-terminal 6xHis-tagged WEEV E1 and E2 were expressed in bacteria as inclusion bodies that were subsequently solubilized with 8M urea, purified by immobilized metal ion affinity chromatography and finally refolded using an arginine system. The purified 6xHis-tagged proteins showed 50kDa bands as revealed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, consistent with the expected sizes of WEEV E1 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 E1 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, which is time-consuming, expensive, laborious, tedious, and hazardous.
Hu
Wei-Gang
WG
Biotechnology Section, Defence Research and Development Canada-Suffield, Box 4000, Station Main, Medicine Hat, Alberta, Canada T1A 8K6. wei-gang.hu@drdc-rddc.gc.ca
Chau
Damon
D
Wong
Christina
C
Masri
Saad A
SA
Fulton
R Elaine
RE
Nagata
Les P
LP
D000818:Animals; D000914:Antibodies, Viral / Q000097:blood; D000956:Antigens, Viral / Q000276:immunology*; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel / Q000662:veterinary; D004667:Encephalitis Virus, Western Equine / Q000235:genetics*; D004683:Encephalomyelitis, Equine / Q000662:veterinary* / Q000821:virology; D004797:Enzyme-Linked Immunosorbent Assay / Q000662:veterinary; D015967:Gene Expression Regulation, Viral; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D012698:Serologic Tests / Q000662:veterinary; D014709:Vero Cells; D014759:Viral Envelope Proteins / Q000235:genetics*
D000914:Antibodies, Viral; D000956:Antigens, Viral; D012367:RNA, Viral; D014759:Viral Envelope Proteins