A truncated hepatitis E virus ORF2 protein expressed in tobacco plastids is immunogenic in mice.
OBJECTIVE
To cost-effectively express the 23-ku pE2, the most promising subunit vaccine encoded by the E2 fragment comprising of the 3'-portion of hepatitis E virus (HEV) open reading frame 2 (ORF2) in plastids of tobacco (Nicotiana tabacum cv. SR1), to investigate the transgene expression and pE2 accumulation in plastids, and to evaluate the antigenic effect of the plastid-derived pE2 in mice.


METHODS
Plastid-targeting vector pRB94-E2 containing the E2 fragment driven by rice psbA promoter was constructed. Upon delivery into tobacco plastids, this construct could initiate homologous recombination in psaB-trnfM and trnG-psbC fragments in plastid genome, and result in transgene inserted between the two fragments. The pRB94-E2 was delivered with a biolistic particle bombardment method, and the plastid-transformed plants were obtained following the regeneration of the bombarded leaf tissues on a spectinomycin-supplemented medium. Transplastomic status of the regenerated plants was confirmed by PCR and Southern blot analysis, transgene expression was investigated by Northern blot analysis, and accumulation of pE2 was measured by ELISA. Furthermore, protein extracts were used to immunize mice, and the presence of the pE2-reactive antibodies in serum samples of the immunized mice was studied by ELISA.


RESULTS
Transplastomic lines confirmed by PCR and Southern blot analysis could actively transcribe the E2 mRNA. The pE2 polypeptide was accumulated to a level as high as 13.27 microg/g fresh leaves. The pE2 could stimulate the immunized mice to generate pE2-specific antibodies.


CONCLUSIONS
HEV-E2 fragment can be inserted into the plastid genome and the recombinant pE2 antigen derived is antigenic in mice. Hence, plastids may be a novel source for cost-effective production of HEV vaccines.
Department of Botany, The University of Hong Kong, China.
Yuan-Xiang
YX
Zhou
Maggie Yuk-Ting
MY
Lee
James Ming-Him
JM
Ng
Mee-Len
ML
Chye
Wing-Kin
WK
Yip
Sze-Yong
SY
Zee
Eric
E
Lam
D000818:Animals; D015152:Blotting, Northern; D015139:Blotting, Southern; D016752:Hepatitis E virus / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D018087:Plastids / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000276:immunology; D014026:Nicotiana / Q000235:genetics*; D014614:Vaccines, Synthetic / Q000276:immunology*; D014761:Viral Hepatitis Vaccines / Q000276:immunology*; D014764:Viral Proteins / Q000235:genetics / Q000276:immunology*
C400971:ORF2 protein, Hepatitis E virus; D011994:Recombinant Proteins; D014614:Vaccines, Synthetic; D014761:Viral Hepatitis Vaccines; D014764:Viral Proteins