Expression of particulate-form of Japanese encephalitis virus envelope protein in a stably transfected Drosophila cell line.
BACKGROUND
Japanese encephalitis virus (JEV), a member of the family Flaviviridae, is an important mosquito-borne human pathogen. Its envelope glycoprotein (E) is the major determinant of the pathogenicity and host immune responses. In the present study, we explored the feasibility of producing recombinant JEV E protein in the virus-free Drosophila expression system.


RESULTS
The coding sequence for the signal sequence of premembrane and E protein was cloned into the Drosophila expression vector pAc5.1/V5-His. A Drosophila cell line S2 was cotransfected with this construct as well as a plasmid providing hygromycin B resistance. A cell line expressing the JEV E protein was selected by immunofluoresence, confocal microscopy, and western blot analysis using three different monoclonal antibodies directed against JEV E protein. This cell line was stable in the yield of JEV E protein during two months in vitro maintenance in the presence of hygromycin B. The results showed that the recombinant E protein had an expected molecular weight of about 50 kilodalton, was immunoreactive with all three monoclonal antibodies, and found in both the cytoplasm and culture supernatant. Sucrose gradient ultracentrifugation analysis revealed that the secreted E protein product was in a particulate form. It migrated to the sucrose fraction with a density of 1.13 g/ml. Balb/c mice immunised with the sucrose fraction containing the E protein particles developed specific antibodies. These data show that functioning JEV E protein was expressed in the stable S2 cell line.


CONCLUSIONS
The Drosophila expression system is a more convenient, cheaper and safer approach to the production of vaccine candidates and diagnostic reagents for JEV.
Zhang
Fuquan
F
Division of Biomedical Sciences, Faculty of Medicine, Imperial College London, London, UK. fuquan.zhang@bbsrc.ac.uk
Ma
Wenyu
W
Zhang
Li
L
Aasa-Chapman
Marlen
M
Zhang
Hongyi
H
D000818:Animals; D000914:Antibodies, Viral / Q000097:blood; D015153:Blotting, Western; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D003470:Culture Media / Q000737:chemistry; D003593:Cytoplasm / Q000737:chemistry; D004330:Drosophila; D004664:Encephalitis Virus, Japanese / Q000235:genetics* / Q000276:immunology; D005822:Genetic Vectors; D007114:Immunization; D051379:Mice; D008807:Mice, Inbred BALB C; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology; D014162:Transfection; D014759:Viral Envelope Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000276:immunology
D000914:Antibodies, Viral; D003470:Culture Media; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins