title: Efficient production of Japanese encephalitis virus-like particles by recombinant lepidopteran insect cells.
abstract: The production of Japanese encephalitis (JE) virus-like particles (VLPs) in stably transformed lepidopteran insect cells was investigated. The DNA fragment encoding the JE virus (JEV) prM signal peptide, the precursor (prM) of the viral membrane protein (M), and the envelope glycoprotein (E) was cloned into the plasmid vector pIHAbla. The pIHAbla contained the Bombyx mori actin promoter downstream of the B. mori nucleopolyhedrovirus (BmNPV) IE-1 transactivator and the BmNPV HR3 enhancer for high-level expression, together with a blasticidin resistance gene for use as a selectable marker. DNA encoding a form of prM with a pr/M cleavage site mutation was used to suppress the cell-fusion activity of VLPs. After transfection with the resultant plasmid, Trichoplusia ni BTI-TN-5B1-4 (High Five) cells were incubated with blasticidin, and cells resistant to the antibiotic were obtained. Western blot analysis and enzyme-linked immunosorbent assay of a culture supernatant showed that transfected High Five cells secreted an E antigen equivalent to the authentic JEV E. Sucrose density-gradient sedimentation analysis of the culture supernatant from recombinant High Five cells indicated that secreted E antigen molecules were produced in a particulate form. VLPs recovered from the supernatant successfully induced neutralizing antibodies in mice, particularly when adsorbed to alum adjuvant. High yields (≈30 μg/ml) of E antigen were achieved in shake-flask cultures. These results indicate that recombinant insect cells may offer a novel approach for efficient VLP production.
authors.affiliation: Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodai, Nada, Kobe, 657-8501, Japan. yamaji@kobe-u.ac.jp
authors.forename: Hideki
authors.initials: H
authors.lastname: Yamaji
authors.forename: Masataka
authors.initials: M
authors.lastname: Nakamura
authors.forename: Miwa
authors.initials: M
authors.lastname: Kuwahara
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Takahashi
authors.forename: Tomohisa
authors.initials: T
authors.lastname: Katsuda
authors.forename: Eiji
authors.initials: E
authors.lastname: Konishi
mesh_terms: D000818:Animals; D057134:Antibodies, Neutralizing / Q000097:blood; D000914:Antibodies, Viral / Q000097:blood; D001709:Biotechnology / Q000379:methods*; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D022321:Japanese Encephalitis Vaccines / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D007915:Lepidoptera; D008297:Male; D051379:Mice; D008809:Mice, Inbred C3H; D013678:Technology, Pharmaceutical / Q000379:methods*; D058425:Vaccines, Virus-Like Particle / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
chemical_list: D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D022321:Japanese Encephalitis Vaccines; D058425:Vaccines, Virus-Like Particle