title: Expression and immunogenicity of recombinant glycoprotein D of herpes simplex virus 1 in Drosophila S2 cells.
abstract: Herpes simplex virus type 1 (HSV-1) is responsible for cold sores in the general population, but also contributes to the development of other more serious diseases in some circumstances. The viral glycoprotein D (gD) is essential for virus entry into host cells. In the present study, the Drosophila melanogaster Schneider 2 (S2) expression system (DES) was evaluated for the expression of recombinant gD1. The DNA sequences encoding the full-length gD1 (369aa, FLgD1) and a truncated gD1 form corresponding to the ectodomain (314aa, EgD1) were cloned into S2 expression vector pMT/BiP/V5-HisA to generate pMT-EgD1 and pMT-FLgD1, respectively. Two forms of gD1 gene were fitted with a hexahistidine tag to facilitate their purification. Cell populations expressing the highest gD1 levels were selected by using a limiting dilution assay. Western blot, flow cytometry (FACS), and confocal immunofluoresence assay demonstrated that the full-length form is restrained in the lipid membranes of the cell and the ectodomain form is secreted into the medium. Recombinant ectodomain gD1 was scaled up and purified from the culture medium using nickel nitrilotriacetic acid affinity chromatography, and a maximum production level of 56.8 mg/L of recombinant gD1 was obtained in a shake-flask culture of S2 cells after induction with 5 µM CdCl2 for 4 days. Mice were then immunized with recombinant purified gD1 and produced high titers of antibody measured by enzyme-linked immunosorbent assay (ELISA; 1:5,120,000) as well as high plaque neutralization titer (1:320). Overall, the data indicated that stable expression in S2 cells is a practical way of synthesizing gD1 for use in structural and functional studies in the further study.
authors.affiliation: a College of Life Science and Technology , Xinjiang University , Urumchi , China.
authors.forename: Hongyan
authors.initials: H
authors.lastname: Mao
authors.affiliation: a College of Life Science and Technology , Xinjiang University , Urumchi , China.
authors.forename: Xiaofei
authors.initials: X
authors.lastname: Zhao
authors.affiliation: a College of Life Science and Technology , Xinjiang University , Urumchi , China.
authors.forename: Hongjuan
authors.initials: H
authors.lastname: Zhu
authors.affiliation: a College of Life Science and Technology , Xinjiang University , Urumchi , China.
authors.forename: Jingxia
authors.initials: J
authors.lastname: Guo
authors.affiliation: a College of Life Science and Technology , Xinjiang University , Urumchi , China.
authors.forename: Zhenghai
authors.initials: Z
authors.lastname: Ma
mesh_terms: D000818:Animals; D004331:Drosophila melanogaster; D005260:Female; D051379:Mice; D008807:Mice, Inbred BALB C; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D014759:Viral Envelope Proteins / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification
chemical_list: D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; C039447:glycoprotein D, Human herpesvirus 1
keywords: Drosophila melanogaster Schneider 2 cells; glycoprotein D; herpes simplex virus type 1; immunogenicity