Expression and immunogenicity of recombinant glycoprotein D of herpes simplex virus 1 in Drosophila S2 cells

Hongyan Mao, Xiaofei Zhao, Hongjuan Zhu, Jingxia Guo, Zhenghai Ma
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引用次数: 2

Abstract

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.
单纯疱疹病毒1型重组糖蛋白D在果蝇S2细胞中的表达及免疫原性
单纯疱疹病毒1型(HSV-1)是导致普通人群唇疱疹的罪魁祸首,但在某些情况下也会导致其他更严重的疾病的发展。病毒糖蛋白D (gD)是病毒进入宿主细胞所必需的。本研究利用果蝇Schneider 2 (S2)表达系统(DES)对重组gD1的表达进行了评价。将编码全长gD1的DNA序列(369aa, FLgD1)和对应外域的截断gD1形式(314aa, EgD1)克隆到S2表达载体pMT/BiP/V5-HisA中,分别生成pMT-EgD1和pMT-FLgD1。两种形式的gD1基因都带有六组氨酸标签,以方便其纯化。使用极限稀释法选择表达最高gD1水平的细胞群。Western blot,流式细胞术(FACS)和共聚焦免疫荧光分析表明全长型被抑制在细胞的脂质膜中,外畴型被分泌到培养基中。重组胞外结构域gD1按比例放大,用镍腈三乙酸亲和层析法从培养基中纯化,用5µM CdCl2诱导S2细胞摇瓶培养4天后,重组胞外结构域gD1的最大产量为56.8 mg/L。然后用重组纯化的gD1免疫小鼠,产生高滴度的抗体,用酶联免疫吸附法(ELISA)测定;1:52,120,000)以及高斑块中和效价(1:20 20)。综上所述,这些数据表明,在S2细胞中稳定表达gD1是一种合成gD1的实用方法,可用于进一步的结构和功能研究。
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