Grabherr R, Ernst W, Doblhoff-Dier O, Sara M, Katinger H
University of Agriculture, Food Science and Forestry, Vienna, Austria.
Biotechniques. 1997 Apr;22(4):730-5. doi: 10.2144/97224rr02.
Based on the method of direct cloning into the baculovirus genome by linearizing and re-ligation in presence of the target insert, we designed viral constructs that express foreign genes on the surface of baculovirus particles. We chose the glycosylated envelope protein gp41 of human immunodeficiency virus type 1 (HIV-1) as a model for displaying recombinant proteins on budded virus. The ectodomain of the envelope protein gp41 of HIV-1 was being fused to the entire baculovirus major coat protein gp64 (Ac-cops41) and to the membrane anchor sequence of gp64 (Acmars41). Two different promoters, the "very late" polyhedrin promoter (Ac-mars41) and the "early and late" gp64 promoter (Ac-promars41) were compared. The expression of gp41 in infected cells and its presence on viral particles was confirmed by enzyme-linked immunosorbent assay (ELISA), Western blot and electron microscopy.
基于在目标插入片段存在的情况下通过线性化和重新连接直接克隆到杆状病毒基因组的方法，我们设计了在杆状病毒颗粒表面表达外源基因的病毒构建体。我们选择1型人类免疫缺陷病毒（HIV-1）的糖基化包膜蛋白gp41作为在出芽病毒上展示重组蛋白的模型。HIV-1包膜蛋白gp41的胞外结构域与整个杆状病毒主要衣壳蛋白gp64（Ac-cops41）以及gp64的膜锚定序列（Acmars41）融合。比较了两种不同的启动子，即“极晚期”多角体蛋白启动子（Ac-mars41）和“早期和晚期”gp64启动子（Ac-promars41）。通过酶联免疫吸附测定（ELISA）、蛋白质印迹法和电子显微镜证实了gp41在感染细胞中的表达及其在病毒颗粒上的存在。
