人细小病毒B19病毒样颗粒的制备

Preparation of human parvovirus B19 virus-like particles

摘要:

采用昆虫杆状病毒表达系统, 制备人细小病毒B19病毒样颗粒(VLPs)。先通过PCR方法合成细小病毒B19衣壳蛋白基因VP2, 将其克隆到pFastBac1质粒, 然后转化含杆状病毒穿梭载体Bacmid的E. coli DH10Bac感受态细胞, 获得重组杆状病毒表达质粒Bacmid-VP2。在脂质体介导下转染Sf9昆虫细胞, 包装重组杆状病毒rBac-VP2。利用rBac-VP2感染Sf9细胞表达B19 VP2蛋白, 通过间接免疫荧光、Western blotting等方法鉴定目的蛋白表达。采用两次超速离心的方法对表达产物进行纯化, 纯化产物在透射电镜下可见直径约22 nm的VLPs。本研究成功制备了人细小病毒B19的VLPs, 为B19感染血清学检测方法的建立提供了参考。

Abstract:

The baculovirus expression system was employed to prepare the virus-like particles (VLPs) of human parvovirus B19. The synthesized VP2 gene of B19 was inserted into the multi-cloning site (MCS) of pFastBac1 vector; the resulting plasmid was transferred to the Escherichia coli DH10Bac competent cells, which contain a baculovirus shuttle vector (Bacmid), to generate Bacmid-VP2 by site-specific transposition. Recombinant baculovirus carrying VP2 gene (rBac-VP2) was then rescued from Bacmid-VP2-transfected Sf9 cells. Indirect immunofluorescence and Western blotting were used to identify the VP2 protein in rBac-VP2-infected Sf9 cells, and the VLPs were observed under transmission electron microscope after being enriched by ultracentrifugation. The B19 VLPs were successfully produced in insect cells with baculovirus expression system, which will facilitate the development of diagnostic reagents to detect the antibody against B19 virus in human serum.