人细小病毒B19病毒样颗粒的制备  

Preparation of human parvovirus B19 virus-like particles

作　　者：邹小辉[1,2] 董流昕[2] 宋敬东[2] 屈建国[2] 于修平[1] 鲁茁壮[2] 洪涛[2] 

机构地区：[1]山东大学医学院微生物学教研室,济南250012 [2]中国疾病预防控制中心病毒病预防控制所,北京100052

出　　处：《生物工程学报》2009年第4期575-579,共5页Chinese Journal of Biotechnology

基　　金：国家科技支撑计划(No.2008BAI56B01)资助~~

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.coliDH10Bac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B19VP2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22nm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。The baculovirus expression system was employed to prepare the virus-like particles （VLPs） of human parvovirus B 19. The synthesized VP2 gene of B19 was inserted into the multi-cloning site （MCS） of pFastBacl 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 ceils, 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 B 19 virus in human serum.

关 键 词：细小病毒 B19 VP2 杆状病毒 病毒样颗粒 

分 类 号：R373[医药卫生—病原生物学]