杆状病毒Ac60基因的原核表达与亚细胞定位研究

Bacterial Expression of Baculovirus Ac60 gene and Its Subcellular Localization

用PCR方法从AcMNPV基因组中扩增到ORF60基因,插入原核表达载体pET-28a(+),构建pET-Ac60质粒,再将该质粒转化大肠埃希菌BL21,在IPTG诱导下表达了分子量约为16 ku的融合蛋白。用纯化的表达产物免疫新西兰大白兔制备了多克隆抗体,应用该抗体检测了AcMNPV感染的昆虫宿主细胞(Sf9)中ORF60基因的表达,结果显示:在感染后的细胞中有2条分子量分别约为33 ku和17 ku蛋白质带能与所制备的抗体发生特异性反应。间接免疫荧光分析发现:在病毒感染的晚期,Ac60蛋白同时存在于所感染宿主的细胞核和细胞质中。

The PCR product was cloned into the expression vector pET-28a（ ＋ ） and transformed into Escherichia colt BL21, and a fusion protein around 16 ku was expressed after induction with IPTG （isopropyhhio-β-D-galactoside）. Purified fusion protein was injected into New Zealand white rabbit to harvest polyclonal antibodies. Western blotting analysis of extracts of AcMNPV-infected Si9 cells showed two specific polypeptides with apparent sizes of 33 ku and 17 ku for the Ac60 antiserum. The indirect immunofluoresecenee analysis demonstrated that the Ac60 protein was distributed in cytoplasm and nucleus of infected cells at the late stage of infection.