杆状病毒AcMNPV orf59基因的克隆表达、抗体制备及亚细胞定位

用PCR方法从苜蓿丫纹夜蛾核多角体病毒（Autographa californica nucleopolyhedrovirus，AcMNPV）基因组中扩增到orf59基因，插入原核表达载体pET-32a(+)，构建pET-Ac59质粒，再将该质粒转化大肠杆菌BL21，在IPTG诱导下表达了分子量约为30kDa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orf59基因的表达，结果显示：在感染后的细胞中有两条分子量分别约为38kDa和25kDa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，Ac59蛋白同时存在于所感染宿主的细胞核和细胞质中。这些研究为下一步深入进行Ac59功能的研究奠定了一定的基础。

Cloning and Expression of Autographa californica nucleopolyhedrovirus orf59 Gene and Its Antibody Preparation and Subcellular Localization

Autographa californica nucleopolyhedrovirus (AcMNPV) orf59 gene was amplified from AcMNPV genomic DNA via PCR. The PCR product was cloned into the expression vector pET-32a(+) and transformed into Escherichia coli BL21, and a fusion protein around 30kDa was expressed after induction with IPTG (isopropylthio-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 Sf9 cells showed two specific polypeptides with apparent sizes of 38kDa and 25kDa for the Ac59 antiserum. The indirect immunofluoresecence analysis demonstrated that the Ac59 protein was distributed in cytoplasm and nucleus of infected cells at the late stage of infection. This study lays a foundation for the further research of the gene function.