Wang Haiyan, Chen Keping, Guo Zhongjian, Yao Qin
Institute of Life Sciences, Jiangsu University, Zhenjiang 212013, China.
Wei Sheng Wu Xue Bao. 2008 Feb;48(2):247-51.
Bombyx mori nucleopolyhedrovirus (BmNPV) orf25 gene was characterized for the first time. The coding sequence of Bm25 was amplified and subcloned into the prokaryotic expression vector pGEX-4T-2 to produce glutathione S-transferase-tagged fusion protein in the BL21 (DE3) cells. The GST-Bm25 fusion protein was expressed efficiently after induction with IPTG. The purified fusion protein was used to immunize New Zealand white rabbits to prepare polyclonal antibody. Temporal expression analysis revealed a 30-kDa protein, which was detected beginning 24 hours post-infection using a polyclonal antibody against GST-Bm25 fusion protein. The transcript of Bm25 was detected by RT-PCR at 18-72 h p.i. In conclusion, the available data suggest that Bm25 encodes a 30kDa protein expressed in the late stage of infection cycle.
首次对家蚕核型多角体病毒(BmNPV)的orf25基因进行了表征。扩增了Bm25的编码序列,并将其亚克隆到原核表达载体pGEX-4T-2中,以在BL21(DE3)细胞中产生谷胱甘肽S-转移酶标记的融合蛋白。用IPTG诱导后,GST-Bm25融合蛋白高效表达。纯化的融合蛋白用于免疫新西兰白兔以制备多克隆抗体。时序表达分析显示出一种30 kDa的蛋白质,使用针对GST-Bm25融合蛋白的多克隆抗体在感染后24小时开始检测到该蛋白质。在感染后18 - 72小时通过RT-PCR检测到Bm25的转录本。总之,现有数据表明Bm25编码一种在感染周期后期表达的30 kDa蛋白质。