家蚕核型多角体病毒orf25基因在病毒感染过程中 编码一个30kDa的晚期蛋白(英文)

Bombyx mori nucleopolyhedrovirus orf25 encodes a 30kDa late protein in the infection cycle

摘要:

首次对家蚕核型多角体orf25基因进行了描述。扩增Bm25基因, 亚克隆到原核表达载体pGEX-4T-2, 在大肠杆菌BL21 (DE3)中表达含有GST标签的融合蛋白。IPTG诱导后高效表达GST-Bm25融合蛋白。纯化的融合蛋白免疫新西兰大白兔制备多克隆抗体。利用制备的抗GST-Bm25融合蛋白的多克隆抗体进行表达时相分析显示: 24 h p.i.检测到30 kDa的蛋白条带。RT-PCR方法, 在18-72 h p.i 检测到Bm25基因的转录本。结论: 以上数据表明Bm25基因编码一晚期表达的30kDa蛋白。

关键词:BmNPV; Bm25; 表达时相; RT-PCR; 多克隆抗体

Abstract:

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.

Key words:BmNPV; Bm25; Expression temporal; RT-PCR; polyclonal antibody