棉铃虫核型多角体病毒(HaSNPV)ORF99基因在昆虫细胞中的表达及亚细胞定位

Expression and subcellular location of ORF99 from Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus(HaSNPV)

棉铃虫核型多角体病毒ORF99基因Ha99是其基因组中特有的一个基因,序列分析发现Ha99基因开放阅读框包含354 bp核苷酸,编码118个氨基酸残基,预测相对分子质量为13 kDa.Ha99基因进一步在Bac-to-Bac系统中进行表达,SDS-PAGE和Western blot分析显示Ha99基因得到了成功表达,表达的相对分子质量为13kDa,与预测的大小相符.亚细胞定位分析表明,ORF99基因的表达产物主要定位在细胞质中.时序表达分析显示Ha99基因在病毒侵染后6 h开始表达,一直持续到侵染后122 h,表明Ha99基因是一个晚期表达的基因.

The ORF99 of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus（HaSNPV）（Ha99） is one of the 20 genes that are unique to HaSNPV.Computer-assisted analysis revealed that open reading frame of Ha99 was 354 bp long in size encoded 118 amino acid residues with predicted 13 kDa molecular weight.SDS-PAGE and Western blot results suggested that Ha99 protein was expressed by using Bac-to-Bac system.The molecular weight of expressed protein is 13 kDa,consisting of the predicted molecular weight.The subcellular location was studied using EGFP-Ha99,which suggested that the Ha99 protein is primarily localized in the cytoplasm.The Ha99 was also detected at 6 h.p.i and remained detectable until 122 h.p.i.by RT-PCR,suggesting that Ha99 is a late expression gene.