本研究利用已知的颗粒体蛋白基因(granulin,gra)设计引物,通过PCR扩增得到ClanGV的gra基因。对 PCR结果序列分析表明,ClanGV的gra基因开放阅读框(ORF)全长747bp,共编码248个aa,预计编码的蛋白质 大小为29．3kDe。在启始密码子ATG上游-24bp处,有一个杆状病毒晚期启动子序列,ATAAG。有两个TATA 框,分别位于ATG上游的-26bp和-65bp位置。基于gra的同源分析和进化树分析表明,ClanGV和茶小卷叶蛾颗 粒体病毒(Adoxophyes orana granulovirus,AoGV)、苹果蠹蛾颗粒体病毒(Cydia pomonella granulovirus,CpGV)、 云杉卷叶蛾颗粒体病毒(Choristoneura fumiferana granulovirus,CfGV)、马铃薯块茎蛾颗粒体病毒(Phthorimaea operculella granulovirus,PoGV)的亲缘关系较近。利用提取的颗粒体蛋白免疫家兔,制备了抗体进行免疫杂交分 析,结果显示ClanGV除了与分月扇舟蛾颗粒体病毒(Clostera anastomosis L．granulovirus,CaLGV)的颗粒体蛋 白有较强的杂交信号外,与棉铃虫核多角体病毒(Helicoverpa armigera nucleopolyhedrovirus,HaNPV)多角体蛋 白也有明显的杂交带出现,与甜菜夜蛾核多角体病毒(Spodoptera exigua nucleopolyhedrovirus,SeNPV)只有非常 微弱的杂交信号。

Primers based on published sequences of granulin genes were synthesized and used to amplify the granulin gene from Clostera anachoreta granulovirus (ClanGV). The generated product was se-quenced. Sequence analysis indicated the entire ORF of ClanGV granulin gene was 747bp, encoding a protein of 248 aa with an estimated molecular weight of 29. 3kDa. A bacluloviral late promoter was present at 24 bp upstream of the ATG condon. Two TATA boxes were at 26 and 65bp upstream of the ATG codon, respectively. Phylogenetic analysis indicated that ClanGV was closely related to Adoxophyes ora-na granulovirus (AoGV)、Cydia pomonella granulovirus (CpGV)、Choristoneura fumiferana granulovirus (CfGV)、and Phthorimaea operculella granulovirus (PoGV). Antibodies prepared against the protein were used in Western blot analysis and indicated that ClanGV granulin reacted positively with occlusion body proten from HaSNPV and CaLGV and reacted weakly with polyhedrin of SeMNPV.