关键词: 斜纹夜蛾, 核型多角体病毒, ORF13, 原核表达, 多克隆抗体

Abstract:

【Objective】The objective of this study is to research the structure and function of a newly found virus strain Spodoptera litura multicapsid nucleopolyhedrovirus II (SpltMNPVII) ORF13. 【Method】 In order to study the function of the ORF13, the primers were designed according to the sequence of SpltMNPVII genome. The promoter of ORF13 was amplified by PCR. The promoter activities analysis and the time course of mRNA transcription analysis were done. The fragment of the ORF13 was then cloned into the vector of pET28a(+) and expressed. The polyclonal antibody was prepared by using the purified fusion protein. Titration determination of anti-ORF13 antibody was evaluated by ELISA. 【Result】 Nucleotide sequence analysis demonstrated that this gene has a baculovirus late promoter motif ATAAG at -84 bp upstream of start codon, early promoter motif was not found in it. Both promoter activities analysis and the time course of mRNA transcription analysis of the ORF13 showed that ORF13 was transcripted in early stage as well as in late stage, which begin it’s transcription at 2 h post infection (hpi) and reached a peak at 18 hpi and then the transcription level was slightly decreased from 24 hpi. pET-28a-ORF13 fusion protein expressed in prokaryotic and purified polyclonal antibody was good specificity, with the titer of 1﹕3 200 or more. 【Conclusion】 SpltMNPV II ORF13 is a composition structure protein, which was expressed at both early and late stage. ORF13 encoding protein may be a membrane protein of BV, which should be a factor in BV infecting host cells. The polyclonal antibody can be used to further study the biological characteristics and functions of proteins.

Key words: Spodoptera litura, nucleopolyhedrovirus, ORF13, prokaryotic expression, polyclonal antibody