斜纹夜蛾核型多角体病毒基因ⅡORF13的克隆、表达与启动子活性分析

摘要

[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.%[目的]研究新发现的斜纹夜蛾核型多角体病毒Ⅱ株基因ORF13的结构与功能.[方法]根据SpltMNPVⅡ ORF13基因序列设计引物,经PCR扩增并克隆PRF13.在生物信息学分析基础上进行启动子活性分析和转录时相分析.构建ORF13片段的原核表达载体,表达并纯化融合蛋白后制备多克隆抗体.[结果]核苷酸序列分析表明,起始密码子上游-84bp处发现杆状病毒晚期启动子基序ATAAG,没有发现明显的早期启动子基序.启动子活性分析和转录时相分析证实,ORF13是一个早期和晚期都表达的基因,在病毒感染2h就开始转录,18h达到最高峰,24h以后转录水平有所下降,但基本维持恒定.pET-28a-ORF13原核表达的融合蛋白经纯化后制备的多克隆抗体特异性高,效价可达1:3200以上.[结论]SpltMNPV Ⅱ ORF13是一个早期和晚期都表达的病毒组成型结构蛋白基因.ORF13编码的蛋白可能是一种BY的膜蛋白,与细胞跨膜转运有关,制备的多克隆抗体可用于深入研究该蛋白的生物学特性与功能.