斜纹夜蛾核型多角体病毒ⅡORF146基因的克隆、表达与启动子活性分析

Cloning and expression of ORF146 gene of Spodoptera litura multicapsid nucleopolyhedrovirus II (SpltMNPVII)

【目的】研究斜纹夜蛾核型多角体病毒II ORF146基因的结构与功能。【方法】根据SpltMNPV IIORF146基因序列设计引物,经PCR扩增克隆ORF146基因。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ORF146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,读码框含1383 bp,编码460个氨基酸的蛋白质,推定分子量为50.4 kDa。启动子活性分析和转录时相分析都表明该基因是个早、晚期都表达的基因,在病毒感染8 h和18 h有两个转录峰,24 h以后转录水平略有下降,但趋于稳定。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 ORF146基因是一个早期和晚期都表达的病毒组成型结构蛋白基因。推测ORF146基因可能与SpltMNPV II病毒感染宿主细胞后病毒DNA复制有关。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。

[Objective]To study the structure and function of a newly found virus strain Spodoptera litura multicapsid nucleopolyhedrovirus II（SpltMNPVII） ORF146 gene.[Methods]The primers were designed according to the sequence of SpltMNPVII genome.The promoter of ORF146 was amplified by PCR.The promoter activities and the time course of mRNA transcription were analyzed.The fragment of the ORF146 gene 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-ORF146 antibody was evaluated by ELISA.[Result]Nucleotide sequence analysis demonstrated that this gene has a 1383 bp ORF,encoding 460 amino acids with a predicted molecular weight of 50.4 kDa.Analysis of both promoter activities and the time course of mRNA transcription of the ORF146 gene showed that ORF146 was transcripted in early stage as well as in late stage.The transcription began at 2 h post infection（hpi） and reached two peaks at 8 and 18 hpi and then the transcription level was slightly decreased from 24 hpi.pET-28a-ORF146 fusion protein expressed in prokaryotic and purified polyclonal antibody was with good specificity,with the titer above 1 ∶ 3200.[Conclusion] SpltMNPV II ORF146 gene is a composition structure protein,which was expressed at both early and late stage.ORF146 might be involved in viral DNA replication.The polyclonal antibody can be used to further study the biological characteristics and functions of proteins.