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【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】 The purpose of this study was to investigate the structure and function of II ORF146 gene of Spodoptera litura nucleopolyhedrovirus. 【Method】 According to the sequence of SpltMNPV IIORF146, primers were designed and ORF146 gene was cloned by PCR. Promoter activity analysis and transcriptional phase analysis were performed on the basis of bioinformatics analysis. Construction of ORF146 prokaryotic expression vector, expression and purification of the fusion protein prepared polyclonal antibody. 【Result】 Nucleotide sequence analysis showed that the reading frame contains 1383 bp and encodes a protein of 460 amino acids with a predicted molecular mass of 50.4 kDa. Both promoter activity analysis and transcriptional phase analysis indicated that the gene was expressed both early and late, with two transcriptional peaks at 8 h and 18 h after virus infection. Transcript levels decreased slightly but remained stable after 24 h. The purified polyclonal antibody of pET-28a-ORF13 prokaryotic fusion protein was highly specific, with a titer of more than 1:3200. 【Conclusion】 The SpltMNPV II ORF146 gene is a viral structural constitutive protein gene expressed both early and late. It is speculated that the ORF146 gene may be related to viral DNA replication after host cell infection with SpltMNPV II virus. The prepared polyclonal antibody can be used to further study the biological characteristics and function of this protein.