›› 2008, Vol. 51 ›› Issue (5): 486-491.

• 研究论文 • 上一篇    下一篇


杨 波,董小敏,蔡大威,刘志刚,胡 征,汪浩勇,张珈敏,胡远扬   

  • 出版日期:2010-07-27 发布日期:2008-05-20
  • 通讯作者: 杨 波

Characterization of the nonstructural gene promoter of Periplaneta fuliginosa densovirus

YANG Bo   

  • Online:2010-07-27 Published:2008-05-20


为了研究蟑螂 Periplaneta fuliginosa 浓核病毒(pfDNV)非结构基因转录调控的机制,用 PCR 的方法从蟑螂浓核病毒基因组 DNA 中扩增了非结构蛋白基因 ns3 翻译起始密码子上游 325 bp 的启动子序列;并进一步以其作为模板,利用聚合酶链式反应技术,得到 6 个不同长度的启动子片段和两个定点突变体片段,构建由其驱动的萤火虫荧光素酶基因报告质粒,在 脂质体介导下转染多种昆虫细胞,体外分析了该启动子的活性。结果表明:该 325 bp DNA 片段在 Sf9,S2,Tn368 及 Ld652 细胞中均具有较高的启动子活性;其转录起始基序 CAGT 对维持该启动子的基本转录活性而言是必需的,而 TATA 盒对此则是非必需的,但它的存在可显著提高启动子的转录活性。同时,我们还发现,病毒非结构蛋白 NS1 对该启动子具有反式激活作用,并且这种激活作用的大小取决于 NS1 蛋白在细胞中的浓度。

关键词: 蟑螂浓核病毒, 启动子, 反式激活, 昆虫细胞, 表达载体


To understand the mechanism of transcriptional regulation of Periplaneta fuliginosa densovirus (PfDNV) nonstructural genes5-flanking sequence of ns3 gene (325 bp), which encompasses the region from the

5-terminus of the viral genome to the translation init iation codon of the ns3 gene, was cloned. This construction was subsequently used as a template for generation of six 5-or 3-end unidirectional truncation mutants and two site directed mutants in the PCR amplification. These amplified products were subcloned into the report plasmid pGL3-basic and the resultant plasmids were transfected into several insect cell lines. The results indicated that the 325 bp DNA sequence possessed promoter activity in insect cell lines including Sf9, Ld652, Tn368, and S2 cells. Subsequent promoter deletion analysis showed that the promoter had a TATA dependent and TATAindependent transcriptional activity. In addition, we found that the tr a nscriptional activity of this promoter could be transactivated by the viral nons tructural protein NS1 in a concentration dependent fashion in S2 cells.

Key words: Periplaneta fuliginosa densov irus (PfDNV), promoter, transactivation, insect cells, expression vector