【摘要】 GP6 4是杆状病毒在其发育循环第一时相所产生的芽生型病毒粒子 (buddedvirus ,BV)的特异性囊膜糖蛋白 ,它在病毒入侵细胞过程中具有重要作用。为了探明杆状病毒gp6 4基因的表达调控 ,从所克隆的BmNPV和AcMNPVgp6 4基因ATG上游 437～ 439bp启动子的序列分析发现 ,该启动子同时具有早期和晚期转录模体。将所构建的该启动子控制下荧光素酶报告基因 (Luc)的非融合表达质粒分别转染Bm N和Sf 2 1细胞进行瞬间表达分析 ,BmNPVgp6 4启动子能被允许宿主Bm N细胞RNA聚合酶II所识别 ,AcMNPVgp6 4启动子既能被允许宿主Sf 2 1又能被非允许宿主Bm N细胞RNA聚合酶II所识别 ,同时 ,这两个启动子的转录活性在各自的允许宿主细胞中被相应的病毒因子所反式激活 2 .4～ 4倍。将家蚕核多角体病毒同源重复序列 3(BmNPVhomologousregion 3,hr3)克隆到该启动子控制下的Luc报告基因下游 ,用所构建的质粒分别转染细胞 ,瞬间表达分析结果表明 ,BmNPVhr3能分别增强该启动子在Bm N和Sf 2 1细胞中的转录活性 13～ 2 2倍和 70 0 0～ 140 0 0倍以上。同时 ,相应的病毒因子能反式激活插入了hr3的gp6 4启动子在各自允许宿主细胞中转录活性 73～ 78倍 ,这暗示着BmNPVhr3除了具有病毒DNA复制原点和增强子的功能外 ,它在病毒的反式激活过程中?更多还原

【Abstract】 Baculovirus GP64 envelope glycoprotein is a specific major component of the envelope of the budded virus and is involved in virus entry into the host cells by endocytosis. For promoter activity analysis in the baculovirus gp64 gene, two DNA fragments containing 437 and 439 bp upstream of 5′ ends of the BmNPV and AcMNPV gp64 ORF were amplified by polymerase chain reaction and cloned, respectively. The sequence analysis indicated that two gp64 genes have both early (CAGT) and late (A/GTAAG) transcriptional start sites. By use of the plasmids with a reporter luciferase gene (Luc) driven by gp64 promoter to transfect insect cells, transient expression assay showed that pBmgp64Luc had high expression levels in permissive Bm-N cells and very low levels in non-permissive Sf-21 cells, while pAcgp64Luc had relatively high expression levels both in permissive Sf-21 cells and in non-permissive Bm-N cells. Furthermore, the transcription of both gp64 promoters appeared to be transactivated by 2.4-4 times in corresponding permissive cells by corresponding viral factors, separately. By inserting BmNPV homologous region-3 (hr3) into the downstream of luciferase reporter gene driven by gp64 promoter, it enhanced transcription from both gp64 promoters by 13-22 times in Bm-N cells and over 7000-14 000 times in Sf-21 cells, respectively. In the presence of BmNPV hr3, correspondingly, the viral factors transactivated the transcriptional activity from two promoters by about 73-78 times in corresponding permissive cells. It suggested that BmNPV hr3 plays an important role in co-activation with viral factors onto the gp64 promoter besides the functions of viral DNA origin and enhancer.更多还原