Tn5转座子插入p95基因不影响AcMNPV的复制  

Insertional Disruption of AcMNPV p95 Gene did not Affect Virus Replication in Sf9 Cells

作　　者：尹隽[1] 胡志鹏[1] 宋大新[1] 钟江[1] 

机构地区：[1]复旦大学生命科学学院微生物学与微生物工程系,上海200433

出　　处：《复旦学报（自然科学版）》2008年第3期301-305,共5页Journal of Fudan University：Natural Science

摘　　要：从Tn5转座子介导的AcMNPV随机插入突变体库中,分离到一株复制正常的突变体AcApra41.突变定位发现Tn5转座子插入了病毒p95基因中.为了排除AcApra41中还有其他突变,利用同源重组法构建了p95基因定点插入突变的重组病毒AcGFP-P95in.PCR确认p95基因中插入了Tn5转座子;Western blot也证实A-cApra41和AcGFP-P95in感染的细胞中,P95蛋白的分子量都因为插入突变而变小,由野生型的95 ku变为55ku.病毒复制动态曲线和荧光显微镜观察证实带有该插入突变的病毒能够在Sf9细胞中正常复制,并表达极晚期基因.这一结果表明完整的P95蛋白对病毒复制是非必须的.A replication-competent mutant virus, AcApra41, was isolated from a library of AcMNPV random insertional mutants constructed with Tn5 transposon mediated mutagenesis. The Tn5 transposon was determined to be located in the p95 gene of AcMNPV genome. To exclude the possibility that AcApra41 contained other mutations, a recombinant virus, AcGFP-P95in, which had targeted insertion of Tn5 in p95 gene in exactly the same way as in AcApra41, was constructed by homologous recombination. The insertion of Tn5 transposon was confirmed with PCR. The insertion of transposon resuited in the appearance of a mmcated form of P95 protein of 55 ku in cells infected with AcGFP-P95in as shown by Western blot using antibody against the N-terminal part of P95 protein, in contrast with the full length P95 protein of 95 ku in ceils infected with wild type virus. Virus replication curve and fluorescent microscopic observation indicated that AcGFP-P95in replicated and expressed viral genes normally as wild type virus in Sf9 cells. The results indicated that a fulllength P95 protein was not essential for virus replication.

关 键 词：杆状病毒 突变体 复制 基因表达 

分 类 号：Q939.4[生物学—微生物学] U461[机械工程—车辆工程]