摘要
ie0基因是家蚕核型多角体病毒(BmNPV)的一个晚期基因,该基因编码氨基酸序列的高度保守性及多种结构域提示其在病毒感染过程中的重要性。利用Red同源重组技术,分别对BmNPV ie0基因编码不同结构域的序列以及完整序列进行敲除构建ie0缺失型病毒,以野生型病毒为对照,研究ie0序列完全或部分缺失对病毒转录、复制及对宿主细胞致病能力等方面的影响。结果表明ie0基因完整序列或编码各结构域的序列缺失后均会对病毒产生不同影响:宿主细胞中完全缺失型和部分缺失型病毒滴度均有不同程度降低;完全缺失型和部分缺失型病毒基因组的拷贝数均与野生型病毒无显著差异;部分缺失型病毒的晚期和极晚期基因转录水平变化与ie0基因完全缺失型病毒相一致,均显著低于野生型病毒,并且缺失锌指结构域编码序列的影响最为明显。MTT检测结果也显示ie0基因完全缺失后病毒对宿主细胞的致病能力有所下降。亚细胞定位显示IE0蛋白在细胞质、细胞核中均有分布,但主要位于细胞质。综上所述,ie0基因为BmNPV病毒增殖、基因组复制的非必需基因,但对病毒晚期和极晚期基因的转录具有调控作用,影响最为显著的为锌指结构域的编码序列,且该基因的缺失会导致病毒对宿主细胞的致病能力有所下降。
ie0 gene is a late gene of Bombyx mori nucleopolyhedrovirus( BmN PV). The high conservation and multiple domains of amino acid sequence encoded by ie0 gene revealed the importance of ie0 gene in the process of viral infection. By Red homologous recombination technology,the coding sequences of different domains and the whole sequence of BmN PV ie0 gene were knocked out to construct ie0 knockout viruses. The effect of whole and partial sequence deletion of ie0 on viral transcription,replication,and host cell infection was studied using wild type virus as control. The results showed that the deletion of the whole sequence and various domain-encoding sequences of ie0 gene had distinct effects on virus. In host cells,the titers of complete-deletion-type and partial-deletion-type viruses were decreased at different degrees. Genome copy number of completedeletion-type and partial-deletion-type viruses had no significant difference when compared with wild type virus.The transcriptional level variation of late and very late genes of partial-deletion-type virus was consistent with those of complete-deletion virus,both of which were significantly lower than wild type virus,and the influence of deleting the coding sequence of zinc finger domain was the most obvious. MTT results also showed that complete deletion of ie0 gene would lead to the decline of viral pathogenicity to host cells. Subcellular localization showed that IE0 protein was mainly located in cytoplasm although it was distributed in both cytoplasm and nucleus. In summary,ie0 gene is inessential for BmN PV proliferation and genome replication,but it has regulatory effect on the transcription of viral late and very late genes,especially the coding sequence of zinc finger domain. The deletion of ie0 gene will lead to the decline of viral pathogenicity to host cells.
出处
《蚕业科学》
CAS
CSCD
北大核心
2016年第4期645-655,共11页
ACTA SERICOLOGICA SINICA
基金
国家高技术研究发展计划"863"项目(No.2011AA10060-3)
浙江理工大学"521"人才培养计划项目(No.1161013-2521506)