家蚕杆状病毒(BmNPV)Bm122基因的缺失影响芽生型病毒粒子的形成  

Influence of Deficiency in BombyxMori Nuclear Polyhedrosis Virus(BmNPV)Gene Bm122 on the Production of Budded Virus

作　　者：朱丽萍[1] 于威[1,2] 陈滨[1,2] 何欢[1,2] 解纯刚[1,2] 

机构地区：[1]浙江理工大学生物化学研究所,杭州310018 [2]浙江理工大学浙江省家蚕生物反应器和生物医药重点实验室,杭州310018

出　　处：《浙江理工大学学报（自然科学版）》2016年第2期277-282,共6页Journal of Zhejiang Sci-Tech University(Natural Sciences)

基　　金：国家高技术研究发展计划“863”项目(2011AA100603);浙江省自然科学基金项目(Y207217)

摘　　要：为研究家蚕杆状病毒（BmNPV）编码的Bm122基因的生物学功能,利用Red重组技术和Bac-to-Bac系统分别构建了Bm122-ko-bacmid和Bm122-re-bacmid,实现对Bm122基因的敲除和异位补回。进而将Bm122-kobacmid、Bm122-re-bacmid、wtbacmid（野生型）分别转染BmN细胞,病毒滴度测定结果显示：Bm122缺失后,病毒无法形成正常水平的芽生型病毒粒子（budded virus,BV）,表明该基因是病毒生成有感染力的BV所必需的基因;透射电子显微镜观察发现,Bm122敲除后细胞内未观察到杆状病毒粒子,进一步证实Bm122的缺失影响了BV的生成。qPCR结果显示,Bm122缺失后病毒DNA复制水平降低。结果表明,Bm122是病毒生成正常水平的BV所必需的基因。To study the biological function of BmNPV-encoded Bm122 gene,we constructed a Bm122 knockout bacmid（Bm122-ko-bacmid）and Bm122 repair bacmid（Bm122-re-bacmid）to achieve knockout and repair of Bm122 gene by using Red recombinat technology and Bac-to-Bac system respectively.Then,transfect BmN cells with Bm122-ko-bacmid,Bm122-re-bacmid and wtbacmid（wild type bacmid）,and virus titer measuring results indicate that,with the Bm122 deficiency,virus is unable to produce infectious BV（budded virus）of normal level.In addition,it also shows that the gene is the necessary gene required by virus to generate infectious BV.No baculovirus particles were found in cells through transmission electron microscopy after Bm122 knockout,further proving that lack of Bm122 affects the generation of BV.qPCR results show that the level of viral DNA replication in Bm122-ko-bacmid is evidently reduced.In conclusion,Bm122 is essential to the production of BV of normal level.

关 键 词：家蚕杆状病毒 Bm122基因 Red重组技术 BAC-TO-BAC系统 病毒复制 

分 类 号：Q812[生物学—生物工程]