Bm59基因缺失对家蚕核型多角体病毒(BmNPV)复制、转录及组装的影响  

Impact of Bm59 Deletion on Viral Replication,Transcription and Assembly of Bombyx mori Nucleopolyhedrovirus

作　　者：蒋磊[1,2] 田星[1,2] 巩成见 于威[1,2] 舒建洪[1,2] 吴银丽 童富淡[1,2] 

机构地区：[1]浙江理工大学生物化学研究所,杭州310018 [2]浙江省家蚕生物反应器和生物医药重点实验室,杭州310018 [3]宁海县动物卫生监督所,浙江宁海315600

出　　处：《蚕业科学》2016年第2期266-273,共8页ACTA SERICOLOGICA SINICA

基　　金：国家高技术研究发展计划"863"项目(No.2011AA10060-3);横向联合项目(No.15040124-J)

摘　　要：Bm59是家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus,Bm NPV)的核心基因之一。利用λRed重组技术和Bac-to-Bac系统构建野生型病毒wt Bacmid-polh-egfp、缺失型病毒Bm59ko-Bacmid-polh-egfp以及补回型病毒Bm59re-Bacmidpolh-egfp,并分别转染家蚕卵巢培养细胞Bm N,研究Bm59基因在病毒侵染、增殖和组装中的功能。对病毒滴度的检测结果表明3种类型病毒都能产生有活力的子代病毒并使细胞感染发病,但Bm59ko-Bacmid-polh-egfp在各个时相的滴度值均显著低于其他2种类型病毒(P<0.05)。电子显微镜下观察Bm59ko-Bacmid-polh-egfp转染的细胞中只有少量细长的杆状病毒粒子,而其他2种类型病毒转染细胞后则能产生大量具有囊膜包裹的成熟病毒粒子。qRT-PCR检测结果显示,Bm59缺失型病毒基因组DNA的复制能力显著降低(P<0.05),早期基因lef-3、晚期基因vp39和极晚期基因p10的转录水平显著低于野生型病毒(P<0.05)。综上所述,Bm59基因虽然是家蚕核型多角体病毒复制的非必需基因,但会显著影响病毒的增殖速度和病毒粒子的组装,对病毒各个时期的基因转录水平具有下调作用。Bm59 gene is one of the core genes of Bombyx mori nucleopolyhedrovirus（ Bm NPV）. In this study,the λRed recombination system and the Bac-to-Bac system were used to create wild type virus wt Bacmid-polh-egfp,knockout type virus Bm59ko-Bacmid-polh-egfp and repair type virus Bm59re-Bacmid-polh-egfp,all of which were transfected into Bm N cells for exploring function of Bm59 gene in viral infection,replication and assembly. Test results of viral titer indicated that all three type viruses were able to produce viable progeny viruses capable of infecting new cells. However,titer of Bm59ko-Bacmid-polh-egfp was significantly lower than the other two viruses in all phases（ P〈0. 05）. Observation under transmission electron microscope showed that there were very few thin and long baculoviral particles in cells transfected by Bm59ko-Bacmid-polh-egfp, whereas a great many mature viral particles wrapped in envelops were produced in cells transfected by the other two type viruses. Test result of qRT-PCR revealed that Bm59 deletion had a strong down-regulatory effect on viral DNA replication（ P〈0. 05） and significantly reduced the tran-scription levels of early gene lef-3,late gene vp39,and very late gene p10 in comparison to wild type virus（ P〈0. 05）.These results indicate that Bm59 is a non-essential gene for viral replication of Bm NPV. However,it significantly influences viral reproduction and assembly and has a down-regulatory effect on gene transcription levels in all phases.

关 键 词：家蚕核型多角体病毒 Bm59基因 λRed重组系统 BAC-TO-BAC系统 QRT-PCR 

分 类 号：Q939.4[生物学—微生物学] Q527.5