【摘要】 晚期表达因子12(late expression factor 12,LEF-12)是昆虫核型多角体病毒(nucleopolyhedrovirus,NPV)基因组编码的转录调控因子之一。为了研究家蚕核型多角体病毒(BmNPV)LEF-12的生物学功能,通过Red重组技术敲除BmNPV的lef-12基因,构建lef-12缺失型病毒lef12-ko-Bacmid,再利用Bac-to-Bac系统将lef-12重新补回到病毒基因组中,获得补回型病毒lef12-re-Bacmid。将野生型病毒wtBacmid、lef12-ko-Bacmid和lef12-re-Bacmid分别转染BmN细胞,发现3种病毒均可在转染的细胞中产生具有感染活性的病毒粒子,但病毒滴度测定结果显示lef12-ko-Bacmid的病毒粒子产量比wtBacmid下降了3倍左右,而lef12-re-Bacmid的病毒粒子产量基本上恢复到了野生型病毒的水平,表明lef-12的缺失会影响病毒在宿主细胞中的增殖。进一步研究lef-12敲除对病毒基因组复制和基因转录的影响,结果表明当lef-12缺失后,BmNPV DNA复制没有受到明显影响,说明lef-12不是病毒复制的必需基因;但lef-12缺失后,病毒晚期基因vp39、极晚期基因p10的转录水平均明显低于野生型和补回型病毒。透射电子显微镜观察结果也进一步证实lef-12缺失后,病毒仍能进行正常复制和装配,但与野生型病毒相比其增殖数量有所下降。上述研究结果提示:BmNPV lef-12不是病毒基因组复制的必需基因,但其缺失将导致宿主细胞的感病时间延迟,对病毒晚期和极晚期基因的转录也具有显著影响。更多还原

【Abstract】 Late expression factor 12( lef-12) is a transcription factor which generally exist in all the genome of insect nuclepolyhedrovirus( NPV). In order to explore the biological function of BmNPV lef-12,a lef-12 knockout Bacmid( lef12-ko-Bacmid) was generated by Red recombination in E. coli. Additionally,a lef-12 repaired Bacmid( lef12-re-Bacmid) was constructed by Bac-to-Bac system. Then three types of Bacmid DNA wild-type Bacmid( wtBacmid),lef12-ko-Bacmid and lef12-re-Bacmid were transfected into BmN cells, respectively. It was found that all three types of virus could yield infective viralparticles in the transfected cells. However viral titer detection results showed that lef12-ko-Bacmid was three times lower than wtBacmid,but the lef12-re-Bacmid was recovered similar to wtBacmid,indicating that the deletion of lef-12 could affect virus proliferation in host cells. Moreover,the level of virus replication and gene transcription were analyzed in the absence of lef-12. The lef-12 deletion did not show obvious effect on viral genome replication,indicating that it was not essential for viral replication. After lef-12 deletion the transcription levels of viral late gene vp39 and very late gene p10were significantly lower than wild-type virus and repaired virus. Results of transmission electron microscopy also confirmed that the virus could replicate and assemble normally after lef-12 deletion,but the quantity of virus reproduction decreased. In conclusion,lef-12 is not an essential gene for viral genome replication; however,lef-12 deletion will delay the infection process and obviously influence the transcription of viral late and very late genes.更多还原