利用Red/ET系统结合rpsL反向筛选敲除AcMNPV lef-10基因

Disruption of AcMNPV lef-10 by Red Recombination System in combination with rpsL counter-selection

【目的】利用Red/ET系统结合rpsL反向筛选建立苜蓿银纹夜蛾核型多角体病毒(AcMNPV)晚期表达因子lef-10点突变载体,为进一步研究AcMNPVlef-10的功能奠定基础。【方法】利用Red/ET系统结合rpsL反向筛选BAC修饰系统对AcMNPV lef-10进行点突变,由于lef-10和必需基因vp1054有重叠,所以在敲除lef-10时只能通过引进点突变使lef-10失活,避免影响vp1054的正常表达。首先,构建rpsL-AMP筛选盒,然后在野生型AcBacmid中引进点突变(方法是通过2次Red/ET重组:第1次重组在野生型AcBacmid lef-10中引进rpsL-AMP抗性筛选标记,形成一次重组Bacmid,第2次重组是在一次重组Bacmid中引进含点突变的lef-10片段),再利用链霉素反向筛选去除rpsL-AMP抗性筛选标记,同时引入相应的点突变。将从lef-10基因点突变重组菌中提取得到的AcBacmid与质粒pTriEx1.1和pTriEx-innateP-lef10分别共转染草地贪夜蛾Sf9细胞,同时用野生型AcBacmid与质粒pTriEx1.1共转染Sf9细胞作为对照,显微镜下观察病毒的复制情况,确定lef-10基因是否为AcMNPV的必需基因。【结果】通过对重组Bacmid的PCR鉴定和点突变序列的测定,证明AcMNPVlef-10基因已经发生点突变;通过共转染试验发现,随着转染时间的延长,lef-10补回型病毒和野生型病毒一样,均能在Sf9细胞中复制产生具有感染性的子代病毒粒子BV,从而对邻近细胞造成二次感染,使得细胞死亡,且lef-10补回型重组病毒产生感染性病毒粒子的能力与野生型基本一致;而由于lef-10缺失型重组病毒不能产生有感染性的病毒粒子,所以在被lef-10缺失型重组病毒转染的细胞中,细胞数目不会随着转染时间的增加而发生明显改变。【结论】利用Red/ET重组系统结合rpsL反向筛选系统可以在Ac MNPVlef-10基因中引入点突变,且lef-10是杆状病毒生活周期中的一个必需基因,其缺失会影响杆状病毒的复制。

【Objective】To study the function of late expression factor lef-10 of Autographa californica nucleopolyhedro virus（AcMNPV）,the RedET Recombination System in combination with rpsLcounter-selection was used to introduce a single point mutation into the AcMNPVlef-10 gene.【Method】Since the ORF box of the lef-10 gene partially overlaps the upstream gene orf53 and the downstream gene vp1054,a single point mutation was introduced into the AcMNPVlef-10 gene to protect the complete genesequence on both sides.First,we constructed rpsL-AMP screening box.Then introduction of point mutation was done through two RedET Recombination steps：the first Recombination was introduction of rpsLAMPresistance selection marker into the AcMNPVlef-10 gene and the second Recombination was to introduce a single point mutation into the AcMNPVlef-10 gene through rpsLcounter-selection,while removing the rpsL-AMPresistance selection marker.Then,Sf9 cells were co-transfected with lef10-KO-Bacmid DNA and pTriEx1.1as well as lef10-KO-Bacmid DNA and pTriEx-innateP-lef10.The co-transfection with wtBacmid DNA and pTriEx1.1was sued as control.Then the infected cells were observed under the microscope.【Result】Based on PCR identification of recombinant Bacmid and point mutations sequences,it was confirmed lef-10 gene had been disrupted.Co-transfection experiments showed that with the extension of the transfection time,vAclef-10KO-REP and vAcwt replicated in Sf9 cells to produce infectious virions BV.This resulted in secondary infection of neighboring cells and cell death.The production ability of infectious virions of vAclef-10KO-REP was consistent with vAcwt.Because vAclef10-KO can not produce infectious virions,the number of cells did not change significantly with the extension of the transfection time when using vAclef10-KOto transfect Sf9 cells.【Conclusion】AcMNPVlef-10 gene has significant effects on replication of virus.