| 摘要: |
| 【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AcMNPV lef-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Ac-Bacmid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rpsLAMP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Ac-MNPV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AcMNPV lef-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。 |
| 关键词: 苜蓿银纹夜蛾核型多角体病毒(AcMNPV) lef-10 Red/ET重组系统 rpsL反向筛选系统 基因敲除 |
| DOI： |
| 分类号: |
| 基金项目:陕西省自然科学基础研究计划项目(2011JM3002) |
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| Disruption of AcMNPV lef-10 by Red Recombination System in combination with rpsL counter selection |
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BAI Yu,XU Xiao-dong
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| Abstract: |
| 【Objective】To study the function of late expression factor lef-10 of Autographa californica nucleopolyhedro virus (AcMNPV),the RedET Recombination System in combination with rpsL counter-selection was used to introduce a single point mutation into the AcMNPV lef-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 AcMNPV lef-10 gene to protect the complete gene sequence 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 rpsL-AMP resistance selection marker into the AcMNPV lef-10 gene and the second Recombination was to introduce a single point mutation into the AcMNPV lef-10 gene through rpsL counter selection,while removing the rpsLAMP resistance selection marker.Then,Sf9 cells were co-transfected with lef10-KO-Bacmid DNA and pTriEx1.1 as well as lef10-KO-Bacmid DNA and pTriEx-innateP-lef10.The co-transfection with wtBacmid DNA and pTriEx1.1 was 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-KO to transfect Sf9 cells.【Conclusion】lef-10MNPV lef-10 gene has significant effects on replication of virus. |
| Key words: Autographa californica multiple nucleopolyhedrovirus (AcMNPV) lef-10 RedET recombination system rpsL counter selection BAC modification system gene disruption |
