| 摘要: |
| 【目的】对苜蓿银纹夜蛾核型多角体病毒（AcMNPV）晚期表达因子LEF-10的第21位保守氨基酸残基进行饱和点突变，研究该位点突变对AcMNPV活性的影响，为进一步研究LEF-10朊病毒特性及其在病毒复制中的作用奠定基础。【方法】同源序列比对发现，LEF-10第21位亮氨酸残基高度保守。对pTriEx质粒载体上LEF-10第21位的亮氨酸残基通过异位回补进行饱和突变，将构建好的重组质粒与线性化的BacmidΔlef-10共转染草地贪夜蛾Sf9细胞构建重组病毒，研究LEF-10第21位上不同氨基酸残基对病毒活性的影响。同时联用Red/ET基因敲除系统和rpsl反向筛选系统对AcMNPV Bacmid的LEF-10基因进行原位突变，突变后的线性化Bacmid与pTriEx-p(p10)-dsRed质粒共转染草地贪夜蛾Sf9细胞构建原位突变型病毒，研究点突变对重组病毒活性的影响，并通过流式细胞术检测突变型病毒对晚期基因表达的影响。【结果】异位回补点突变重组病毒和原位点突变重组病毒转染和感染Sf9细胞的结果均表明，当LEF-10第21位亮氨酸残基突变为异亮氨酸残基、丙氨酸残基、缬氨酸残基、脯氨酸残基、半胱氨酸残基和甲硫氨酸残基时，可以拯救LEF-10缺陷型病毒，并产生具有感染性的重组病毒，但其他突变型重组病毒均为致死突变。将7种原位突变重组病毒分别以高MOI（MOI=10）和低MOI（MOI=1）感染Sf9细胞，结果表明低MOI感染Sf9细胞时，7种重组病毒外源蛋白的表达水平相当；高MOI感染Sf9细胞时，7种重组病毒外源蛋白的表达水平差异明显，其中突变为丙氨酸残基的重组病毒将外源蛋白的表达维持在较高水平。【结论】LEF-10第21位氨基酸残基对其功能起着至关重要的作用，该位点氨基酸残基的性质影响AcMNPV的活性。 |
| 关键词: 苜蓿银纹夜蛾核型多角体病毒 晚期表达因子LEF-10 饱和突变 基因敲除 |
| DOI： |
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| 基金项目:陕西省国际科技合作与交流计划项目“用于亚单位疫苗生产的杆状病毒表达载体的优化”（2016KW-022） |
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| Effect of LEF-10 21 mutation on AcMNPV activity |
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WANG Xin,XU Xiaodong
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| Abstract: |
| 【Objective】A point saturation mutation of the 21st conserved amino acid residues of the late expression factor LEF-10 of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) was performed and its effect on AcMNPV activity was studied to provide foundation for further research on characteristics of LEF 10 prion and role of prion in viral replication.【Methods】Based on homology sequence alignment,it was found that the leucine residue at position 21 of LEF-10 was highly conserved.The leucine residue at position 21 of LEF-10 on pTriEx plasmid vector was subjected to saturation mutation by ectopic backfill.The constructed recombinant plasmid was co-transfected with linearized BacmidΔlef-10 into Sf9 cells of Spodoptera frugiperda to construct recombinant virus,and the effect of different amino acid residues at the 21 position of LEF-10 on virus activity was studied.The LEF-10 gene of AcMNPV Bacmid was in situ mutated using the Red/ET gene knockout system and the rpsl reverse sieving system.The mutant linearized Bacmid and pTriEx-p(p10) dsRed plasmid were co-transfected into Sf9 cells of Spodoptera frugiperda to construct an in situ mutant virus,and the effect of point mutation on activity of the recombinant virus was clarified.The effect of mutant viruses on late gene expression was also detected by flow cytometry.【Results】The results of transfection and infection of Sf9 cells with ectopic backfill point mutation recombinant virus and in situ mutation recombinant virus showed that the mutation of leucine residue at position 21 of LEF-10 to an isoleucine residue,alanine residues,valine residues,proline residues,cysteine residues and methionine residues would rescue LEF-10-deficient viruses and produce infectious recombinant viruses,while other mutations recombinant viruses were lethal.Seven in situ mutant recombinant viruses were used to infect Sf9 cells with high and low MOI,respectively.The results showed that expression levels of foreign proteins were equivalent with low MOI,while they were significantly different with high MOI.The recombinant virus mutated to an alanine residue maintained the highest expression level of foreign proteins.【Conclusion】The amino acid residue at position 21 of LEF-10 plays a vital role in the function of LEF-10,and its properties affect AcMNPV activity. |
| Key words: Autographa californica multiple nucleopolyhedrovirus (AcMNPV) late expression factor LEF-10 saturation mutation gene knockout |
