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敲除Ac148-150对苜蓿银纹夜蛾核型多角体病毒复制和外源蛋白表达的影响
赵凯霞, 陈红英
西北农林科技大学 生命科学学院
摘要:
【目的】同时敲除苜蓿银纹夜蛾核型多角体病毒(AcMNPV)基因组的Ac148、Ac149、Ac150(Ac148-150)基因,检测敲除Ac148-150基因片段对AcMNPV复制和外源蛋白表达能力的影响,为基因功能鉴定和缩小杆状病毒基因组提供参考。【方法】利用Red/ET同源重组系统和rpsL AMP反向筛选系统对Ac148-150基因进行敲除,得到敲除型杆状病毒质粒(KOAc148-150杆状病毒质粒),提取AcMNPV KOAc148-150杆状病毒质粒和野生型杆状病毒质粒,将其分别与报告基因表达载体共转染Sf9细胞,成功构建重组杆状病毒(野生型病毒vAcMNPV/GFP和缺失突变病毒vAc△148-150/GFP),测定2种重组杆状病毒的一步生长曲线并比较其差异。将2种重组杆状病毒感染Sf9细胞后,采用荧光显微镜观察GFP荧光细胞分布,用流式细胞仪检测GFP荧光强度,再通过SDS-PAGE和Western blot 检测Ac148-150缺失突变体表达的GFP产量。【结果】菌落PCR结果表明,AcMNPV中Ac148-150基因敲除成功。缺失突变病毒vAc△148-150/GFP与野生型病毒vAcMNPV/GFP的一步生长曲线基本一致,说明Ac148-150缺失不影响病毒的复制。荧光显微镜观察和流式细胞仪分析结果表明,敲除Ac148-150对外源蛋白GFP分布和荧光强度未产生影响。SDS-PAGE和Western blot检测结果显示,缺失突变病毒vAc△148-150/GFP和野生型病毒vAcMNPV/GFP表达GFP蛋白产量基本一致。【结论】从AcMNPV基因组中同时敲除Ac148、Ac149、Ac150基因后,对病毒的复制和外源蛋白的表达能力无影响。
关键词:  苜蓿银纹夜蛾  核型多角体病毒;Ac148-150;杆状病毒;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)
Influence of deletion of Autographa californica multiple nucleopolyhedrovirus Ac148-150 on viral replication and foreign protein expression
ZHAO Kaixia, CHEN Hongying
Abstract:
【Objective】The effect of Ac148,Ac149 and Ac150(Acc148-150) deletion on AcMNPV replication and expression of foreign proteins was investigated to provide foundation for identification of baculovirus gene functions and shortening baculovirus genome.【Method】Ac148-150 genes were knocked out by Red/ET homologous recombination and rpsL-AMP counter-selection systems.The recombinant baculoviruses (wild type virus vAcMNPV/GFP and deletion mutant virus vAc△148-150/GFP) were successfully constructed by co-transfection with KOAc148-150 bacmid and wild type bacmid and reporter expression vector in Sf9 cells,and the one-step growth curve of two recombinant baculoviruses was determined.In Sf9 cells infected with recombinant baculoviruses,GFP expression were then observed by fluorescence microscopy,and GFP fluorescence intensity was detected by flow cytometry.The expression of GFP in Ac148-150 deletion mutant was examined by SDS PAGE and Western blot.【Result】The knockout of Ac148-150 was verified by PCR.Compared with the wild type virus vAcMNPV/GFP,the one-step growth curve of the deletion mutant virus vAc△148-150/GFP was basically the same,indicating that Ac148-150 deletion did not affect viral replication.Fluorescence microscopy and flow cytometry results showed that the deletion of Ac148-150 had no effect on the expression of foreign proteins.As detected by SDS-PAGE and Western blot,there was no significant difference in GFP yield between Ac148-150 deletion mutant and wild-type virus.【Conclusion】The deletion of Ac148,Ac149,Ac150 from the AcMNPV genome had no effects on virus replication and the ability of expressing foreign proteins.
Key words:  Autographa californica  multiple nucleopolyhedrovirus  Ac148-150  baculovirus  gene knockout

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