为构建一种重组乙型肝炎病毒(hepatitis B virus,HBV)复制子模型,使其能够在病毒感染的细胞中表达可视化报告基因蛋白,本研究删除HBV基因组核心蛋白(HBV core,HBc)编码区部分序列,构建HBV1.1-ΔHBc113复制子载体。利用内含肽(intein)介...为构建一种重组乙型肝炎病毒(hepatitis B virus,HBV)复制子模型,使其能够在病毒感染的细胞中表达可视化报告基因蛋白,本研究删除HBV基因组核心蛋白(HBV core,HBc)编码区部分序列,构建HBV1.1-ΔHBc113复制子载体。利用内含肽(intein)介导蛋白拼接的特性,选取加强绿色荧光蛋白(enhanced green fluorescent protein,EGFP)和超级折叠绿色荧光蛋白(super folder green fluorescent protein,sfGFP)作为外显肽,建立EGFP^(N1-8)/EGFP^(C9-11)和sfGFP^(N1-10)/sfGFP^(C11)蛋白分裂系统。基于ΔHBc113载体,分别构建EGFP^(C9-11)和sfGFP^(C11)重组HBV复制子;应用DNA印迹法(Southern blotting)验证两种rHBV载体的细胞内复制能力。结果显示,构建的HBV1.1-ΔHBc113复制子载体能够在HBc反式互补条件下在转染细胞中形成病毒复制。构建的EGFP^(C9-11)和sfGFP^(C11)重组HBV复制子能够支持HBc互补条件下的细胞内病毒复制,并产生子代病毒颗粒;HBV重组复制子表达的EGFP^(C9-11)或sfGFP^(C11)蛋白在共表达氮端蛋白EGFP^(N)/sfGFP^(N)细胞中,通过intein介导的蛋白质高效拼接,能够形成完整的、有功能的GFP。结果表明,构建的荧光蛋白重组HBV复制子系统能够为HBV高通量药物筛选和HBV易感性研究提供实验工具,具有重要的病毒学意义和应用前景。展开更多
Transgenic safety issues cause more and more controversies with the planting area of transgenie crops increased year by year. Gene flow from transgenie crops to wild relatives through pollen dispersal is one of the fo...Transgenic safety issues cause more and more controversies with the planting area of transgenie crops increased year by year. Gene flow from transgenie crops to wild relatives through pollen dispersal is one of the focus problems. Gene splitting technique provides a new strategy for the control of transgene flow by bio-logical containment. The construction of gene splitting technique is based on protein trans-splicing mediated by intein. Currently, it has been proved in Arabidopsis, tabaoco, wheat, etc. that active and functional proteins can be reassembled by intein mediated protein trans-splicing after gene splitting, which provides theoretical basis and experimental supporting for the limit of transgene flow by gene splitting technique. The theoretical basis of gene splitting technique and research progresses of its application on the control of transgene flow were reviewed in this paper.展开更多
基金Supported by National Natural Science Foundation of China(No.30800186 and 31070698)National High Technology Research and Development Program of China(863 Program,No.2006AA03Z451)+2 种基金Shanghai Key Project of Basic Program(No.10JC1400300)Ph.D. Programs Foundation of Ministry of Education of China(No.200802551026)Special Funds of Basic Scientific Research Operating Expenses from Chinese Universities(No.11D10530)~~
文摘为构建一种重组乙型肝炎病毒(hepatitis B virus,HBV)复制子模型,使其能够在病毒感染的细胞中表达可视化报告基因蛋白,本研究删除HBV基因组核心蛋白(HBV core,HBc)编码区部分序列,构建HBV1.1-ΔHBc113复制子载体。利用内含肽(intein)介导蛋白拼接的特性,选取加强绿色荧光蛋白(enhanced green fluorescent protein,EGFP)和超级折叠绿色荧光蛋白(super folder green fluorescent protein,sfGFP)作为外显肽,建立EGFP^(N1-8)/EGFP^(C9-11)和sfGFP^(N1-10)/sfGFP^(C11)蛋白分裂系统。基于ΔHBc113载体,分别构建EGFP^(C9-11)和sfGFP^(C11)重组HBV复制子;应用DNA印迹法(Southern blotting)验证两种rHBV载体的细胞内复制能力。结果显示,构建的HBV1.1-ΔHBc113复制子载体能够在HBc反式互补条件下在转染细胞中形成病毒复制。构建的EGFP^(C9-11)和sfGFP^(C11)重组HBV复制子能够支持HBc互补条件下的细胞内病毒复制,并产生子代病毒颗粒;HBV重组复制子表达的EGFP^(C9-11)或sfGFP^(C11)蛋白在共表达氮端蛋白EGFP^(N)/sfGFP^(N)细胞中,通过intein介导的蛋白质高效拼接,能够形成完整的、有功能的GFP。结果表明,构建的荧光蛋白重组HBV复制子系统能够为HBV高通量药物筛选和HBV易感性研究提供实验工具,具有重要的病毒学意义和应用前景。
基金Supported by Major Project of China on New Varieties of GMO Cultivation(2013zx08010-003)National Natural Science Foundation of China(31100408)
文摘Transgenic safety issues cause more and more controversies with the planting area of transgenie crops increased year by year. Gene flow from transgenie crops to wild relatives through pollen dispersal is one of the focus problems. Gene splitting technique provides a new strategy for the control of transgene flow by bio-logical containment. The construction of gene splitting technique is based on protein trans-splicing mediated by intein. Currently, it has been proved in Arabidopsis, tabaoco, wheat, etc. that active and functional proteins can be reassembled by intein mediated protein trans-splicing after gene splitting, which provides theoretical basis and experimental supporting for the limit of transgene flow by gene splitting technique. The theoretical basis of gene splitting technique and research progresses of its application on the control of transgene flow were reviewed in this paper.
