Tang Yizhe, Le Long P, Matthews Qiana L, Han Tie, Wu Hongju, Curiel David T
Division of Human Gene Therapy, Department of Medicine, and the Gene Therapy Center, University of Alabama at Birmingham, 901 19th Street South, BMR2-502, Birmingham, AL 35294, USA.
Virology. 2008 Aug 1;377(2):391-400. doi: 10.1016/j.virol.2008.04.023.
Adenoviral capsid protein IX (pIX) has been shown to be a potential locale to insert targeting, imaging-related and therapeutic modalities by genetic modification. Recent evidences suggested that capsid protein mosaicism could be a promising strategy for improving the utility of Ad vector. In this study, we explored a method to genetically generate triple pIX mosaic Ad serotype 5 (Ad5) displaying three types of pIX on a single virion. pIXs were modified at their carboxy termini with a Flag sequence, a hexahistidine sequence (His(6)) or a monomeric red fluorescent protein (mRFP1), respectively. Western blotting analysis and fluorescence microscopy of the purified recombinant viruses indicated that all three modified pIXs were incorporated into the viral particles. Immuno-gold electron microscopy (EM) further confirmed that three types of pIX indeed co-existed on an individual virion. These results firstly validated a triple mosaic capsid configuration on pIX, and demonstrated the possibility of further radical design.
腺病毒衣壳蛋白IX（pIX）已被证明是通过基因改造插入靶向、成像相关和治疗方式的潜在位点。最近的证据表明，衣壳蛋白镶嵌可能是提高腺病毒载体效用的一种有前景的策略。在本研究中，我们探索了一种通过基因方法产生三重pIX镶嵌腺病毒血清型5（Ad5）的方法，该病毒在单个病毒粒子上展示三种类型的pIX。分别在pIX的羧基末端用Flag序列、六组氨酸序列（His(6)）或单体红色荧光蛋白（mRFP1）进行修饰。对纯化的重组病毒进行的蛋白质免疫印迹分析和荧光显微镜检查表明，所有三种修饰的pIX都被整合到病毒颗粒中。免疫金电子显微镜（EM）进一步证实，三种类型的pIX确实共存于单个病毒粒子上。这些结果首先验证了pIX上的三重镶嵌衣壳结构，并证明了进一步进行根本性设计的可能性。
