Riikonen Reetta, Matilainen Heli, Rajala Nina, Pentikainen Olli, Johnson Mark, Heino Jyrki, Oker-Blom Christian
University of Jyvaskyla, Dept. of Biological and Environmental Science, PO Box 35, FIN-40351 Jyvaskyla, Finland.
Technol Cancer Res Treat. 2005 Aug;4(4):437-45. doi: 10.1177/153303460500400411.
The use of baculovirus vectors shows promise as a tool for gene delivery into mammalian cells. These insect viruses have been shown to transduce a variety of mammalian cell lines, and gene transfer has also been demonstrated in vivo. In this study, we generated two recombinant baculovirus vectors displaying an integrin-specific motif, RKK, as a part of two different loops of the green fluorescent protein (GFP) fused with the major envelope protein gp64 of Autographa californica M nucleopolyhedrovirus. By enzyme linked immunosorbent assays, these viruses were shown to bind a peptide representing the receptor binding site of an alpha2 integrin, the alpha2I-domain. However, the interaction was not strong enough to overcome binding of wild type gp64 to the unknown cellular receptor(s) on the surface of alpha2 integrin-expressing cells (CHO-alpha2beta1) or enhance the viral uptake. After treatment of these cells with phospholipase C, internalization of all viruses was blocked or decreased significantly. However, one of the RKK displaying viruses, AcGFP(K)gp64, was still able to internalize into CHO-alpha2beta1 cells, although at a lower level as compared to non-treated cells. This may indicate the possible utilization of a PLC independent alternative route via, in this case, the alpha2beta1 integrin.
杆状病毒载体作为一种将基因导入哺乳动物细胞的工具显示出了前景。这些昆虫病毒已被证明能转导多种哺乳动物细胞系，并且在体内也已证实了基因转移。在本研究中，我们构建了两种重组杆状病毒载体，它们在与苜蓿银纹夜蛾多核型多角体病毒主要包膜蛋白gp64融合的绿色荧光蛋白（GFP）的两个不同环区中展示了整合素特异性基序RKK。通过酶联免疫吸附测定，这些病毒被证明能结合代表α2整合素受体结合位点（α2I结构域）的肽段。然而，这种相互作用不够强，无法克服野生型gp64与表达α2整合素的细胞（CHO-α2β1）表面未知细胞受体的结合，也无法增强病毒摄取。用磷脂酶C处理这些细胞后，所有病毒的内化均被阻断或显著减少。然而，其中一种展示RKK的病毒AcGFP(K)gp64仍能够内化进入CHO-α2β1细胞，尽管与未处理的细胞相比水平较低。这可能表明在这种情况下，可能利用了一条不依赖磷脂酶C的替代途径，即通过α2β1整合素。
