Massa Silvia, Paolini Francesca, Curzio Gianfranca, Cordeiro Marcelo Nazario, Illiano Elena, Demurtas Olivia Costantina, Franconi Rosella, Venuti Aldo
a ENEA - Italian National Agency for New Technologies, Energy and the Environment , Department of Sustainability, Laboratory of Biotechnologies , Rome , Italy.
b Regina Elena National Cancer Institute HPV-UNIT , RIDAIT Dept , Rome , Italy.
Hum Vaccin Immunother. 2017 Feb;13(2):271-282. doi: 10.1080/21645515.2017.1264766. Epub 2017 Jan 24.
Signal sequences (ss) play a critical role in the sorting of nascent secretory and membrane proteins. This function has been conserved from bacteria through eukaryotes, although ss appear diverse in length and amino acid composition. Sorting of proteins is also critical to instruct antigens for a proper immunological response. Thus, a plant ss was used to drive Human Papillomavirus (HPV) model antigens into the human secretory pathway: the HPV16 E7 oncoprotein, its chimera with the coat protein (CP) of the Potato Virus X (PVX), the first 200 amino acids of the HPV16 minor capsid protein L2 (known to harbour cross-reacting epitopes) and its chimera with E7 gene. These genes were used to transfect HEK-293 cells and to immunize C57BL/6 mice. The ss-provided genes were expressed, and proteins detected by immunofluorescence and immunoblotting. Mouse immunization with DNA constructs carrying the ss elicited a strong humoral response against both E7 and L2 and a weak cell-mediated immunity. To our knowledge this is the first demonstration that a signal sequence derived from a plant can modulate the sorting of a heterologous protein in mammalian cells. This activity in mammalian cells may be responsible for the observed increased humoral response to DNA-based vaccines that are generally weak inducers of IgG response. This might open new perspectives in the design of DNA vaccines, especially to counteract infections where a strong humoral response is needed.
信号序列（ss）在新生分泌蛋白和膜蛋白的分选过程中发挥着关键作用。尽管信号序列在长度和氨基酸组成上呈现出多样性，但从细菌到真核生物，这一功能一直得以保留。蛋白质的分选对于指导抗原引发适当的免疫反应也至关重要。因此，一种植物信号序列被用于将人乳头瘤病毒（HPV）模型抗原驱动至人类分泌途径：HPV16 E7癌蛋白、其与马铃薯X病毒（PVX）外壳蛋白（CP）的嵌合体、HPV16次要衣壳蛋白L2的前200个氨基酸（已知含有交叉反应表位）及其与E7基因的嵌合体。这些基因被用于转染HEK - 293细胞并免疫C57BL / 6小鼠。含有信号序列的基因得以表达，通过免疫荧光和免疫印迹检测到了蛋白质。用携带信号序列的DNA构建体对小鼠进行免疫，引发了针对E7和L2的强烈体液反应以及微弱的细胞介导免疫。据我们所知，这是首次证明源自植物的信号序列能够调节哺乳动物细胞中异源蛋白的分选。在哺乳动物细胞中的这种活性可能是观察到的对基于DNA的疫苗体液反应增强的原因，而这类疫苗通常是较弱的IgG反应诱导剂。这可能为DNA疫苗的设计开辟新的前景，特别是在应对需要强烈体液反应的感染方面。
