Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Kyushu University, Maidashi, Higashi-ku, Fukuoka, Japan.
Analyst. 2011 May 21;136(10):2056-63. doi: 10.1039/c1an15027h. Epub 2011 Mar 25.
A fluorescent single-domain antibody (fluobody), a chimera of a green fluorescent protein (AcGFP) with a single chain variable fragment antibody (scFv), against plumbagin (5-hydorxy-2-methyl-1,4-naphthoquinone; PL) was successfully expressed in the hemolymph of silkworm larvae using a Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system to develop a rapid, simple, and sensitive fluorescence-linked immunosorbent assay (FLISA). In this study, two kinds of fluobody, in which the PL-scFv was fused at the N-terminus (N-fluobody) or C-terminus of AcGFP (C-fluobody), were expressed in silkworm larvae for comparative purposes. Interestingly, both fluobodies expressed in the BmNPV bacmid DNA system retained both of their original functions as an AcGFP and a PL-scFv, although the functions of the N-fluobody were found to be inferior to those of C-fluobody when they were expressed in Escherichia coli. Moreover, an improvement in the limit of quantification for PL measurement was observed in FLISA (24 ng mL(-1)) compared with conventional ELISA (0.2 µg mL(-1)). Since both the C-fluobody and N-fluobody are useful probes for FLISA and the time-, cost-consuming refolding step required in the conventional bacterial expression system can be avoided when they are expressed in the BmNPV bacmid DNA system, the silkworm expression system is useful for expressing fluobodies when developing FLISA.
一种针对白花丹醌（5-羟基-2-甲基-1,4-萘醌；PL）的荧光单域抗体（fluobody），由绿色荧光蛋白（AcGFP）与单链可变片段抗体（scFv）组成的嵌合体，成功地通过家蚕杆状病毒（BmNPV） bacmid DNA 系统在蚕幼虫的血淋巴中表达，从而开发出一种快速、简单、灵敏的荧光酶联免疫吸附测定法（FLISA）。在这项研究中，为了进行比较，在蚕幼虫中表达了两种 fluobody，一种是将 PL-scFv 融合到 AcGFP 的 N 端（N-fluobody），另一种是融合到 C 端（C-fluobody）。有趣的是，尽管在大肠杆菌中表达时，N-fluobody 的功能不如 C-fluobody，但在 BmNPV bacmid DNA 系统中表达的两种 fluobody 都保留了它们作为 AcGFP 和 PL-scFv 的原始功能。此外，与传统的酶联免疫吸附测定法（0.2 µg mL(-1))相比，FLISA 中 PL 测量的定量限得到了改善（24 ng mL(-1))。由于 C-fluobody 和 N-fluobody 都是 FLISA 的有用探针，并且可以避免在传统细菌表达系统中需要耗时、高成本的复性步骤，因此当在家蚕杆状病毒 bacmid DNA 系统中表达时，家蚕表达系统对于表达 fluobody 开发 FLISA 是有用的。
