Peipp Matthias, Saul Domenica, Barbin Karin, Bruenke Joerg, Zunino Susan J, Niederweis Michael, Fey Georg H
Chair of Genetics, University of Erlangen, Nuremberg, Staudtstrasse 5, D 91058 Erlangen, Germany.
J Immunol Methods. 2004 Feb 15;285(2):265-80. doi: 10.1016/j.jim.2003.12.001.
Two sets of expression vectors were constructed that permitted the efficient expression of single-chain Fv fragments (scFvs) fused N-terminally to an enhanced mutant of the green fluorescent protein GFP+ or the red fluorescent protein DsRed in insect and mammalian cells. The vectors allowed rapid cloning of scFv fragments and secretion of the fusion proteins in a native conformation. Fluorescent scFv fusion proteins directed against a series of cluster of differentiation (CD) antigens were efficiently secreted by transiently transfected mammalian cells and insect cells infected with baculoviral expression constructs. Yields of the secreted proteins varied from 100 microg/l to 3 mg/l. The purified proteins were functionally active in flow cytometry, immunofluorescent microscopy, and competition binding experiments performed to delineate the epitopes recognized by different monoclonal antibodies against the same polypeptide. The use of two different scFv fragments fused with red and green fluorescent proteins and reacting with T- and B-cell lineage markers (CD7 and CD19), respectively, allowed a simplified quantitation of both subsets in two-color flow cytometry experiments with mixed populations of T- and B-lymphoid cells. Due to the lack of Fc domains in the scFv proteins, the fluorescent fusion proteins showed more than 20-fold reduced background fluorescence compared with whole antibodies of the same specificity in experiments with effector cells expressing the high affinity FcgammaRI receptor CD64. Thus, for a number of analytical applications, fluorescent scFv fusion proteins offer advantages over the use of complete primary antibodies and chemically labeled fluorescent secondary antibodies.
构建了两组表达载体，可在昆虫和哺乳动物细胞中高效表达N端融合绿色荧光蛋白增强突变体GFP+或红色荧光蛋白DsRed的单链Fv片段（scFv）。这些载体允许快速克隆scFv片段，并以天然构象分泌融合蛋白。针对一系列分化簇（CD）抗原的荧光scFv融合蛋白可由瞬时转染的哺乳动物细胞和感染杆状病毒表达构建体的昆虫细胞有效分泌。分泌蛋白的产量从100μg/l到3mg/l不等。纯化后的蛋白在流式细胞术、免疫荧光显微镜以及为确定针对同一多肽的不同单克隆抗体所识别的表位而进行的竞争结合实验中具有功能活性。使用分别与红色和绿色荧光蛋白融合并与T细胞和B细胞谱系标记物（CD7和CD19）反应的两种不同scFv片段，可在T淋巴细胞和B淋巴细胞混合群体的双色流式细胞术实验中简化对两个亚群的定量分析。由于scFv蛋白中缺乏Fc结构域，在与表达高亲和力FcγRI受体CD64的效应细胞进行的实验中，荧光融合蛋白的背景荧光比具有相同特异性的完整抗体降低了20多倍。因此，对于许多分析应用而言，荧光scFv融合蛋白比使用完整的一抗和化学标记的荧光二抗具有优势。
