Display of functional antibody Fab fragment on the baculovirus surface

外源肽或蛋白质在病毒颗粒表面的展示已被证明对于从不同文库中选择编码新功能的基因是有价值的。最近，杆状病毒（苜蓿银纹夜蛾核型多角体病毒，AcNPV）已成功地用于展示异源蛋白的病毒颗粒的表面上的融合蛋白的主要杆状病毒包膜糖蛋白gp64。在用这种重组杆状病毒感染昆虫细胞时，gp64融合蛋白以及gp64被表达并转运到细胞膜，在出芽过程中它们被子代病毒拾取。在这项研究中，产生的重组杆状病毒展示抗体Fab片段在其表面上进行了研究。设计重组杆状病毒，使得编码Fab片段的轻链的基因表达为与gp64的N末端的融合物，同时Fab片段的Fd片段的基因表达为分泌蛋白。用重组杆状病毒感染Sf9昆虫细胞后，通过酶联免疫吸附测定（ELISA）分析感染细胞的培养液，表明培养液具有抗原结合活性。通过流式细胞术使用特异于Fab片段的荧光素异硫氰酸酯（FITC）缀合的抗体成功地在用重组杆状病毒感染的Sf9细胞的表面上检测到Fab片段。这些结果表明，抗体Fab片段可以以活性形式展示在杆状病毒颗粒的表面上，并且展示所需Fab片段的杆状病毒可以通过使用具有荧光标记抗原的荧光激活细胞分选仪（FACS）来选择。功能性抗体Fab段的成功展示为高效筛选特异性抗体提供了新的途径。

Display of foreign peptides or proteins on the surface of virus particles has proven to be valuable for selection of genes encoding novel functions from diverse libraries. Recently, baculovirus (Autographa californica nucleopolyhedrovirus, AcNPV) has been successfully used for displaying heterologous proteins on the surface of viral particles by fusing the protein to the major baculoviral envelope glycoprotein gp64. On infection of insect cells with such a recombinant baculovirus, gp64-fusion proteins as well as gp64 are expressed and transported to the cell membrane where they are picked up by progeny viruses during the budding process. In this study, generation of recombinant baculovirus displaying an antibody Fab fragment on its surface was investigated. Recombinant baculovirus was designed so that the gene encoding the light chain of the Fab fragment was expressed as a fusion to the N-terminus of gp64, while at the same time the gene of the Fd fragment of the Fab fragment was expressed as a secretion protein. Following infection of Sf9 insect cells with the recombinant baculovirus, culture broth of infected cells was analyzed by enzyme-linked immunosorbent assay (ELISA), indicating that the culture broth has an antigen-binding activity. The Fab fragments were successfully detected on the surface of Sf9 cells infected with the recombinant baculovirus by flow cytometry using a fluorescein isothiocyanate (FITC)-conjugated antibody specific to the Fab fragment. These results suggest that antibody Fab fragments can be displayed on the surface of baculovirus particles in an active form and that baculoviruses displaying desired Fab fragments can be selected by the use of fluorescence-activated cell sorter (FACS) with a fluorescence-labeled antigen. The successful display of a functional antibody Fab fragment may offer a novel approach for efficient selection of specific antibodies.