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Display of functional antibody Fab fragment on the baculovirus surface

杆状病毒表面功能性抗体Fab片段的展示

基本信息

批准号：
17560690
负责人：
YAMAJI Hideki
金额：
$ 2.24万
依托单位：
Kobe University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17560690/
关键词：
Insect cell Baculovirus Antibody

项目摘要

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.

在病毒颗粒表面展示外源肽或蛋白质已被证明对从不同文库中选择编码新功能的基因是有价值的。近年来，杆状病毒（加利福尼亚Autographa nucleopolyhedrovirus， AcNPV）通过融合杆状病毒包膜糖蛋白gp64，成功地在病毒颗粒表面展示了异源蛋白。在用这种重组杆状病毒感染昆虫细胞后，gp64融合蛋白和gp64被表达并运输到细胞膜上，在出芽过程中被后代病毒获取。在这项研究中，研究了在其表面显示抗体Fab片段的重组杆状病毒的产生。设计重组杆状病毒，使编码Fab片段轻链的基因融合到gp64的n端表达，同时将Fab片段Fd片段的基因作为分泌蛋白表达。重组杆状病毒感染Sf9昆虫细胞后，用酶联免疫吸附试验（ELISA）对感染细胞的培养液进行分析，结果表明培养液具有抗原结合活性。利用FITC抗体在重组杆状病毒感染的Sf9细胞表面成功检测到Fab片段。这些结果表明，抗体Fab片段可以以活性形式显示在杆状病毒颗粒表面，并且可以使用荧光活化细胞分选器（FACS）与荧光标记抗原选择显示所需Fab片段的杆状病毒。功能抗体Fab片段的成功展示可能为特异性抗体的有效选择提供一种新的途径。