关键词: FMDV, 耐酸性改造, 昆虫细胞, 组装, 空衣壳结构

Abstract:

【Objective】 Foot-and-mouth disease virus (FMDV), a non-enveloped picornavirus, is sensitive to acidic conditions. At pH below 7 the icosahedral virus capsid dissociates into 12 pentamers. It is difficult to synthesize FMDV capsids in insect cells for the influence of low pH of insect cell medium. This study aimed at assembling empty capsid-like particles of AsiaⅠFMDV in insect cells by site-specific mutation of related amino acid residues to improve pH-stability of empty capsids. 【Method】 Two amino acid residues were site-specifically mutated to get modified P12A gene named mP12A (H140L and H143L in VP3). Both P12A and mP12A were then inserted into a transfer vector pFastBacTM Dual under the control of PH promoter, respectively. The 3C gene was placed under the control of P10 promoter. Two recombinant baculoviruses were generated by transfecting recombinant bacmids into Sf9 cells. High Five? cells were infected with two recombinant baculoviruses after multiplication in Sf9 cells. 【Result】 The target proteins were successfully expressed in insect cells by Western blotting, and that expressed capsid proteins could be processed by expressed viral 3C proteinase. The capsid proteins were mainly expressed near the inner membrane of insect cells as revealed by immunofluorescent assay, and that small amounts were released into culture medium as showed by sandwich ELISA. Furthermore, empty capsid-like particles were observed in lystate of High Five? cells infected with recombinant baculoviruses with mutated P12A under electron microscope. Whereas, many small-sized particles were observed in lystate of High Five? cells infected with recombinant baculovirus without mutation in P12A. The observed empty capsid-like particles were about 25-30 nm in diameter.【Conclusion】 This is the first report on the observations of empty capsid-like particles of FMDV in insect cells by microscopy. The potential utility of these recombinant, non-infectious, FMDV empty capsids for diagnostic and vaccine purposes is apparent.

Key words: FMDV, acid-resistant modification, insect cells, assembly, empty capsid structures