Expression of human papillomavirus 6b L1 protein in silkworm larvae and enhanced green fluorescent protein displaying on its virus-like particles

Abstract

Human papillomavirus (HPV) 6b L1 capsid protein was expressed using the Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid expression system in silkworm larvae. Two constructs, full-length L1 (500 a.a) and C-terminal-deleted short L1 (479 a.a), and three PCR-manipulated antigenic loops at amino acids 55–56, 174–175, and 348–349 regions were incorporated with whole enhanced green fluorescent protein (EGFP). Expressed in full, short L1 proteins and variants were purified in heparin affinity column chromatography and confirmed by SDS-PAGE and western blot. The presence of self-assembled virus-like particles (VLPs) and EGFP incorporation on the surface of VLPs were confirmed by the observation of transmission electron and immunoelectron microscopies, respectively. HPV 6b L1 major capsid protein was successfully expressed in silkworm, and effective manipulation on the antigenic regions showed the path to versatile vaccine development based on HPV L1-VLPs.

Production of human papillomavirus 6b L1 virus-like particles incorporated with enhanced green fluorescent whole protein in silkworm larvae

“…With immuno-labeling with Alexa Fluor® 594, we can confirm that its red fluorescence was coinciding with the EGFP fluorescence, indicating that each expressed L1:EGFP chimeric VLPs had EGFP fluorescence. This result is contradictory to previous report, which mentions these L1:EGFP chimeric VLPs did not show EGFP fluorescence [14].…”

“…Previously, we had showed that the expression of full length HPV 6b L1 capsid protein could form efficiently VLPs when expressed in silkworm-bacmid system [14]. In this work, we present incorporation of whole EGFP into two antigenic loops of HPV 6b L1, expression using bacmid technology, and characterization of the VLP assembly.…”

“…Two different surface loops incorporated with EGFP in HPV 6b L1 capsid protein was constructed as described previously [14]. Briefly, each HPV 6b L1:EGFP fusion gene was obtained by two-step PCR.…”

“…Each amplified fusion gene was inserted at EcoRI-KpnI site in pFastBac1. The recombinant BmNPV CPbacmid containing each HPV 6b L1:EGFP fusion gene was constructed according to the protocol described previously [14,15]. The fifth instar Bombyx mori larvae were individually injected with 4 µg of chimeric HPV 6b L1:EGFP bacmids and reared in 25°C incubator [15].…”

Study of intact virus‐like particles of human papillomavirus by capillary electrophoresis

“…Capsomers assembly into HPV‐VLP requires disulfide bridges . HPV‐VLP production has been made using various expression systems, such as insect, yeast, silkworms, and plant cells . In this study, HPV16‐VLP were produced using the baculovirus‐insect cells expression system that is one of the two most commonly used systems with yeast cells.…”