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KMID : 1094720130180030514
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Biotechnology and Bioprocess Engineering
2013 Volume.18 No. 3 p.514 ~ p.519
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Production of human papillomavirus 6b L1 virus-like particles incorporated with enhanced green fluorescent whole protein in silkworm larvae
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Muthukutty Palaniyandi
Kato Tatsuya
Park Enoch Y.
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Abstract
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Using human papillomavirus (HPV) as a subunit vaccine and its manipulation of surface loops is current trending research. Since the atomic model of L1 protein conformations were deciphered, their manipulations of epitopes bring multivalent vaccines. Here, in the present study, we have manipulated antigenic loops of HPV 6b L1 capsid proteins in the amino acid regions 174 ∼ 175 (L1:174EGFP) and 348 ∼ 349 (L1:348EGFP) with whole enhanced green fluorescent protein(EGFP), expressed in the silkworm larva using Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid technology. The expressed proteins were partially purified using sucrose density-gradient centrifugation and size-exclusion chromatography (SEC). The display of EGFP in virus-like particles (VLPs) was confirmed by immuno-fluorescence microscopy, Western blots and immune-transmission electron microscopy (immuno-TEM). There was higher expression of EGFP incorporated L1:174EGFP than L1:348EGFP. Hydrodynamic diameter of VLPs was corroborated by dynamic light scattering, confirming the size of expected range of around 160 nm and substantiating the incorporation of EGFP. From immuno-TEM, each L1:EGFP VLP formed small particles, suggesting that small particles of L1:EGFP fusion protein were aggregated. Our study illustrates that incorporation of whole protein can efficiently form chimeric VLPs, without hindering the conformation. HPV L1 protein accommodated a whole protein on its antigenic loop as a small particle, but an inserted whole protein was unstable.
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KEYWORD
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virus-like particle, human papillomavirus, L1 protein, silkworm, BmNPV bacmid, display of protein
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