Production of scFv-displaying BmNPV in silkworm larvae and its efficient purification

被引:5
作者
Dojima, Takashi [1 ]
Nishina, Takuya [2 ]
Kato, Tatsuya [2 ]
Ueda, Hiroshi [3 ]
Park, Enoch Y. [1 ,2 ]
机构
[1] Shizuoka Univ, Grad Sch Sci & Technol, Integrated Biosci Sect, Suruga Ku, Shizuoka 4228529, Japan
[2] Shizuoka Univ, Biotechnol Lab, Dept Appl Biol Chem, Fac Agr,Suruga Ku, Shizuoka 4228529, Japan
[3] Univ Tokyo, Sch Engn, Dept Chem & Biotechnol, Bunkyo Ku, Tokyo 1138656, Japan
关键词
bacmid; baculovirus; silkworm larva; single-chain variable fragment (scFv); surface display; NUCLEAR POLYHEDROSIS-VIRUS; GP64 ENVELOPE GLYCOPROTEIN; MAMMALIAN-CELLS; BACULOVIRUS VECTORS; IMMUNE-RESPONSE; FUSION PROTEIN; EXPRESSION; INFECTION; INSECT; GENE;
D O I
10.1042/BA20100173
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
070307 [化学生物学]; 071010 [生物化学与分子生物学];
摘要
Baculovirus-display technology utilizing the gp64 envelope protein has been developed. A simple and efficient process to separate the virus from the majority of the protein contaminants may be needed for the future demand of pure and functional baculovirus vectors ideal for vaccine- and gene-delivery applications. In the present study, using Bombyx mori (silkworm) larvae as a host, scFv (single-chain variable fragment)-surface displaying recombinant baculovirus production and its purification from silkworm larval haemolymph by SEC (size-exclusion chromatography) were demonstrated. The amounts of scFv were 4-8 mu g/ml in the haemolymph. The scFv gp64 fusion protein was confirmed to be incorporated into the cell membrane and the BmNPV (B. mori nucleopolyhedrovirus) surface by immunofluorescence microscopy and Western blotting. rBmNPV (recombinant BmNPV) was purified to higher purity by SEC using Sephacryl S-1000 column chromatography than by sucrose-density-gradient centrifugation. The recovery of purified rBmNPV was 22.2%, and the virus purity in the SEC fraction was increased 269-fold compared with its purity in haemolymph. judging from the results of ELISA, approx. 0.9% of the total baculovirus-particle proteins were occupied by scFv on their surface. A BmNPV-based silkworm-larval system is suitable for large-scale production of baculovirus-surface-displayed proteins or peptides in comparison with a cell-culture system. The present study will be useful for future BmNPV-application studies for gene delivery and vaccine trials.
引用
收藏
页码:63 / 69
页数:7
相关论文
共 29 条
[1]
Involvement of the toll-like receptor 9 signaling pathway in the induction of innate immunity by baculovirus [J].
Abe, T ;
Hemmi, H ;
Miyamoto, H ;
Moriishi, K ;
Tamura, S ;
Takaku, H ;
Akira, S ;
Matsuura, Y .
JOURNAL OF VIROLOGY, 2005, 79 (05) :2847-2858
[2]
Baculovirus induces an innate immune response and confers protection from lethal influenza virus infection in mice [J].
Abe, T ;
Takahashi, H ;
Hamazaki, H ;
Miyano-Kurosaki, N ;
Matsuura, Y ;
Takaku, H .
JOURNAL OF IMMUNOLOGY, 2003, 171 (03) :1133-1139
[3]
Importance of a CDR H3 basal residue in VH/VL interaction of human antibodies [J].
Aburatani, T ;
Ueda, H ;
Nagamune, T .
JOURNAL OF BIOCHEMISTRY, 2002, 132 (05) :775-782
[4]
Induction of antibodies in mice by a recombinant baculovirus expressing pseudorabies virus glycoprotein B in mammalian cells [J].
Aoki, H ;
Sakoda, Y ;
Jukuroki, K ;
Takada, A ;
Kida, H ;
Fukusho, A .
VETERINARY MICROBIOLOGY, 1999, 68 (3-4) :197-207
[5]
LOCATION, SEQUENCE, TRANSCRIPTIONAL MAPPING, AND TEMPORAL EXPRESSION OF THE GP64 ENVELOPE GLYCOPROTEIN GENE OF THE ORGYIA-PSEUDOTSUGATA MULTICAPSID NUCLEAR POLYHEDROSIS-VIRUS [J].
BLISSARD, GW ;
ROHRMANN, GF .
VIROLOGY, 1989, 170 (02) :537-555
[6]
BACULOVIRUS GP64 ENVELOPE GLYCOPROTEIN IS SUFFICIENT TO MEDIATE PH-DEPENDENT MEMBRANE-FUSION [J].
BLISSARD, GW ;
WENZ, JR .
JOURNAL OF VIROLOGY, 1992, 66 (11) :6829-6835
[7]
EUKARYOTIC VIRUS DISPLAY - ENGINEERING THE MAJOR SURFACE GLYCOPROTEIN OF THE AUTOGRAPHA-CALIFORNICA NUCLEAR POLYHEDROSIS-VIRUS (ACNPV) FOR THE PRESENTATION OF FOREIGN PROTEINS ON THE VIRUS SURFACE [J].
BOUBLIK, Y ;
DIBONITO, P ;
JONES, IM .
BIO-TECHNOLOGY, 1995, 13 (10) :1079-1084
[8]
Comparison of the N-linked glycosylation of human β1,3-N-acetylglucosaminyltransferase 2 expressed in insect cells and silkworm larvae [J].
Dojima, Takashi ;
Nishina, Takuya ;
Kato, Tatsuya ;
Uno, Btsuyoshi ;
Yagi, Hirokazu ;
Kato, Koichi ;
Park, Enoch Y. .
JOURNAL OF BIOTECHNOLOGY, 2009, 143 (01) :27-33
[9]
Comparison of the efficiencies of different affinity tags in the purification of a recombinant secretory protein expressed in silkworm larval hemolymph [J].
Dojima, Takashi ;
Nishina, Takuya ;
Kato, Tatsuya ;
Ueda, Hiroshi ;
Park, Enoch Y. .
BIOTECHNOLOGY AND BIOPROCESS ENGINEERING, 2009, 14 (03) :281-287
[10]
Baculovirus stimulates antiviral effects in mammalian cells [J].
Gronowski, AM ;
Hilbert, DM ;
Sheehan, KCF ;
Garotta, G ;
Schreiber, RD .
JOURNAL OF VIROLOGY, 1999, 73 (12) :9944-9951