Efficient production of canine interferon-alpha in silkworm Bombyx mori by use of a BmNPV/Bac-to-Bac expression system.
We exploited the silkworm Bombyx mori for the production of recombinant canine interferon-alpha (CaIFN-alpha). The recombinant baculovirus harboring canine interferon gene was rapidly generated by the BmNPV/Bac-to-Bac system that was recently developed. In B. mori-derived cell lines, the expression of the recombinant protein reached maximal levels around 72-96 h post-infection. For the isolation of the expressed recombinant protein from B. mori larvae, the whole bodies of the infected larvae were homogenized, and the expressed protein was purified by affinity chromatography. Based on the fact that the recombinant CaIFN-alpha showed two bands on the sodium dodecyl sulfate polyacrylamide gel electrophoresis pattern, the expressed protein was thought to be glycosylated. The rCaIFN-alpha yield was about 528 microg per larva, showing that the expression in silkworm was successful. Furthermore, the recombinant protein was proven to be able to inhibit the infection of Madin-Darby canine kidney cells by the vesicular stomatitis virus, indicating that it is biologically active in vitro. The method established in this study provides an efficient way to produce a large amount of CaIFN-alpha and paves the way for further utilization of this protein as a therapeutic agent or vaccine adjuvant in dogs.
Na
Zhao
Z
College of Animal Sciences, Zhejiang University, Huajiachi Campus, Hangzhou, 310029, China.
Huipeng
Yao
Y
Lipan
Lan
L
Cuiping
Cao
C
Umashankar
M L
ML
Xingmeng
Lu
L
Xiaofeng
Wu
W
Bing
Wang
W
Weizheng
Cui
C
Cenis
J L
JL
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D012831:Bombyx / Q000235:genetics* / Q000378:metabolism*; D002460:Cell Line; D002470:Cell Survival; D002846:Chromatography, Affinity; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D006031:Glycosylation; D016898:Interferon-alpha / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D007814:Larva / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D018353:Rhabdoviridae Infections / Q000276:immunology; D017422:Sequence Analysis, DNA; D018116:Vesiculovirus / Q000276:immunology
D016898:Interferon-alpha; D011994:Recombinant Proteins