|
KMID : 0613820140240090995
|
|
Journal of Life Science
2014 Volume.24 No. 9 p.995 ~ p.1000
|
|
|
Production of Red-spotted Grouper Nervous Necrosis Virus (RGNNV) Capsid Protein Using Saccharomyces cerevisiae Surface Display
|
|
Park Mi-Rye
Suh Sung-Suk
Hwang Jin-Ik
Kim Dong-Giun
Park Jong-Bum
Chung Young-Jae
Lee Taek-Kyun
|
|
|
Abstract
|
|
|
|
The studies of marine viruses in terms of viral isolation and detection have been limited due to the high mutation rate and genetic diversity of marine viruses. Of the modern methods currently used to detect marine viruses, serological methods based on enzyme-linked immunosorbent assay (ELISA) are the most common. They depend largely on the quality of the antibodies and on highly purified suitable antigens. Recently, a new experimental system for using viral capsid protein as an antigen has been developed using the yeast surface display (YSD) technique. In the present study, the capsid protein gene of the red-spotted grouper nervous necrosis virus (RGNNV) was expressed and purified via YSD and HA-tagging systems, respectively. Two regions of the RGNNV capsid protein gene, RGNNV1 and RGNNV2, were individually synthesized and subcloned into a yeast expression vector, pCTCON. The expressions of each RGNNV capsid protein in the Saccharomyces cerevisiae strain EBY100 were indirectly detected by flow cytometry with fluorescently labeled antibodies, while recognizing the C-terminal c-myc tags encoded by the display vector. The expressed RGNNV capsid proteins were isolated from the yeast surface through the cleavage of the disulfide bond between the Aga1 and Aga2 proteins after β-mercaptoethanol treatment, and they were directly detected by Western blot using anti-HA antibody. These results indicated that YSD and HA-tagging systems could be applicable to the expressions and purification of recombinant RGNNV capsid proteins.
|
|
|
KEYWORD
|
|
|
Capsid protein, flow cytometry, recombinant protein, RGNNV, yeast surface display
|
|
|
FullTexts / Linksout information
|
|
|
|
|
|
Listed journal information
|
|
|