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KMID : 0545120020120030463
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Journal of Microbiology and Biotechnology
2002 Volume.12 No. 3 p.463 ~ p.469
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Expression of Rotavirus Capsid Proteins VP6 and VP7 in Mammalian Cells Using Semliki Forest Virus-Based Expression System
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CHOI, EUN-AH
KIM, EUN/OH, YOON-I/SHIN, KWANG-SOON/KIM, HYUN-SOO/KIM, SHUL-JOONG
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Abstract
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Rotaviruses are the world-wide leading causative agents of severe dehydrating gastroenteritis in young children and animals. The outer capsid glycoprotein VP7 and inner capsid glycoprotein VP6 of rotaviruses are highly antigenic and immunogenic. An SFV-based expression system has recently emerged as a useful tool for heterologous protein production in mammalian cells, exhibiting a much more efficient performance compared to other gene expression systems. Accordingly, the current study adopted an SFV-based expression system to express the VP7 of a group A human rotavirus from a Korean isolate, and the VP6 of a group B bovine rotavirus from a Korean isolate, in mammalian cells. The genes of the VP6 and VP7 were inserted into the SFV expression vector pSFV-1. The RNA was transcribed in vitro from pSFV-VP6 and pSFV-VP7 using SP6 polymerase. Each RNA was then electroporated into BHK-21 cells along with pSFV-helper RNA containing the structural protein gene without the packaging signal. The expression of VP6 and VP7 in the cytoplasm was then detected by immunocytochemistry. The recombinant virus was harvested by ultracentrifugation and examined under electron microscopy. After infecting BHK 21 cells with the defective viruses, the expressed proteins were separated by SDS-PAGE and analyzed by a Western blot. The results indicate that an SFV-based expression system for the VP6 and VP7 of rotaviruses is an efficient tool for developing a diagnostic kit and/or preventive vaccine.
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