Expression and Characterization of the Infectious Bursal Disease Virus Protein 4 Mutants.

• Main Authors: Lin, Hsiang-Min, 林湘敏
• Other Authors: Lai, Su-Yuan
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/94753084171502175806

碩士 === 中臺科技大學 === 食品科技研究所 === 101 === Infectious bursal disease virus （IBDV）is the causative agent of a highly contagious immunosuppressive disease among young chickens. Its genome contains two segments of double-stranded RNA. The viral protease VP4 is a serine protease which catalyzes the hydrolysis of the polyprotein （VP2-VP4-VP3）. It has been shown to contain a Ser/Lys catalytic dyad in the active site. In this study, plasmids HVP4-S652A, HVP4-K692A and HVP4-S652K692A were constructed. Mutant proteins HVP4-S652A, HVP4-K692A, HVP4-S652K692A were expressed in E. coli and purified. Our results showed that the activities of the active site mutants were dramatically decreased. When subjected to TEM observation, HVP4-S652A, HVP4-K692A and HVP4-S652K692A exhibit a tubular structure with 100~300 nm in length. Mutant proteins HS1VP4-S652A, HS1VP4-K692A and HS1VP4-S652K692A were also expressed and analyzed by SDS-PAGE and Western blotting. The results demonstrated that these proteins have lost the self-cutting activity in E. coli cells.