The study of shrimp white spot syndrome virus – wssv 044 gene expression

• Main Authors: Chia-Yi Tsai, 蔡佳怡
• Other Authors: Nai-Yaeh Tien
• Format: Others
• Language: zh-TW
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/89763811417482627534

碩士 === 嘉南藥理科技大學 === 生物科技系暨研究所 === 96 === White spot syndrome virus (WSSV), genus Whispovirus, family Nimaviridae, is a major shrimp pathogen that is highly virulent in penaeid shrimp and can also infect most species of crustacean. Because of the large size of WSSV genome and the uniqueness of viral proteins , only a few WSSV genes have been studied beyond sequence analysis. In this study, the ORF of wssv044 gene contained 201 nucleotides, encoding 67 a.a. The wssv044 gene was expressed in E. coli with His6-WSSV044 fusion protein ( 11-kDa ). The His6-WSSV044 were confirmed using Western bolt analysis with mouse anti-His antibodies. After 1mM IPTG and 37℃ induction for 3 hours, we found the His6-WSSV044 fusion proteins were express with inclusion body form. Subsequently, the expression of WSSV genes by fusing enhanced green fluorescent protein (EGFP) gene as a reporter gene were investigated in the insect Sf9 cells. First, we constructed and isolated the recombinant plasmids containing different WSSV genes respectively, which fused with N-terminal or C-terminal of EGFP. Then these recombinant plasmids were transfected into Sf9 cells and observed under a inverted fluorescence microscope. The results revealed that the fusion proteins containing different WSSV genes might be expressed diversely in transfected insect cells. A His pull-down assay has been used further to confirm the interactions between WSSV044 and shrimps proteins.