Functional Analysis of Cathepsin L (CTSL) in Orange-Spotted Grouper (Epinephelus coioides)

• Main Authors: You-HanCai, 蔡侑翰
• Other Authors: Han-You Lin
• Format: Others
• Language: zh-TW
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/22240321999500028184

碩士 === 國立成功大學 === 生物科技研究所碩博士班 === 100 === Cathepsin L plays a very important role in animal physiological defend mechanism. In previous studies, this proteolysis enzyme was present in the lysosome, and degradated the cell waste. But in the immune cell, the same mechanism of cathepsin L in the lysosome was help in the foreign antigens digest, and antigen presentation. Since there were little informations of the teleost cathepsin L, the cathepsin L gene of orange spotted grouper (Epinephelus coioides) was cloned and the recombinant was expressed by E. coli expression system, the recombinant teleost cathepsin L was used to ananlysis the biochemical properties, and whether cathepsin L has ability to against pathogens in this study. Meanwhile, the cathepsin L mRNA expression pattern in various organs at different time was detected by real-time polymerase chain reaction after immune stimulation. The results showed the grouper cathepsin L was strictly work between pH 3 to 6, between 4 65℃and has maximal activity at 40℃; and it retain 80% maximal activity at 25℃. The half-life of this enzyme is about 2.07 hrs under the working condition. In the bactericidal test, the enzyme degradated bacterial protein while culture together under working condition, and that will kill the bacterial in this situation. In the tissues tropism, cathepsin L can be detected at all the organs, the mRNA expression raised significanly in the liver after Vibrio anguillarum stimulation. Since the relative mRNA expression will highly expression in the infectious situation and the bactericidal bioactive we observed. We speculate that orange-spotted grouper cathepsin L may play a specific role in immune mechanisms and against pathogen.