Cloning,Expression and Biological Effect of Pineapple Cu/Zn-SOD Superoxide Dismutase

• Main Authors: Fu Ming Tzu, 傅明慈
• Other Authors: Lin Chi Tsai
• Format: Others
• Language: zh-TW
• Published: 2000
• Online Access: http://ndltd.ncl.edu.tw/handle/74811647007849830996

碩士 === 國立海洋大學 === 水產生物技術研究所 === 88 === A full-length cDNA clone of 820 bp encoding a putative copper / zinc-superoxide dismutase ( Cu/Zn-SOD ) from pineapple fruit was cloned by PCR approach. Nucleotide sequence analysis of this cDNA clone revealed that it comprised a complete open reading frame coding for 152 amino acid residues. The deduced amino acid sequences showed high similarity ( 62.5 % ~ 81.6 % ) with the sequences of the Cu/Zn-SODs from other plant species. Computer analysis of the residues required for coordinating copper ( His-45, 47,62 and 119 ) and zinc ( His-62, 70,79 and Asp-82 ), as well as the two cysteines (56 and 145) that form a single disulfide bond, were well-conserved among all reported Cu/Zn-SOD sequences in present study. To further characterize the pineapple Cu/Zn-SOD, we subcloned its cDNA into an expression vector, pGEX-4T-1, and transformed into Escherichia coli XL-1blue.The expression of the recombinant Cu/Zn-SOD cDNA was confirmed by enzyme activity staining on a native acrylamide gel. In addition, the biological effect of the pineapple Cu/Zn-SOD was also investigated by transfecting pcDNA3-Cu/Zn-SOD into the microglial BV-2 cells which were treated lipopolysaccharide ( LPS, from the cell wall of gram-negative bacteria ) after transfection.. We found that expressed pineapple Cu/Zn-SOD in BV-2 cells could change the intracellular redox state and decrease the production of NO·, IL-1 and TNF- . These result suggested that the plant Cu/Zn-SOD may be useful in the reduction of the cellular immune reactions.