Studies on phospholipase signal peptide of Photobacterium damselae

• Main Authors: Shuo-Chieh Li, 李碩傑
• Other Authors: Cheng-Hui Lin
• Format: Others
• Language: zh-TW
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/69995467309552222717

碩士 === 國立臺灣海洋大學 === 水產養殖學系 === 96 === Abstrat Using the ability of the signal peptide to secrete the target protein out of cell, we try to construct a new expression vector which can express target protein out of cell. DNA of the signal peptide was synthesized by chemical synthesis method. The restriction enzyme sites of NdeI and NcoI were desiged in the end of DNA and ligated with pET32a(+)(NdeI/NcoI) to construct a plasmid called p32asp. EGFP gene digested from pT2KXIG△in by restriction enzyme and ligated with p32asp digested with the same restriction enzyme to construct p32aspGFP. Plasmid p32aspGFP was transformed into E.coli BL21(DE3). The recombinant bacteria were cultured and induce with IPTG. The spGFP protein was expressed in high level after 5 hour induction. The size of spGFP protein is 28.8 kDa in the soluble protein, and 26.6 kDa in culture medium. NNV coat protein gene was digested from pBScoat by restriction enzyme EcoRI and NcoI and ligated with p32asp digested with the same restriction enzyme to construct p32aspc. Plasmid p32aspc was transformed into E.coli BL21(DE3). The recombinant bacteria were cultured and induced with IPTG. The spc protein was expressed the 38.8kDa in 1mM IPTG iduced soluble protein but no expression in insoluble protein. The size 36.6 kDa of spc protein is in culture medium.