Production and functional analysis of engineered proteins using baculovirus expression system

• Main Authors: Lin Yi-ting, 林怡婷
• Other Authors: Dr.Yu-Chan Chao
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/74450048750240713283

碩士 === 臺北醫學大學 === 細胞及分子生物研究所 === 93 === Baculovirus expression system is a conventional tool for production of large amounts of engineered proteins, which may be subsequently purified for further studies in their biological and enzymatic functions. In this study, two baculovirus expression systems, AcMNPV and BmNPV, were used to produce 3 recombinant proteins: porcine lactoferrin, phytase of E. coli, and apisimin, which is a small component of royal jelly. The use of BmNPV expression system is for subsequent massive production of recombinant protein in silkworm, which, after purification, will be added in feeding to increase porcine immunity and alleviate pollutions caused by porcine feces. We’ve successfully produce recombinant porcine lactoferin, phytase of E. coli, and apisimin of royal jelly by AcMNPV and BmNPV expression systems. Recombinant apisimin protein was further purified for studies for its biological and enzymatic activities. Purified phytase protein produced by baculovirus expression system exhibited higher enzymatic activity compared to phytase produced by plant. Purified apisimin protein produced by baculovirus expression system did not cause cell toxicity in immunological experiment. From ELISA experiment, productions of TNF-, IL-1- and IL-6 increased proportionally with the concentration of apisimin, indicating activation of immune responses by apisimin. Under inflammatory condition, e.g., stimulation by LPS, production of TNF- decreased inversely with the concentration of apisimin. On the contrary, productions of IL-1- and IL-6 show initial increase, followed by a steady decline as the concentration of apisimin increased. Therefore, it’s proposed that apisimin could regulate immune response bi-directionally. In test for antibiotic function, apisimin did not suppress bacterial growth in 11 bacteria strains tested. However, it exhibited ability in cleaning up free radicals, and this function was concentration-dependent. In conclusion, purified recombinant proteins produced by baculovirus expression system were shown to be biologically functional. Therefore, baculovirus expression system is a useful tool in studying functions of proteins whose functions are still unknown.