| Summary: | 碩士 === 南臺科技大學 === 生物科技系 === 104 === Protein expression is an essential issue for various biomedical studies. Both high level protein expression, and correct folding and post-translational modifications are needed.Nerve Growth Factor protein has many important functions, for example, it binds with different receptor binding, which have different cellular regulation. Consequently, it promotes neural differentiation, protect and promote nerve regeneration in nerve injury condition. Moreover, it plays an important role in neurological disease processes. The purpose of this study is using Bac-to-Bac system to construct recombinant baculovirus, and resulted a recombinant baculovirus, which contains NGF gene for proteins expression in insect and mammalian. This system is originally for producing foreign proteins in insect cells, in order to express protein in mammalian cells, CMVie promoter is required to drive the target gene in these cells. Moreover, in order to verify whether a gene is able to expression in mammalian cells, we used jellyfish green fluorescent protein gene (EGFP) as a reporter and a recombinant baculovirus , named vAcBac-CMVie-EGFP was constructed. The recombinant virus which named vAcBac-CMVie-NGF-10xHis, produced recombinant protein with poly-histidine tag in C-terminal for affinity labeling to facilitate protein detection and purification. Results showed that the use of baculovirus expression system in Sf-9 cell can properly express NGF protein, at 72 h post cell infection. These results showed that we have succeeded in establishing a recombinant protein expression system for NGF expression. Thus, this is a system easy for detection and purification of recombinant protein for future manufacture and purification protein for the study of protein activity and structure analysis.
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