| Summary: | 碩士 === 國立清華大學 === 生物科技研究所 === 94 === The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) is important for vaccine development. A truncated SARS-CoV TW1 S protein, STR2 (88 kDa), carrying three S fragments (S-74-253, S-294-739, and S-1129-1255) could express and show the major form as the Endo H-sensitive (~115 kDa) in CHO cells. To establish stably expressing cell clones, the CHO/dhFr- cells were transfected with two amplifiable vectors, ISID (IRES-driven dhfr) and ISIZ (SV40-driven dhfr), for stepwise MTX selection. The enhancement of the ~115 kDa glycoform generation was observed through gene amplification. After stepwise MTX selection, we can compare the difference of gene amplification between the two vectors in CHO cell chromosome engineering. This study can provide information for development of mammalian cell-based SARS-CoV subunit vaccines, and for amplifiable vectors design to produce recombinant proteins.
Because post-translational modification could improve the efficiency and stability of the recombinant proteins in mammalian cells, mammalian cells are the most common used as host cells to produce biopharmaceuticals. CHO cells, which can amplify target gene following dhfr gene amplification to increase productivity, are used as host cells in this study.
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