| Summary: | 碩士 === 國立清華大學 === 生物科技研究所 === 93 === The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) is important for vaccine development. The truncated SARS-CoV TW1 S protein, SNHRI (88 kDa), carrying three S fragments (S74-253, S294-739, and S1129-1255) was expressed in mammalian cells. Mammalian cell expression of SNHRI glycoprotein with the use of a 138bp intron was found to increase by 1.9 folds, 2.5 folds, and 4.1 folds in Vero E6, QBI-293A cells, and CHO/dhFr- cells (dihydrofolate reductase [dhfr] gene deficient CHO cells), respectively. The expressed SNHRI glycoprotein was mainly Endo H-sensitive (~115 kDa glycoproteins) in these three mammalian cell lines. A minor form of Endo H-resistant glycoproteins (~130 kDa) was also found in CHO/dhFr- cells and VeroE6 cells. Expression vectors using the exon splicing enhancers, such as bidirectional splicing enhancer (BSE) or an exon splicing enhancer derived from the EDA alternative exon of the fibronectin gene (EDA ESE), was also found to increase SNHRI protein expression by 1.7 and 2.6 folds as compared to the intronless expression vector. Coupling BSE or EDA ESE with the 138bp intron leaded to suppression of the intron-enhancing effect. In order to establish stably expressing CHO cell clones, a stepwise methotrexate (MTX) selection was conducted in the CHO/dhFr- cells transfected with two amplifiable expression vectors, pSID (without intron) and pISID (with intron), containing a dhfr gene placed after an internal ribosome entry sites. Twelve stable cell clones expressing higher amount of ~115 kDa SNHRI protein were selected for gene amplification. This study provides useful information for future development of mammalian cell based SARS-CoV subunit vaccines.
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