Influence of intron and exon splicing enhancers on mammalian cell expression of a truncated spike protein of SARS-CoV and its implication for subunit vaccine development

The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) is important for vaccine development. A truncated S protein of the TW1 strain, S(TR2) (88 kDa), carrying three S fragments (S74–253, S294–739, and S1129–1255) was investigated to study the influences of intron and exon splicing enhancers to improve S(TR2) protein expression in mammalian cells. Our results showed that S(TR2) protein expression with the use of an 138 base-pair intron addition increased by 1.9-, 2.5-, and 4.1-fold in Vero E6, QBI-293A cells, and CHO/dhFr− cells (dihydrofolate reductase [dhfr] gene deficient CHO cells), respectively. Using the exon splicing enhancers, including a bidirectional splicing enhancer (BSE) or an exon splicing enhancer derived from the EDA alternative exon of the fibronectin gene (EDA ESE), were also found to increase S(TR2) protein expression in CHO/dhFr− cells by 1.7- and 2.6-fold. Nevertheless, combination of the intron and the exon splicing enhancers resulted in suppressing the intron-enhancing e S(TR2) protein expression in in CHO/dhFr− cells. Our studies also demonstrated the S(TR2) protein was mainly as the Endo H-sensitive glycoprotein (115 kDa) expressed in Vero E6, QBI-293A, and CHO/dhFr− cells. However, only a minor form of the Endo H-resistant glycoproteins (∼130 kDa) was detected in CHO/dhFr− cells. Taken together, our results indicated that intron had a better enhancing effect on S(TR2) protein expression than exon splicing enhancers, and the expression of ∼130 kDa S(TR2) glycoprotein was enhanced by the intron addition into the expression vector construct. Results of the present study can provide an optimal strategy to enhance SARS-CoV S protein expression in mammalian cells and may contribute to the development of SARS-CoV subunit vaccine.