Construction of an Eukaryotic Expression Vector for SARS-CoV Sars7a and EGFP Fusion Protein Expression
Abstract: Partial overlapping primers were designed based on the severe acute respiratory syndrome coronavirus (SARS-CoV) sars7a gene and were chemically synthesized. The sars7a gene fragment was obtained by two-round of PCR and this fragment was used as template for a further round of PCR by using a pair of primers to introduce Kozak sequence and to delete the stop codon. The mammalian cell expression vector for Sars7a and enhanced green fluorescent protein (EGFP) fusion protein was generated by inserting the PCR product into pEGFP-N1 vector, with sars7a gene upstream to EGFP gene. K562 cells were transfected by the expression vector and the green fluorescence of EGFP could be detected with flow cytometry and confocal microscopy, indicating the expression of Sars7a-EGFP fusion protein. This fusion protein was distributed in the whole cells, which suggested that Sars7a was probable a cytosolic protein rather than a membrane protein. Besides, the expression of Sars7a had no significant effect on the apoptotic cell death of K562 cells.