Sars-cov S1 Protein In Mammalian Cells, Expression And Purification,

Aim: Severe acute respiratory syndrome(SARS) has been link to a new coronavirus called SARS-associated coronavirus (SARS-CoV). The spike (S) protein of SARS-CoV is an important viral structural protein. The S protein is large type I transmembrane glycoprotein that are responsible for receptor binding and membrane fusion. It was the primary target for the host's immune responses and induced neutralizing antibodies. The S protein could be functionally differentiated into the corresponding domains: N-terminal S1 and C-terminal S2. The S1 domain involved in viral attachment with the cellular receptors and hence determines the host cell tropism. S2 domain involved in host cell entry and cell-to-cell fusion. We performed construction of eukaryotic expression vector, expression and purification the recombinant SI protein in mammalian cell for genetic engineering for the research SARS vaccine and pathologic analysis.Methods: The DNA fragment of S1 gene was amplified from PUC18 S plasmid encoding full length of SARS-CoV S gene by polymerase chain reaction(pcr) using Oligonucleotide primers. The amplified fragment, CD5 leader sequence and human IgG Fc fragment were then inserted into pEAK12 to create a new expression plasmid. 293E cells were transfected with this new plasmid encoding a codon-optimized form of SARS-CoV S1 protein. Cell clones which stable and high expressed recombinant protein were selected by screening using puromycin. The expressed protein was detected by western blotting and purified by Protein A affinity chromatography. Biological activity of recombinant protein after purification was analyzed by FACS. Results: the target gene were inserted into pEAK 12 vector with correct sequencing and confirmed by the results of the digestion with restriction endonucleases and sequence analysis. Recombinant protein after purification can binding VeroE6 cells with SARS-CoV S receptor.Conclusions:c Eukaryotic expression vector encoding SARS-CoV S1 protein gene was constructed successfully and SARS-CoV S1 protein with biological activity was expressed efficiently and stably in 293E cells, we made successfully the mammalian cell lines which stably expressed SARS-CoV S1 protein.