The Expression, Purification And Functional Analysis Of SARS-CoV Recombinant Spike Protein In Mammalian Cells

The expression, purification and functional analysis of SARS-CoV recombinant spike protein in mammalian cells.Severe acute respiratory syndrome (SARS) has been linked 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 typeâ… transmembrane glycoprotein which is responsible for receptor binding. It was the primary target for the host's immune responses and induced neutralizing antibodies. Because of the S protein is glycoprotein, mammalian cells is the best system to express and purify S protein for its biological activity function.The recombinant full length S protein and its mutants can be expressed and purified in high level in the mammalian cell system in this thesis. The recombinant S protein can bind to the cellular receptor angiotensin converting enzyme 2 (ACE2), and down-regulate the receptor ACE2 which has critical relationship with acute lung injury. ACE2 protects mice from severe acute lung injury induced by acid aspiration or sepsis in the paper. These data identify a critical function for ACE2 in acute lung injury, and the recombinant human ACE2 protects mice from severe acute lung injury which suggesting a novel therapy for an often lethal and previously untreatable syndrome that affects millions of people worldwide every year.In addition, the high yield of the recombinant S protein in mammalian cell system make it possible to use this protein to be SARS genetic engineering vaccine generally.Establishment of high-throughput drug screening model targeting retinoic acid receptor in cells.To screen new drug for the treatment of acute promyelocytic leukemia, psoriasis and acne, high-throughput drug screening cell models marked by green fluorescent protein (GFP) have been established. Eight repeats of retinoic acid response element (RARE) were synthesized and cloned into a GFP expression vector. This construct was stably transfected into cells in vitro. Stable and sensitive Cell clones with high copy numbers of RARE were selected by retinoic acid (RA) using fluorescence-activated cell sorting (FACS). A cell line has been chosen to be high-throughput drug screening cell model. This model was shown with low background, high sensitive and good reproducibility, and was convenient and inexpensive. This drug screening cell model can be used for retinoic acid receptor target high-throughput drug screening.